AU4676000A - Anti-microbial agents, diagnostic reagents, and vaccines based on unique apicomplexan parasite components - Google Patents

Description

WO 00/66154 PCT/US00/1 1478 -1 ANTIMICROBIAL AGENTS, DIAGNOSTIC REAGENTS, AND VACCINES BASED ON UNIQUE APOCOMPLEXIAN PARASITE COMPONENTS Inventors: Rima L.W. McLeod et al. 5 The U.S. government may have rights in this patent by means of partial support under: NIH NIAID TMP R01 Al 16945; NIH NIAID TMP R01 AI 27530, and NIH ROI AI 43228. 10 This invention relates uses of components of plant-like metabolic pathways not including psbA or PPi phosphorfructokinase and not generally operative in animals or encoded by the plastic DNA, to develop compositions that interfere with Apicomplexan growth and survival. Components of the pathways include enzymes, transit peptides and nucleotide sequences encoding the enzymes and peptides, or promoters of these 15 nucleotide sequences to which antibodies, antisense molecules and other inhibitors are directed. Diagnostic and therapeutic reagents and vaccines are developed based on the components and their inhibitors. A cDNA sequence that encodes chorismate synthase expressed at an early state of Apicomplexan development, is disclosed and may be altered to produce a "knockout" organism useful in vaccine production. 20 BACKGROUND Apicomplexan parasites cause the serious diseases malaria, toxoplasmosis, sryptosporidiosis, and eimeriosis. Malaria kills more than 2 million children each year. Toxoplasmosis is the major opportunistic brain infection in AIDS patients, causes loss 25 of life, sight, hearing, cognitive and motor function in congenitally infected infants, and considerable morbidity and mortality in patients immunocompromised by cancer, transplantation, autoimmune disease and their attendant therapies. Cryptosporidiosis is WO 00/66154 PCT/US00/11478 -2 an untreatable cause of diarrhea in AIDS patients and a cause of epidemics of gastrointestinal disease in immunocompetent hosts. Eimeria infections of poultry lead to billions of dollars in losses to agricultural industries each year. Other Apicomplexan infections, such as babesiosis, also cause substantial morbidity and mortality. Although 5 there are some methods for diagnosis and treatment of Apicomplexan caused diseases, some of these treatments are ineffective and often toxic to the subject being treated. The tests available to diagnose Apicomplexan infections include assays which isolate the parasite, or utilize light, phase, or fluorescence microscopy, ELISAs, agglutination of parasites or parasite components to detect antibodies to parasites, or 10 polymerase chain reaction (PCR) to detect a parasite gene. Most of the assays utilize whole organisms or extracts of whole organisms rather than recombinant proteins or purified parasite components. In many instances, the available assays have limited ability to differentiate whether an infection was acquired remotely or recently, and are limited in their capacity to diagnose infection at the outpatient or field setting. 15 The primary antimicrobial agents used to treat toxoplasmosis are pyrimethamine (a DI-HFR inhibitor) and sulfadiazine (a PABA antagonist). The use of pyrimethamine is limited by bone marrow toxicity which can be partially corrected by the concomitant administration of folinic acid. 7 gondii cannot utilize folinic acid but mammalian cells can. Another problem is that pyrimethamine is potentially teratogenic 20 in the first trimester of pregnancy. The use of sulfonamides is limited by allergy, gastrointestinal intolerance, kidney stone formation and Stevens-Johnson syndrome. There arc a small number of antimicrobial agents utilized less frequently to treat toxoplasmosE, These include clindanmycin. spiramycin, azithromycin, clarithrom'cini WO 00/66154 PCT/US00/11478 -3 and atovaquone. Usefulness of these medicines for treatment of toxoplasmosis is limited by toxicities including allergy and antibiotic-associated diarrhea, (especially Clostridium difficile toxin associated colitis with clindamycin use). Lesser or uncertain efficacy of macrolides such as spiramycin, azithromycin, and clarithromycin 5 also limits use of these antimicrobial agents. Atovaquone treatment of toxoplasmosis may be associated with lack of efficacy and/or recrudescent disease. There are no medicines known to eradicate the latent, bradyzoite stage of T. gondli, which is very important in the pathogenesis of toxoplasmosis in immunocompromised individuals or those with recurrent eye disease. 10 Medicines used to treat malaria include quinine sulfate, pyrimethamine, sulfadoxine, tetracycline, clindamycin, chloroquine, mefloquine, halofantrine, quinidine gluconate, quinidine dihydrochloride, quinine, primaquine and proguanil. Emergence of resistance to these medicines and treatment failures due to resistant parasites pose major problems in the care of patients with malaria. Toxicities of mefloquine include 15 nausea, vomiting, diarrhea, dizziness, disturbed sense of balance, toxic psychosis and seizures. Melfoquine is teratogenic in animals. With halofantrene treatment, there is consistent, dose-related lengthening of the PR and Qt intervals in the electrocardiogram. Halofantrene has caused first degree heart block. It cannot be used for patients with cardiac conduction defects. Quinidine gluconate or 20 dihydrochloride also can be hazardous. Parenteral quinine may lead to severe hypoglycemia Primaquine can cause hemolytic ancmia. especially in patients whose red blood cells are deficient in Ulucose 6-phosphate dehydrogenase Unfortunately. there are no medicines known to he clTecti e in the treatment of cryptosporidiosis WO 00/66154 PCT/US00/11478 -4 To more effectively treat Apicomplexan infections, there is an urgent need for discovery' and development of new antimicrobial agents which are less toxic than those currently available, have novel modes of action to treat drug resistant parasites that have been selected by exposure to existing medicines, and which are effective against 5 presently untreatable parasite life cycle stages (e.g., Toxoplasma gondli bradyzoites) and presently untreatable Apicomplexan parasites (e.g., Ctyptosporidium parvumn). Improved diagnostic reagents and vaccines to prevent these infections are also needed. Information available on Apicomplexan parasites has not yet provided keys to solutions to health problems associated with the parasites. Analogies to other 10 organisms could provide valuable insights into the operations of the parasite. There are reports of Apicomplexan parasites having plastids, as well as the nuclear encoded proteins, tubulin, calmodulin, PPi phosphofructokinase and enolase, which are reported to be similar in part to, or homologous with, counterparts in plant-like, lower life forms and higher plants. There are reports of a plastid genome and components of a protein 15 synthetic system in a plastid-like organelle of Apicomplexans. Plasmodium and T gondii plastid DNA sequences were reported to have homologies to algal plastid DNA sequences. The plastid membrane of T. gondii was reported to be composed of multiple membranes that appear morphologically similar to those of plant/algal chloroplasts. except for the presence of twvo additional membranes in the 7: gondii 20 plastid, suggesting that it may have been an ancient algal endosymbiont. Some of these Apicomplexan proteins such as tubulin. calmodulin and enolase with certain plant-like feCatuLres also are f und in animals, and therefore may appear in the host as well as the WO 00/66154 PCTIUS00/11478 -5 parasite. A homologue to a gene, psbA encoding a plant protein important for photosynthesis, also was said to be present in Apicomplexans. . Certain herbicides have been reported to inhibit the growth of Apicomplexans. The herbicides which affect growth of Apicomplexans are known to affect plant 5 microtubules or a plant photosynthetic protein. In addition, a compound, salicylhydroxamic acid, (SHAM), had been found to inhibit Plasmodiumnfalciparumi (malaria) and Babesia microti. Techniques of medicinal chemistry and rational drug design are developed sufficiently to optimize rational construction of medicines and their delivery to sites 10 where Apicomplexan infections occur, but such strategies have not yet resulted in medicines effective against Apicomplexans. Rational development of antimicrobial agents has been based on modified or alternative substrate competition, product competition, change in enzyme secondary structure, and direct interference with enzyme transport, or active site. Antisense, ribozymes , catalytic antibodies, disruption 15 of cellular processes using targeting sequences, and conjugation of cell molecules to toxic molecules are newly discovered strategies employed to interrupt cellular functions and can be utilized to rationally develop novel antimicrobial compounds, but they have not yet been utilized to design medicines effective against Apicomplexans. Large scale screening of available compounds with recombinant enzymes is used to 20 identify potentially effective anti-microbial agents. Reagents to diagnose Apicomplexan parasite infections have been developed tarligeting components of Apicomplexans or immuneC responses to the parasites. using ELISA\. wes ern blot, and lPCR technologies. but improved diagnostic reaglenlts, WO 00/66154 PCT/US00/11478 -6 especially those that establish duration of infection or that can be used in outpatient settings are needed to diagnose Apicomplexan infections. No vaccines to prevent Apicomplexan infections are available for humans and only a live vaccine prepared for prevention of toxoplasmosis in sheep is available for livestock. 5 To summarize, Apicomplexan parasites cause substantial morbidity and mortality, and treatments against the parasites are suboptimal or non-existent. Improved antimicrobial compounds that attack Apicomplexan parasites are needed. Because the diseases Apicomplexan parasites cause in some instances are due to recrudescence of latent parasites, an especially pressing clinical problem is that there are 10 no effective antimicrobial agents effective for treatment of these latent parasite life cycle stages, especially in sequestered sites such as the brain or eye. New approaches and drug targets are required. Better in vitro and in vivo assays for candidate compounds are also needed. Better diagnostic and therapeutic methods, reagents and vaccines to prevent these infections are needed. 15 SUMMARY OF TIlE INVENTION This invention relates uses of components of plant-like metabolic pathways (not usually associated with animals, not encoded in the plastid genome, and not including psbA or PPi phosphofructokinase) to develop compositions that interfere with 20 Apicomniplexan growth and survival. Components of the pathways include enzymes, transit peptides and nucleotide sequences encoding the enzymes and peptides, or promoters of these nucleotide sequences. to \which antibodies. antisense molecules and other inhibitors are directed. Diagnostic and therapeutic reagents and vaccines are WO 00/66154 PCT/US00/11478 -7 developed based on the components and their inhibitors. Attenuation of live parasites through disruption of any of these components or the components themselves provide vaccines protective against Apicomplexans. Transit peptides are used to identify other proteins and their organelle targeting 5 sequences that enter and exit from unique Apicomplexan organelles. The identified components are potential for production of medicines, reagents and assays, and vaccines. The protein which includes the transit peptide is not necessarily an enzyme in a biochemical pathway. The methods and compositions of the present invention arise from the 10 inventors' discovery that metabolic pathways, and targeting signals similar to those found in plants and algae, especially, but not exclusively those encoded within the nucleus, are present in Apicomplexan parasites. These plant-like pathways in Apicomplexan parasites are targetable by inhibitors, as measured by determining whether the inhibitors, either singly or in combination, are effective in inhibiting or 15 killing Apicomplexan parasites in viitro and/or in vivo. The present invention includes new methods and compositions to treat, diagnose and prevent human and veterinary disease due to Apicomplexan infections. The invention is based on applications and manipulations of components of algal and higher plant-like metabolic pathways discovered in Apicomplexan parasites. "Plant 20 like" means that products of the pathways. enzymes and nucleotide sequences encoding enzymes in the pathways, are homologous or similar to products, enzymes and nucleotide secI'ences known in plants. << herein plants include algae. As used herein, plant-like e:,:c!udes metabolic pathways generally operative in or identical to those in WO 00/66154 PCTUS00/11478 -8 animals and pathways involving psbA or phosphofructokinase and those encoded by the plastid genome. The limits of a "pathway" are defined as they are generally known to those of skill in the art. Methods to detect plant counterparts in Apicomplexan include: a) immunoassays using antibodies directed to products and enzymes known in plants, 5 b) hybridization assays using nucleotide probes that hybridize to specific sequences in plants; c) determining homologies of Apicomplexan nucleotide or protein sequences with plant nucleotide or protein sequences; and/or d) substrate tests for specific enzymatic activity. The "plant-like" pathways of the present invention are identified by: 10 a) identification of metabolic pathways characteristic of plants but not generally present in animals; b) identification and characterization of Apicomplexan enzymes, nucleic acids and transit sequences as components similar or homologous to those in a); c) identification and development of compounds (inhibitors) which abrogate the 15 effect of the components of the pathways inll vitro and in vivo, singly or in a plurality, against one or more types of Apicomplexan parasites and in conjoint Apicomplexan, bacterial and fungal infections. The identified pathways are then used for: a) rational design or selection ofcompounds more active than thile known 2- compounds (inhibitors), with good absorption following oral administration, with appropriate tissue distribution and without toxicity or carcinogenicity; b) testing of such rationally designed compounds alone and together for safety, etlicacv and appropriate absorption and tissue distributor in ,vitroY) and inl i'luo WO 00/66154 PCT/US00/11478 -9 c) development and testing of diagnostic reagents and assays; d) development and testing of live attenuated and component based vaccines. By locating new targets in Apicomplexan pathways, doors now are open for development of more effective antimicrobial agents to treat Apicomplexan parasites in 5 humans and agricultural animals. In addition, enzymes in these plant-like pathways provide improved diagnostic tests for diseases caused by Apicomplexans. Vaccines against infectious diseases caused by Apicomplexan parasites are derived from the novel compositions of the invention. A method for inhibiting an Apicomplexan parasite, includes selecting the 10 metabolic pathway of the present invention and interfering with the operation of the pathway in the parasite. The Apicomplexan parasite is preferably selected from the group that includes Toxoplasma, Plasmodium, Ciyptosporidia, Eimeria, Babesia and 7heileria. The pathway may utilize a component encoded by an Apicomplexan nuclear gene. 15 Suitable metabolic pathways or components include a) synthesis of heme from glutamate and tRNA glu by the plant-like, henme synthesis (5 carbon) pathway (hereinafter the "heme synthesis pathway"), b) synthesis of C4 acids (succinate) by the breakdown oflipids into fatty acids and then acetyl CoA, and their use in the glyoxylate cycle (hereinafter the 20 "glvoxvlate cycle"). c) synthesis of chorismate from phosphoenolpyruvate and erythrose 4 phosphate by the shikimniate pathwvav (hereinafter thie "shikimatc pathway ). d) svn-hcsis of tetrahydroflate from clihorisminate by the shikimate pathway.

WO 00/66154 PCT/US00/11478 -10 e) synthesis ofubiquinone from chorismate by the shikimate pathway; f) electron transport through the alternative pathway with use of the alternative oxidase (hereinafter the "alternative oxidase pathway"); g) transport of proteins into or out of organelles through the use of transit 5 sequences; h) synthesis of aromatic amino acids (phenylalanine, tyrosine and tryptophan) from chorismate by the shikimate pathway; i) synthesis of the menaquinone, enterobactin and vitamin KI from chorismate by the shikimate pathway; 10 j) synthesis of the branched chain amino acids (valine, leucine and isoleucine) from pyruvate and ketobutyrate by the plant-like branched chain amino acid synthesis pathway; k) synthesis of the "essential" (i.e.. not synthesized by animals) amino acids, histidine. threonine, Ivsine and methionine by the use of plant-like amino 15 acid synthases; I) synthesis of linoleneic and linoleic acid; m) synthesis of amrnylose and amylopectin with starch synthases and Q (branching) enzymes and their degradation; n) synthesis of auxin growth regulators from indoleacetic acid derived from 20 chorismate; o) synthesis ofisoprenoids (diterpenes. 5 carbon units with some properties of lipids) such as giberellins and abscidic acid by the mevalonic acid to gibhelli pat hway.

WO 00/66154 PCT/US00/11478 -11 The interfering compositions are selected from the group consisting of enzyme inhibitors including competitors; inhibitors and competitive or toxic analogues of substrates, transition state analogues, and products; antibodies to components of the pathways; toxin conjugated antibodies or components of the pathways; antisense 5 molecules; and inhibitors of transit peptides in an enzyme. In particular, the interfering compositions include gabaculine, 3-NPA, SHAM, 8-OH-quinoline, NPMG. Interfering with the operation of the metabolic pathway is also accomplished by introducing a plurality of compositions to the pathway, wherein each of the compositions singly interferes with the operation of the metabolic pathway. In certain instances, the 10 plurality of compositions inhibits the parasite to a degree greater than the sum of the compositions used singly, that is, exhibits a synergistic effect. Embodiments of a plurality of compositions include gabaculine and sulfadiazine; NPMG and sulfadiazine; SHAM and gabaculine; NPMG and pyrimethamine; NPMG and cycloguanil (which inhibits Apicomplexan DHFR [TS]), and other inhibitors and competitors of interrelated 15 cascades of plant-like enzymes. Wherein the effect of inhibitors together is greater than the sum of the effects of each alone, the synergistic combination retards the selection of emergence of resistant organisms and is more effective than the individual components alone. In various embodiments, the interfering composition acts on a latent bradyzoite 20 form of the parasite, or multiple infecting Apicomplexan parasites simultaneously, or on conjoint infections with other pathogenic microorganisms which also utilize the plant like metabolic pathway.

WO 00/66154 PCT/US00/11478 -12 A method of determining the effectiveness of a composition in reducing the deleterious efftYects of an Apicomplexan in an animal, include: a).identifying a composition that inhibits growth or survival of an Apicomplexan parasite in vitro by interfering with a plant-like metabolic pathway and b) determining a concentration of 5 the composition in an animal model that is non-toxic and effective in reducing the survival of the parasite in the animal host and/or the deleterious effects of the parasite in the animal. Developing a lead compound that inhibits an Apicomplexan parasite is accomplished by a) identifying a plant-like metabolic pathway in an Apicomplexan 10 parasite and b) identifying a composition that interferes with the operation of the pathway as a lead compound. A composition which inhibits a specific life cycle stage of an Apicomplexan parasite by interfering with a plant-like metabolic pathway that utilizes a component encoded by a nuclear gene includes gabaculine; a composition including an enzyme in a 15 metabolic pathway in an Apicomplexan parasite that is selectively operative in a life cycle stage of the parasite includes the enzymes alternative oxidase, and UDP glucose starch glycosyl transferase. A composition comprising SHAM and 8-OH-quinoline inhibits the alternative oxidase in the latent bradyzoite form of an Apicomplexan parasite. 20 A method to identify a plant-like gene encoding a component of a plant-like metabolic pathway in an Apicomplexan parasite is a) obtaining a strain of E. coli that is deficient for a component of the metabolic pathway, said deficiency causing the strain to require suLpplecImented media lbr grov.th, b) complementing the E. co/i with a gene WO 00/66154 PCTIUS00/11478 -13 or portion of the gene encoding a component of the metabolic pathway in the Apicomplexan parasite; and c) determining whether the complemented E. coli is able to grow in unsupplemented media, to identify the gene. Another method for identifying a plant-like gene product of a metabolic 5 pathway in an Apicomplexan parasite is a) contacting the parasite with a gene probe; and b) determining whether the probe has complexed with the parasite from which the identity of the gene product is inferred. A method for identifying a plant-like gene product of a metabolic pathway in an Apicomplexan parasite also includes: a) cloning and sequencing the gene; and b) 10 determining whether the gene is homologous to a plant gene which encodes a plant enzyme with the same function. A method for identifying a plant-like gene product in a metabolic pathway in an Apicomplexan parasite is a) contacting the parasite or its enzyme with a substrate for the plant-like enzyme; b) measuring enzyme activity; c) determining whether the 15 enzyme is oper.-ative; and d) inhibiting activity of the enzyme in vitro with an inhibitor. Identiring a gene or gene product in an Apicomplexan parasite which possesses an organelle transit sequence which transports a protein, wherein the protein is not necessarily an enzyme in a metabolic pathway, but is identified because it has a characteristic ,rganelle transit sequence is also within the scope of the invention. 20 The invention also relates to a diagnostic reagent for identifying the presence of an Apicomple xan parasite in a subject, where the subject includes a domestic or livestock aniral or a human The reagent may include all or a portion of a component of the plant-Uke pathway, an antibody specific l'or an enzyme that is a componcnit ola WO 00/66154 PCT/US00/11478 -14 plant-like metabolic pathway in the parasite. or all or part ofa nucleotide sequence that hybridizes to a nucleic acid encoding a component of the pathw.ay. A diagnostic assay that identifies the presence ofan Apicomplexan parasite or specific life-cycle stage of the parasite may use the diagnostic reagents defined herein. 5 A diagnostic reagent for identifying thile presence of an Apicomplexan parasite, includes an antibody specific for an enzyme that is part of a plant-like metabolic pathway. A diagnostic assay for the presence of an Apicomplexan parasite in a biological sample includes: a) contacting the sample with an antibody selective for a product of a 10 plant-like metabolic pathway that operates in an Apicomplexan parasite; and b) determining whether the antibody has complexed with the sample, from which the presence of the parasite is inferred. Alternatively, the assay is directed towards a nucleotide sequence. In both these cases, appropriate antibody or nucleotide sequences are selected to distinguish infections by different Apicomplexans. 15 An aspect of the invention is a vaccine for protecting livestock animals, domestic animals or a human against infection or adverse consequences of infection by an Apicomplexan parasite. The vaccine may be produced for an Apicomrnplexan parasite in which a gene encoding a component of a plant-like metabolic pathway in the parasite is manipulated, for example, deleted or modified. \When the gene is 20 deleted or modified in the live vaccine, the component of the pathway may be replaced by the presence of the product of an enzymatic reaction in tissue culture medium. The vaccine strain can then be cultivated in vitr to make the vaccine WO 00/66154 PCT/US00/11478 - 15 A vaccine for protecting animals against infection by an Apicomplexan parasite is based on an Apicomplexan parasite in which the parasite or a.component of a metabolic pathway in the parasite is used. The vaccine may use a component of the pathway that is operative at a 5 particular life stage of the parasite. A suitable component is the AroC gene from 7T. gondli or P. falciparum. A method of treatment for an infection in a subject by an Apicomplexan parasite includes the following steps: a) obtaining an inhibitor of a plant-like metabolic pathway in an Apicomplexan parasite; and b) administering an effective amount of the 10 inhibitor to the subject. BRIEF DESCRIPTIONS OF DRAWINGS FIG. IA-C illustrates the hemne synthesis pathway and the effect of GSAT in 7' gondii. 15 FIG. IA diagrams the hemne synthesis pathway. FIGS. IB and IC show that uptake of tritia'ed uracil by tachyzoites (RH strain) is inhibited by gabaculine, an inhibitor of GSA aminotransferase. P/S = pyrimethamine and sulfadiazine. Note that ALA synthase is also present in 7' godii and constitutes an alternative pathway for hemne synthesis 20 FIG. 2A-B shows unique lipid degradation in the glyoxylate cycle in 7' gudt. FIG. 2A is a schematic representation of the glyoxylate cycle. FIG. 21 shows uptake oftritiaied uracil by tachyzoites (RH strain) is inhibited by 3-NPA (0 005 to 5 WO 00/66154 PCT/US00/11478 - 16 mg: G/ML). Note this inhibitor also effects succinate dehydrogenase, so its inhibitory effect does not unequivocally support presence of the glyoxylate pathway. FIG. 3A is a schematic representation of a pathway which demonstrates alternative oxidase as an alternative pathway for generation of energy in Apicomplexan 5 parasites. FIG. 3B shows that uptake of tritiated uracil by tachyzoites (RH strain) is inhibited by SHAM. FIG. 4A is a schematic representation of the pathway for conversion of shikimate to chorismate in T. gondii.. The inhibitor of EPSP synthase is NMPG. FIG. 4B shows uptake of tritiated uracil by tachyzoites (RH strain) is inhibited by 10 NPMG. Toxicity of NPMG was assessed by its ability to prevent growth of human foreskin fibroblasts (HFF) after 4 days, as measured by tritiated thymidine uptake and microscopic evaluation. FIG. 4C shows product rescue of NPMG's inhibitory effect on EPSP synthase by PABA. The effect of PABA on sulfadiazine is similar, but the effect on pyrimethamine, as predicted reduces thile enzyme to the levels that were 15 present when media alone was utilized, as measured by the uracil uptake. S = sulfadiazine PYR = pyrimethamine PABA = para amino benzoic acid. FIG. 4D shows functional and enzymatic evidence for the shikimnate pathway in 7: 20 gondii with inhibition of EPSP synthase enzyme activity by I mM glyosate. Squares, without glyphosate. Circles, with glyphosate FIG. 4E shows evidence for the shikimate path-.%ay in P.fudcipanmi with litnctional evidence for the shikimnate pathwav ill P..idcipn' GlyphvIosate inhibition olin vi/rlo .rowth of asexual erythrocytic WO 00/66154 PCTIUS00/11478 - 17 forms and PABA and folate antagonism of growth inhibition. Effect of NPMG on C. parvum was not abrogated by PABA. This suggests that either uptake of PAPA by C. parvum differs or effect of NPMG is on a different branch from the shikimate pathway in C. parvum. 5 FIG. 5 is a schematic representation of interrelationships of metabolic pathways in Apicomplexan parasites. FIG. 6 shows inhibitory effects of NPMG, gabaculine, SHAM 8-OH-quinoline on Cryptosporidia. 3NPA also inhibited Cryptosporidia. FIG. 7 shows the effects of gabaculine (20 mM) on growth of 10 tachyzoites/bradyzoites (R5) in human foreskin fibroplasts, over 8 days as determined by uracil uptake. Note increased uptake of uracil by the 8th day. FIG. 8 shows the effect of NPMG, pyrimethamine, and pyrimethamine plus NPMG on survival of mice following intraperitoneal infection with 500 tachyzoites of the RH strain of T. gondii. Dosage of NPMG was 200mg/kg/day and pyrimethamine 15 was 12.5 mg/kg/day). FIG. 9 shows nucleotide and deduced amino acid sequences of T. gondii chorismate synthase cDNA. The asterisk indicates the stop codon. FIG. 10 shows results of CLUSTAL X alignments of the deduced amino acid sequences if the putative T. gondii, chorismate synthase with the corresponding 20 sequences from Synechocystites, S. cerevisiae, S. lypocersicum, N. crassa and H. influenza. Dashes were introduced maximize alignment. Amino acids which are identical in all 6 organisms are underlined. The percent identity of the chorismate synthase from each organism with the T. gondii protein was calculated to be as follows WO 00/66154 PCT/US00/ 1478 - s18 Synechocysits (51.4%). S. cerevisiae (49.6%), S. lycopersicmnt (47.2%), N. crassa (45.0%) and H. influenza (44.5%). The large internal regions in the 7" gondii sequence which have no counterparts in the chorismate synthases of other organisms, were not included in this calculation. 5 FIG. 11 shows the transit sequences of Zea mays and T. gondii chorismate synthases. The sequences of the transit peptide directing the transport of the wx+ protein into maize amyloplasts and chloroplasts and the portion of the T. gondii chorismate synthase sequence which is homologous are aligned. The amino acid sequence is given in one letter code. * indicates an identical amino acid in the Wx Zea 10 mays and T. gondii sequences. * indicates homologous amino acids in the Wx Zea mays and T. gondii sequences. The transit sequence in the Wx Zea mays protein (UDP-glucose-starch-glycosyl transferase) begins at amino acid number I and ends at amino acid number rit-riiir CLJl IT IPII 11 F C11I WO 00/66154 PCT/US00/11478 -'9 72. The portion (amino acids 359 to 430) of P. falciparum AroC which corresponds to the novel internal sequence of the T. gondii AroC which includes the amino acids homologous to the maize protein, is as follows: IPVENMSTKKESDLLYDDKGECKNMSYHSTIQNNEDQILNSTKGFMPPKNDKNFNNIDDYNVTFNNNEEKLL 5 The T. gondii portion of the AroC (chorismate synthase) sequence which demonstrates 30% homology begins at amino acid number 330 and ends at amino acid number 374. The first (single) arrow indicates the processing site of Zeamays UDP glucose Gylcosyl transferase transit peptide and the second (double) arrow indicates the location at which the mature protein begins. 10 FIG. 12 shows P. falciparum, chorismate synthase cDNA and deduced amino acid sequences. FIG. 13 shows a genomic sequence of T. gondii chorismate synthase. FIG. 14 shows (A) a T. gondii cDNA chorismate synthase DNA construct which is useful to produce antibody or a vaccine; (B) a Western blot (arrows mark 15 chorismate synthase). FIG. 15 shows green fluorescent (gfp) protein expression in a stably transfected tachyzoite; this tachyzoite has a reporter construct, a chorismate synthase gfp; gfp is cytoplasmic (grey) and a defined structure in the area of the plastid is the white dot; the nucleus is the larger red area; gfp is in the cytoplasm. 20 FIG. 16 shows life cycle stage associated expression and localization of chorismate synthase in T. gondii. (A) Tachyzoites: (1) 15 days - - Double stained with tachyzoite surface antigen 1 (SAG 1) (perimeter raised, top and bottom left) and DNA stain (DAPI) (bottom right) and chorismate synthase (top right, white); (2) Double stained with 25 dense granule protein 4 (granular stain, top left), chorismate synthase (white); p30, lower right panel, (perimeter raised) rhoptry probe (raised grey, rhop); (3) Double stained chorismate synthase-punctate white, SAG1 (P30, perimeter raised). (Note discrete punctate white area of chorismate synthase staining in perinuclear area, the customary subcellular location of the plastid). 30 (B) Bradyzoites: (1) Abbreviations are the same as in A; Note diffuse granular appearing cytoplasmic staining of bradyzoite chorismate synthase (top e'-t"rin Q1-1CIT II F 1 Q1 I WO 00/66154 PCT/US00/11478 -20 fight); (2)Immunoperoxidase stain with antibody to recombinant chorismate synthase shows diffuse cytoplasmic darker staining. (C) Microgametes (Mi), Macrogametes (Ma); Note darker immunoperoxidase staining of these forms but not schizonts (s) in cat intestine. 5 (D) Chorismate synthase mRNA production in tachyzoites and with bradyzoites; Note SAG1 message for a tachyzoite protein, BAG 1-5 message for a bradyzoite protein and constitutively expressed mRNA for tubulin. FIG. 17 shows: (a) schematic illustration of glyoxylate cycle, (b) inhibitors of isocitrate lyase (ICL), (c) T. gondii isocitrate lyase enzyme activity, (d) inhibition of 10 ICL enzyme activity by 3NPA, and (e) inhibition of tachyzoites in tissue culture. FIG. 18 shows a T. gondii isocitrate lyase (ICL) cDNA sequence. FIG. 19 shows a T. gondii isocitrate lyase (ICL) amino acid sequence. FIG. 20 shows (a) T. gondii isocitrate lyase (ICL) binding pocket and active site inside box, and (b) comparison with the published sequence of yeast isocitrate 15 lyase with mutated lysine (K) which inactivated the enzyme (arrows). FIG. 21 shows a T. gondii isocitrate lyase genomic DNA sequence (ICL). FIG. 22 shows T. gondii isocitrate lyase in bradyzoites; Note brown areas in immunoperoxidase stain preparation. FIG. 23 shows isocitrate lyase (a) in a western blot of tachyzoites (b) during 20 stage conversion, and (c) mRNA during stage conversion. (Abbreviations are the same as in FIG. 16A and D legends). FIG. 24 shows enzymatic, genetic, functional activity of Apicomplexan parasites and its inhibition and show T. gondii acetyl coA carboxylase is inhibited by fop herbicides: 25 (A) Acetyl coA carboxylase enzyme activity is inhibited by -fop herbicides; (B) T. gondii growth in tissue culture inhibited by compounds that inhibit acetyl coA carboxylases; (Note the inhibitor activity is parallel to that in FIG. 24A. Clodinafop is a lead 30 compound. T. gondii uptake of 3H uracil is inhibited by fop herbicides.) (C) Effect of clodinafop on T. gondii with 4 days in culture then removal of the herbicide for 2 days. Note plaques (A) and (C) higher view of r -,-r-rr_rr- C. teCT 11:2 II 5= 011 WO 00/66154 PCT/US00/11478 -21 replicating parasites in these plaque controls and complete eradication of parasites in clodinafop (10(M) treated cultures; Related sequences of Apicomplexan acetyl coA carboxylases; sequences of acetyl coA carboxylase biotin carboxylase domains from 5 apicomplexan parasites are as in Genbank Accession Numbers AF 157612-16. Also, a domain swap yeast with the T. gondii active site and recombinant enzymes made from a fragment of the T. gondii gene are amenable to high throughput screens; Phylogeny of biotin carboxylase domains of apicomplexan accases; 10 Structures of herbicides that inhibit acetyl coA carboxylases. r-a-nsen u=C:T tal II I= 011 WO 00/66154 PCT/US00/ 1478 - 22 DESCRIPTION OF THE PREFERRED EMBODIMENT 5 This invention uses components of plant-like interrelated metabolic pathways that are essential for growth or survival of Apicomplexan parasites. The pathways are generally not operative in animals and do not include psbA or PPi phosphofructokinase and are not encoded in the plastid. Components include enzymes, products, targetting. peptides, nucleotide sequences encoding the enzymes or peptides, and promoters, as 10 targets for specific inhibitors. Use of these pathways provide a rational and novel framework to discover, characterize and develop medicines, diagnostic reagents and vaccines for Apicomplexan parasites. Medicines, diagnostic reagents and vaccines are based upon interrelated plant like enzyme cascades involved in the synthesis or metabolism or catabolism of 15 Apicomplexan nucleic acids, amino acids, proteins, carbohydrates or lipids, energy transfer and unique plant-like properties of these enzymes which are shared with, and provide a basis for, discovery of other parasite proteins which have unique organelle targeting signals or unique promoter regions of the genes which encode the proteins. Synergistic combinations of inhibitors of the enzymes or proteins or nucleic acids 20 which encode them are particularly useful in medicines. To select pathways for use in the invention: a) plant textbooks and the published literature are reviewed for properties characteristic n' plants. but uenerallh not animals, databases such as Genbank or ithe WO 00/66154 PCT/US00/11478 - 23 Apicomplexan ESTs are reviewed to identify homologous Apicomplexan and plant-like genes; and b) Western, northern and southern analyses, PCR, and ELISAs are used to recognize, or are based upon, for example, plant proteins and genes, to determine 5 whether components of the pathways are present in Apicomplexans; c) cloning, isolation and sequencing of genes and creation of gene constructs are used to identify Apicomplexan plant-like genes and their functions; d) assays of enzyme activity are used to determined the operation of plant like systems; 10 e) functions of parasite enzymes or part of a parasite enzyme are demonstrated by complementation of a yeast or bacteria deficient in the enzyme, or product rescue, or other methods to demonstrate enzyme activity; f) activity of compounds, (i.e., inhibitors) known to abrogate effect of the plant-like enzyme, protein, or nucleic acid which encodes them in vitro and in vivo, 15 are tested singlyv or in a plurality, for ability to abrogate the enzyme activity and against Apicomplexan parasites alone or together, and in conjoint Apicomplexan, bacterial and fungal infections. The general compositions of this invention are: A. Inhibiori-v compounds based on: 20 a) targeting proteins by 1i) substrate competition and transition state analogtucs i) product coim etition WO 00/66154 PCT/US00/11478 - 24 (iii) alteration of active site directly or by modification of secondary structure or otherwise altering function of the active site (iv) interfering with protein function with antibody (v) targeting an organelle or protein within an organelle using a toxic 5 compound linked to a targeting sequence. b) targeting nucleic acids encoding proteins (antisense, ribozymes) c) targeting a component of the protein or nucleic acid (as above) B. Diagnostic reagents (genes, proteins, antibodies) in ELISAs, western blots, DNA, RNA assays 10 C. Vaccines (live knockout, live mutated, components - genes, proteins, peptides, parts of genes constructs, etc.) Specific examples of components of plant-like Apicomplexan pathways are in Table I. Compounds known to inhibit these enzymes or properties in Apicomplexans and/or other microorganisms are listed in Table 1, as are novel ways to target them in 15 Apicomplexans.

WO 00/66154 PCTUS00/11478 - 25 Table I A. Apicomplexan plant-like metabolic pathways, components and inhibitors Function Gene Enzyme or property Known inhibitors of Basis for novel name enzymes or property inhibitor HEME SYNTHESIS HemL glutamate- -semialdehyde 3-amino-2,3-dihydrobenzoic SAS,R aminotransferase (GSAT) acid (Gabaculine); 4-amino 5-hexynoic acid; 4-amino-5 fluoropentanoic acid; 4 amino-5-hexynoic acid (Y acetylenic GABA); 2-amino 3-butanoic acid (vinyl glycine); 2-amino-4 methoxy-trans-3-butanoic; 4-amino-5-fluoropentanoic acid GIVt( glutamyl-tRNA synthase HemA glutamyI-tRNA reductase SHIKIMATE PATHWAY Chorismate AroA 3-enolpynirvyshikimate N-(phosphonomethyl) S.ASR synthesis phosphate synthase (3- glycine (glyphosphate), phosphoshikimate-1 sulfosate, EPSP synthase carboxyvinyltransferase) Inhibitors 4 and 5, hydroxymaonate inhibitors of EPSP synthase " AroB dehydroquinate synthase (5 dehydroquinate dyhdrolase) AroC chorismate synthase 5 enolpyruvylshikimate 3 phosphate phospholyase) AroC-ts AroC transit sequence AroO dehydroquinate dehydratase AroE shikimate dehydrogenase AroF 3-deoxy-d-arabino heptulosonate 7 phosphate synthase AroG chonsmate mutase (7-phospho 2-dehydro-3-deoxy-arabino heptulate aidolase) AroH 3-deoxy.d-arabino.heptulosante 7 phosphate synthase Arol shikimate 3. phcspholransferase (sh;Wimate kina3e ) WO 00/66154 PCT/US00/11478 - 26 Function Gene Enzyme or property Known inhibitors of Basis for novel name enzymes or property inhibitor Ubiqinone synthesis UbiA 4-hydroxybenzoate S,AS,R octaprenyltransferase UbiB 3-oxtaprenyl-4-hydroxybenzoate carboxylyase UbiC chorismate synthase Tyrosine synthesis TyrA prephenate dehydrogenase S.AS.R TyrB aromatic acid aminotransferase (aromatic transaminase) TyrC cyclohexadienyl dehydrogenase Tryptophan TrpA tryptophan synthase alpha sub S,AS.R synthesis unit Trps tryptophan synthase beta sub unit TrpC indole-3-glycerol phosphate synthase (anthranilateisome rase) (indoleglycerol phosphate synthase) TrpO anthranilate phosphoribosyltransferase TrpE anthranilate synthase component I TrpF phosphoribosyl anthranilate isomerase TrpG anthranilate synthase component II Phenylatinine PheA Prephenate dehydratase S.AS,R Synthesis (phenol 2-mono-oxygenase), chorismate mutase PheB Catechof 1.2-deoxygenase (phenol hydrox'ylase) SPheC Cyclohexadienyl dehydrataseU Folate Synthesis pabA 4.amino-4-deoxy chorismate S.AS.R synthase II, amidotransferase pab8 4-amino-4-deoyr chorismate synthase I. binding component pabC 4.amino.4.deo,i chonsmate lyase WO 00/66154 PCT/US00/11478 - 27 Function Gene Enzyme or property Known inhibitors of Basis for novel name enzymes or property inhibitor Menaquinone. Enta Isochorismate synthase SAS,R enterobactin synthesis Entb 2.3 dihydro 2,3 dihydroxy benzoate dehydrogenase Entc 2.3 dihydro 2,3 dihydroxy benzoate synthetase ORGANELLE AroC-ts Transport into plastid, organelle S,AS,R TRANSIT targeting ALTERNATIVE AOX Altemrnative oxidase 8-hydroxyquinoline. S,ASR,D RESPIRATION 3-hydroxyquinone, salicythydroxamic acid, monoctone. benzhydroaxamic acid, m-Chlorohydroxamic acid, propylgallate, disulfuram, and others GLYOXYLATE MS Malate synthase S.AS.R CYCLE ICL Isocitrate lyase 3NPA, itaconic acid, 3 nitro propanol Key: S, mcdified substrate competitor; AS, antisense; R, ribozyme; Directed at active site, D; None known, *EPSP synthese inhibitor 4 refers to 3-(phosphonooxy)-4-hydroxy-5-(N-(phosphonomethyl-2 ' oxoethyl)aminc-t-cyclohexene-1l-carboxylic acid (3cx, 4(z, 503), compound with diethyl ethanamide EPSP synthase inhibitor 5 refers to shortened R phosphonate. "A new aromatic analogue of the EPSP synthase enzyme reaction intermediate 1 has been identified, which contains a 3- hydroxymalonate moiety in place of the usual 3-phosphate group. This simplified inhibitor was readily prepared in five steps from ethyl 3,4-dihydroxybenzoate. The resulting 10 tetrahedral intermediate mimic is an effective, competitive inhibitor versus S3P with an apparent K(i) of 0.57 +/- 0.06 muM. This result demonstrates that 3- hydroxymalonates exhibit potencies comparable to aromatic inhibitors containing the previously identified 3-malonate ether replacements and can thus function as suitable 3-phosphate mimics in this system. These new compounds provide another example in which a simple benzene ring can be used effectively in place of the more complex 1I shikimate rinc int the design of EPSP synthase inhibitors. Furthermore, the greater potency of the tetraheral intermediate mimic versus the glycolate derivative and the 5- deoxy analog, again confirms the requirement for multiple anionic charges a: ;he dihydroxybenzoate 5-position in order to attain effective inhibition of this enzyme. The following were identified: inhibition of Toxopflasma gondii (Tg), Plasmodium falciparum (PI). and 21 - Cryptosporidiury carvum (Cp) EPSP synthase by i.-phosphonomethylglycine (NPMG); Tg and Pf chorismate syntha.se (AroC) cDNA and deduced a-rirno acid sequences; a novel sequence in the Tg chorismate synthase gene (AroC-ts) a portion of which is homologous with the plastid transit sequence of Zea mays (sweet corn) The Pfchonrismate syvrase (AroC) also has a corresponding novel and unique internal region Cp. Einmenao bovis (Eb) gencmic DNA which hybridizes with Tg AroC 2 (chorismate synthase) Inhcition of Tg in wtro cy LNPMG abrogated by para-aminobenzoate (PABA) Inhibition of Pfn ,vero by t..MG abrogated by FATA and folate Inhibition of Tg EPSP synthese activity by NFMG i 1 vitro Synergism of NPMG r.;th pyrimethamine, with sulfadiazine and with SHAM WO 00/66154 PCT/US00/11478 -28 for Tg in vitro; Synergy of NPMG with pyrimethamine against Tg in vivo; SHAM and 8-hydroxyquinoline inhibited Tg. Pf. Cp in vitro; reactivity of Tg protein of -66Kd with 5 antibodies (monoclonal and polyclonal to VooDoo lily and T brucei alternative oxidases) and reduction to monomer similar to VooDoo lily and T. brucei alternative oxidases on a reducing gel; Identification of Tg cONA and 5 genomic DNA PCR products using primers based on conserved sequences in other alternative oxidases which are probed and sequenced; Tg, Pf, Cp inhibited by high concentrations of gabaculine. Reactivity of Tg protein of ~40Kd with 3 antibodies to GSAT (polyclonal a soybean, barley and synechococcus GSATs and not preimmune sera). Reactivity of Cp protein of ~40Kd with a barley GSAT. Inhibition of Tg, Pf, Cp in vitro by 3NPA; Reactivity of Tg protein with polyclonal antibodies to 10 cotton malate synthase and cotton isocitrate lyase but not preimmune sera. In screening Tg cONA library a GSAT antibody reactive clones are identified and are sequenced. Tg chorismate synthase and dehydroquinase enzymatic activities are demonstrated.

WO 00/66154 PCT/US00/11478 - 29 Table l B. Components of Plant-Like Metabolic Pathways and Inhibitors Function Gene Enzyme or Known inhibitors of enzyme or Basis for name property property novel inhibitor BRANCHED- ahas acetyhydroxy Imidazolinones imazquin=2-(4,5- S,AS,R CHAIN AMINO acid synthase dihydro-4-methyl-4-(1 ACID methylethyl)-5-oxo-1lH-imidazol SYNTHESIS 2-yl]-3-quinolinecarboxylic acid; (VALINE, imazethapyr-2-[4,5-dihydro-4 LEUCINE, methyl-4-(1 -methylethyl)-5-oxo ISOLEUCINE) 1 H-imidazol-2-yll-3 pyridinecarboxylic acid; imazapyr=()-2-(4,5-dihydro-4 methyl-4-(1-methylethyl)-5-oxo 1H-imidazol-2-ylj-3 pyridinecarboxylic acid, Sulfonyluteas chlorimuron=2 [[[[(4-chloro-6-methoxy-2 pyrimidinyl)amino]carbonyl]amino ]sulfonyl]benzoic acid; chlorsulfuron=2-chloro-N-{[(4 methoxy-6-methyl-1,3,5-triazin-2 yl) amino]carbonyl] benzene sulfonamide; nicosulfurn=2-[[[[(4,6-dimethoxy 2-pyrimidinyl) amino] carbonyl] amino]sulfonyl]-N,N-dimethyl-3 pyridinecarboxamide; primisulfuron=2-([[[[(4,6 bis(difluoromethoxy)-2 pyrimidinyl) amino] carbonyl] amino]suffonyl]benzoic acid; thifensulfuron=3-[[([(4-methoxy-6 methyl-1,3,5-triazin-2-yl) amino] carbonyl]amino] sulfonyl]-2 thiophene-carboxylic acid; tribenuron=2-[[[(4-methoxy-6 methyl-1,3,5-triazin-2 yl)methylamino]carbonyl]aminojs ulfonyl]benzoic acid; sulfometuron=2-[[[[(4,6-dimethyl 2-pyrimidinyl) amino] carbonyl] amino]sulfonyl]benzoic acid; metsulfuron=2-([[[(4-methoxy-6 methyl- 1,3.5-triazin-2 yl)amino]carbonyl]amino]sulfonyl] benzoic acid. halosulfuron=, Sulfonanilides flumetsulam=N (2,6-difluorophenyl)-5 methyl(1,2.4jtriazolo(1,.5 a[pyrimidine-2-sulfonamide Kar Keto-acid HOE 704 reducto isomerase ipd isopropylmalate 0-oisobuter!-, oxalhydrc:.armate S dehydrooenase WO 00/66154 PCT/US00/11478 -30 Function Gene Enzyme or Known inhibitors of enzyme or Basis for name property property novel inhibitor SYNTHESIS OF S,A.R,D ADDITIONAL "ESSENTIAL" AMINO ACIDS (e.g. histidine. methionine, lysine, threonine) Histidine gpde glycerol phosphon c acid derivatives of synthesis phosphate 1,2,4 triazole dehydratase methionine ms + methionine synthesis synthesis+ lysine synthesis Is+ lysine synthesis+ inhibitors of lysine synthesis+ Threonine ts + threonine synthesis synthesis+ GLUTAMINE gs+ glutamine glufosinate=2-amino- 4 - hydroxy S,AS,R,D GLUTAMATE synthase, methyl phosphinyl, butaonic acid SYNTHESIS gts + glutamate synthetase* LIPID acc+ acetyl co A Arloxyphenoxypro-pionates S,AS,R,D SYNTHESIS carboxylase fenoxaprop=()-2-4-((6-chloro- 2 benzoxazolyl)oxy] phenoxy]propanoic acid; fluazifop-P=(R)-2-[4-[[5 (trifluoromethyl)-2 pyridinyl]oxy]phenoxy]propanoic acid: quizalofop=()-2-[4-[(6-chloro-2 quinoxalinyl)oxy]phenoxyl propanoic acid, Cyclohexanediones clethodim=(EE)-()-2-[ 1-[[(((3 chloro-2-propenyl)oxyimino] propyl]-5-[2-(ethyithio)propyl]- 3 hydroxy-2-cyclchexen-1 -one; sethoxydim=2- i-(ethoxyimino) butyl]-5-[2-(ethylthio)propyl] 3 hydroxy-2-cyclohexen- 1-one ps palmitic synthase oas oleic acid synthase las linoleic acid synthase icas linoleneic acid _synthase

.--

WO 00/66154 PCT/US00/11478 -31 Function Gene Enzyme or Known inhibitors of enzyme or Basis for name property property novel inhibitor STARCH wx, UDP glucose S,AS,R SYNTHESIS gbss. starch glucosyl sss transferase (a starch synthase) other starch synthases be. Q or branching glig, enzyme Igc, sbel. II. II AUXIN Auxin analogue Phenoxyaliphatic acid S.AS,R GROWTH (2,4-D=(2,4-dich l orophenoxy) REGULATORS acetic acid; 2,4-DB=4-(2,4-dichlorophenoxy) butanoic acid; MCPP=; MCPA=(4-chloro-2 methylphenoxy) acetic acid; 2,4-DP =) Benzoic acids dicamba=3,6-dichloro-2 methoxybenzoic acid, Picolinic acids [Pyridines] picloram=4-amino-3,5,6-trichloro 2-pyridinecarboxylic acid; clopyralid=3,6-dichloro-2 pyridinecarboxylic acid; triclopyr-[(3,5,6-trichloro-2 pyridinyl)oxy]acetic acid; fluroxypry=[(4-amino-3. 5 dichloro-6-fluoro-2-pyridinyl) oxy]acetic acid; ias indoleacetic acid synthase GIBBERELLIN coaps copalylpyrophos Phosphon 0, S,AS,R SYNTHESIS phate synthase Amo-1618 ks kaurene Cycocel synthase kox kaurene oxidase Phosphon 0, kaox kaurene acid Ancymidol, Paclobutrazol oxidase gas giberellic acid synthase Key S, modtfied substrate competitor; AS, antisense; R, ribozyme; O, direct inhibitor, alteration of target. These are suitable because they are unique to Apicomplexans. Unique to Apicomplexans means that either they do not exist in animals (e.g., acetohydroxyacid synthase, linoleic acid synthase. starch-amylose cr amylopectin synthase, Q or branching enzyme, UDP glucose, starch glycosyl transferase or have unique antigenic or biochemical properties distinct from those of animals (e.g. acetylco A carbcyytase) *Also present in animals +Other enzymes in these pathways unique to Ac:complexans + Enzymes invol.'cd in the synthesis of those essential amino acids include the following: WO 00/66154 PCT/US00/11478 - 32 Lysine: homocitrate synthase, homocitrate dehydrase (Euglena. fungi); aspartokinase, aspartate semialdehyde dehydrogenase, dihydropicolinate synthase, dihydropicolinate reductase, a piperideine - 2. 6 - dicarboxylate transferase, N - succinyl - c-keto- a aminopimelate transaminase, N - succinyl L. L, ac - c-diaminopimelate desuccinylase, L. L a - c diaminopimelate epimerase, meso-a c 5 diaminopimelate decarboxylase. Inhibitors of lysine synthesis include: +2-4-Amino-4-carboxybutyl azridine-2-carboxylic acid(3) (aziridino-diaminopimelate (DAP], aziDAP); N-Hydroxy.OAP4; N-amino DAP5; 4 methyelene DAP 6; 3,4 didehydro DAP; 4 methylene DAP 4. Methionine: L-homoserine acyltransferase, o-succinylhomoserine sulfhydrolase, L-homocysteine 10 transferase, (to activate methionine - but not exclusively in plants: S-adenosylmethonine [SAM] synthase, SAM-methyltransferase, SAM decarboxylase, S-adenosylhomocysteine hydrolase) Threonine: L homoserine kinase, O-phospho-L-homoserine (threonine) synthase Isoleucine, valine: L-threonine deaminase, acetohydroxy acid synthase, acetohydroxy acid isomeroreductase, dihydroxy acid dehydrase, branched-chain amino acid glutamate transaminase. 15 Leucine: isopropylmalate synthase, a-isopropylmalate isomerase, s-isopropylmalate dehydrogenase, a ketoisocaproate transaminase. Histidine: phosphoribulosyl formimino-5-aminolmidazol-4-carboxamide ribotide amidocyclase, imidazol glycerol phosphate dehydrase, imidazole acetol phosphate transaminase, histidinol phosphate phosphatase, L-histidinol dehydrogenase. 20 Additional herbicides which disrupt cell membranes include Diphenyl ethers [nitro phenyl ethers=) (acifluorfen=5-(2-chloro-4-(trifluoromethyl)phenoxy)-2-nitrobenzoic acid;. fomeasafen=5-[2 chloro-4-(trifluoromethyl)phenoxy)-N-(methylsulfonyl)-2-nitrobenzamide, lactofen=()-2-ethoxy-1 methyl-2-oxoethyl 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoate; oxyflurfen=2-chloro-1l-(3 25 ethoxy-4-nitrophenoxy)-4-(trifluoromethyl)benzene), Other bentazon=3-(1-methylethyl)-(1H)-2,1,3 benzothiadiazin-4(3H)-one 2,2-dioxide above. Additional herbicides which disrupt pigment production include clomazone=2-[(2-chlorophenyl)methyl]-4,4-dimethyl -3 isoxazolidinone ; amitrole=1H-1,2,4 triazol-3-amine; norflurazon=4-chloro-5-(methyl amino)-2-(3-(trifluoromethyl) phenyl)-3(2H) pyridazinone; fluridone= 1-methyl-3-phenyl-S-[3-(trifluoromethyl)phenyl]-4(1H)-pyridinone. 30 Enzymes in the heme synthesis [with a default ALA synthase pathway], shikimnate pathway, alternative generation of energy and glyoxylate cycle are exemplified (Table I A) and the others (Table I B) are suitable for the practice of the invention. 35 As outlined succinctly above, the present invention includes new methods and compositions to treat, diagnose and prevent human and veterinary disease due to Apicomplexan parasites. Apicomplexan infections include those due to Toxoplasnia golundlii (toxoplasmosis), I'asmodia (malaria), Cipbrosporidia (cryptosporidiosis). E'imeria (eimeriosis). Bahe'sia (babesiosis), ThLeleria (theileriosis), Neo.)or'a c(imI11. 4( and others An .A\picomplexan parasite. Torop/asma ori is a representative of other A,\picomplexa; parasites because Apicomplexan parasites appear to be phylogeneticall WO 00/66154 PCT/US00/11478 -33 related and have organelles and enzymes which are critical for their growth and survival. The presence of plant-like pathways/enzymes is confirmed in Apicomplexans by a) the effect of known inhibitors of the pathways in plants using in vitro and in vivo assays; b) Western, Northern and Southern hybridization analyses; c) isolation and 5 comparison of relevant genes; d) demonstration of enzymatic activity; e) demonstration of immunologically reactive proteins which cross-react with proteins in plants; f) complementation of organisms which lack a gene or part of the gene encoding an enzyme with a parasite gene which encodes.the enzyme; and/or g) recognition of plant like transit sequences. In vitro assays include product rescue (i.e., complete or partial . 10 abrogation of the effect of an inhibitor by providing the product of the reaction and thus bypassing the need for the enzyme which catalyzes the reaction. The assays are based on inhibition of the parasite i.e. restriction of growth, multiplication or survival of the parasite. Another measure of infection is "parasite burden" which refers to the amoun: (number) of parasites present as measured in vivo in tissues of an infected host. 15 Another measure of infection is destruction of host tissues by the parasites. Inhibitors reduce parasite burden and destruction of host tissues caused by the parasites. Preferably the inhibitors must not be toxic or carcinogenic to the parasites' host and for in vitro assays not be toxic to cells in culture. Enzymes of the newly detected plant-like pathways provide novel, unique and 20 useful targets for antimicrobial therapy. These unique pathways and enzymes are within the plastid, glyoxosomes, cytoplasm or mitochondria. In addition, not suggeste bel'ore for these parasites, some enzymes used in these pathways are encoded by genes within the nucleus WO 00/66154 PCT/US00/11478 - 34 Plant-like pathways detected in Apicomplexan parasites include a) the 5-carbon herne biosynthesis pathway that utilizes glutamate as a carbon skeleton for synthesis and requires the unique enzyme glutamate-1-semialdehyde aminotransferase; b) the mobilization of lipids in the glyoxylate cycle which is a unique pathway that includes the 5 enzymes isocitrate lyase and malate synthase; c) the generation of energy by an alternative pathway which includes a unique alternative oxidase and/or other unique pathways and enzymes for generating energy in the mitochondria or plastid; and, d) the conversion of shikimate to chorismate utilized in the synthesis of ubiquinone,-aromatic amino acids and folate by plants, but not humans. The shikimate pathway includes the 10 enzyme 3-phospho-5-enolpyruvylshikimate (EPSP) synthase, chorismate synthase, and chorismate lyase, as well as a number of enzymes unique to plants, fungi, bacteria, and mycobacteria, but not to animals. Inhibitors of some of these enzymes also provide information about the functioning and targeting of the enzymes. The hemne synthesis pathway involves enzymes encoded in the nucleus and I imported to the plastid. This pathway is present in Apicomplexans including T. goldii. P1. falciparwn, and Cryptosporidia parvum. Inhibitors of the enzyme GSAT in the pathway include gabaculine (3-amino-2,3-dihydro benzoic acid), 4-amino-5-hexanoic acid, and 4-amino-5-fluropentanoic acid. The glyoxylate cycle, reported to be present in plants, fungi, and algae, is also S present in 7 gmdli. The cycle uses lipids and converts them to C4 acids through a series of biochemical reactions. One of the last steps in this series of reactions is dependent on the isocitrate lyase enzyme and another on the mnalate svnthase enzymes Inhibitors of thicse enzymes include 3-nitropropionic acid and itaconic acid WO 00/66154 PCT/US00/11478 - 35 The alternative respiratory pathway, present in a range of organisms including some bacteria, plants, algae and certain protozoans (trypanosomnes), is present in 7: gondii, Cryptosporidia parvum, and Plasmodiuntfa/ciparunt (in the latter parasite. two clones designated W2 and D6 were inhibited). The pathway is inhibited by a range 5 of compounds including salicylhydroxamic acid, 8-hydroxyquinoline, Benzyhydroxamic acid (BHAM), m-Chlorohydroxamic acid (m-CLAM), Propylgallate, Disulfuram and others. Enzymes involved in the synthesis of chorismate, including those which convert shikimate to chorismate, and enzymes which generate folate, aromatic amino acids.and 10 ubiquinone from chorismate in plants, are present in T. gondii, Plasmoditumfalciparwn. Ciyptosporidiun parvumnt, and Eimneria. Inhibitors include N-(phosphonomethyl) glycine (glyphosate, sulfosate and others). A full-length T. gondii cDNA sequence encoding a chorismate synthase from this pathway and the deduced amino acid sequence provide information useful in developing novel antimicrobial agents. The 15 7: gondii chorismate synthase has features in common with other chorismate synthases and entirely unique features as well. The unique features are novel sequences not shared with chorismate synthases from other organisms but with homology to an amyloplast/chloroplast transit sequence of Zea mays (sweet corn). A P. falciparum cDNA sequence encoding chorismate synthase and its deduced amino acid sequence 20 also provide information useful for developing novel antimicrobial agents. The genomic sequences provide information about regulation of the gene (e.g. unique promoter regions) and such unique regions enable targeting their regulatory clements with antisetnse.

WO 00/66154 PCT/US00/11478 -36 A part of the novel internal sequence (i.e., SCSFSESAASTIKHERDGSAATLSRE RASDGRTTS-RHEEVZ.G) in the 7 gouidii AroC (chorismate syvnthase) gene has homology with the chloroplast/amyloplast targeting sequence of Zea nmays (sweet corn) i'x (ULTDP. glucose-starch-glycosyl transferase) protein (i.e., MAALATSQLVATRAGLGVPDASTFRRG 5 AAQGLRGAP.ASA.ADTLSMRTARAAPRHQQQARRGGRFPSLvvc). This transit sequence provides a novel way to target T. gondii enzymes that move from the cytoplasm into the plastid and is generally applicable to targeting any subcellular organelle. The P. falciparumn AroC (chorismate synthase) has a corresponding novel internal sequence. Additional pathways found in Apicomplexan parasites include the synthesis of 10 branched chain amino acids (valine, leucine and isoleucine) and acetohydroxy acid synthase is the first enzyme in the branched chain amino acid synthesis pathway, inhibited by sulfonylureas and imidazolinones, as well as the synthesis of other "essential" amino acids, such as histidine, methionine, lysine and threonine. Starch synthesis, including starch synthases, the UDP-glucose-starch glycosyl transferase, and 15 debranching enzymes and enzymes of lipid, terpene, giberellin and auxin synthesis, are part of other pathways in Apicomplexan parasites. Down modulation of the UDP glucose starch glycosyl transferase pathway leads to a switch from amylose to amylopectin synthesis and thus the bradyzoite phenotype. Demonstration of presence ofone enzyme or the gene that encodes it in a 20 known pathway implies presence of the full pathway. Thus. enzymes in parasite metabolic pathwa'as that can be inhibited include: glutanimyl-tRNA synthetase: glutamyl IRNA reductasc: prephenate dehydrogenase; aromatic acid aminotranslerase (aromatic tiransalminase). cvclohexmdienyl deihydrogenase; typtophan synthase alpha subunit; WO 00/66154 PCTIUS00/11478 -37 tryptophan svnthase beta subunit; indole-3-glycerol phosphate synthase (anthranilate isomerase), (indoleglycerol phosphate synthase); anthranilate phosphoribosyltransferase; anthranilate synthase component I; phosphoribosyl anthranilate isomerase; anthranilate synthase component II; prephenate dehydratase 5 (phenol 2-monooxygenase); catechol 1,2-deoxygenase (phenol hydroxylase); cyclohexadienyl dehydratase; 4-hydroxybenzoate octaprenyltransferase; 3-oxtaprenyl-4 hydroxybenzoate carboxylyase; dehydroquinate synthase (5-dehydroquinate hydrolase); chorismate synthase (5-enolpyruvyishikimate 3-phosphate phosph-lyase); dehydroquinate dehydratase;-shikimate.dehydrogenase; 3-deoxy-d-arabino 10 heptuloonate 7 phosphate synthase; chorismate mutase (7-phospho-2-dehydro-3 deoxy-arabino-heptulate aldolase); 3-deoxy-d-arabino-heptuloonate 7 phosphate synthase; shikimate 3-phosphotransferase (shikimate kinase); UDP glucose starch glycosyl transferase, Q enzymes; acetohydroxy acid synthase; glutamate-1 semialdehyde 2, l -aminotransferase; chorismate lyase; malate synthase; isocitrate lyase. 15 and 3-enolpyruvylshikimate phosphate synthase (3-phosphoshikimate-1 carboxyvinyltransferase). Recombinant protein produced by constructs with genes encoding these enzymes in E coli or in other expression systems is useful for producing antibodies and obtaining a crystal structure. Native enzyme is isolated. The expressed and native 20 proteins are used to design and test new inhibitors in enzyme assays. Expressed and native (from varied life-cycle stages) proteins are used and the expressed protein is a source o'f the enzyme, and the enzyme assay is carried out in the presence and absence of l'the inhibitors, either alone or in combination and controls include the buffer for the WO 00/66154 PCTIUS00/11478 -38 enzyme alone. The crystal structure is useful for characterizations of enzyme active site(s), secondary structure, transit sequence, substrate and product interactions. The design of additional inhibitors is carried out using published methods such as modifying substrates as had been done with inhibitors of EPSP synthase as well as high through 5 put screening of available compounds. Certain pathways are shown to be affected by inhibitors which are synergistic in vitro. Examples of synergistic inhibitors in vitro are gabaculine.(heme synthesis) and SHAM (alternative energy generation); NPMG and SHAM; NPMG and sulfadiazine; and NPMG and pyrimethamine: Gabaculine and sulfadiazine are an additive. 10 combination in vitro. An aspect of the invention is identifying potential targets for therapeutic intervention by considering nuclear as well as organellar genes as part of the production of enzymes for unique plant-like pathways. For example, the protein synthesis of plant like proteins that is also demonstrated in Apicomplexan parasites suggests not only 15 conservation of plastid genes but also conservation of nuclear genes which encode enzymes that act inside or outside the plastid, from an ancestor that is common to Apicomplexan parasites and algae. Many vital metabolic pathways of algae (often shared with their evolutionary relatives, higher plants) also are conserved in the ApicomIplexan parasites, whether or not the pathways involve the plastid. 20 Consequently. Apicomplexan parasites are sensitive to inhibitors that block several of these unique pathways. Combined attack on multiple targets retards the emergenceseiection of resistant organisms Considering nuclear and organiellar genes has the dual advantage of rapidly identifying conservation of specific pathways and WO 00/66154 PCT/US00/11478 -39 simultaneously identifying both target sites and lead compounds for therapeutic drug development. An aspect of the invention is a plurality of inhibitors, singly or in combination, directed against enzymes and/or genes encoding a different metabolic pathway. 5 Examples of inhibitors suitable for practice of the present invention include GSAT, 3NPA, SHAM, 8-0H-quinoline, and NPMG, sulfonylureas, imidazolinones, other inhibitors of EPSP synthase or chorismate synthase which include competitive substrate analogues, transitional state inhibitors and direct active site inhibitors as.well as other known compounds (Table I). Some pluralities of inhibitors produce synergistic effects 10 Improved treatments against Apicomplexan parasites result from a variety of options: I. some compositions may inhibit the operation of more than one pathway, thereby producing a strong effect and lessening the probability of resistance to the drug emerging because more than one mutation may be required; 15 2. some compositions may inhibit more than one step in a pathway; 3. some pluralities of compositions may have synergistic effects, producing more effective drugs; and 4. some compositions may target pathways operative exclusively during a life cycle of the parasite, making them more selective e.g. against the latent phase. 2, 5. some compositions may inhibit other microorganisms (including other Apicomplexans.) An additional detail ofthe invention is that representative ApicomIplexan parasites, notably 1'. gtontdii, are used for assaying candidate inhibitors. The invention is WO 00/66154 PCTIUS00/1 1478 - 40 directed at etTects of inhibitors of the unique plant-like pathways in Apicomplexan, alone and in combination. Organisms used for the assays include 7. gondii tachyzoites, bradyzoites and a mutant that expresses 50% tachyzoite and 50% bradyzoite antigens. Unique plant enzymes and pathways that were found to be inhibited by compounds 5 shown to inhibit plant pathways in Apicomplexans include: (1) glutamate-1 semialdehyde amino transferase, an enzyme important in heme synthesis, (2) isocitrate lyase, an enzyme important in utilization of lipids, (3) alternative oxidase enzyme complex, enzymes important in energy production and (4) 3-phospho-5 enolpyruvylshikimate synthase-(EPSP synthase), an enzyme important in. conversion of 10 shikimate to chorismate which is a precursor for synthesis of folate, ubiquinone, and certain amino acids essential for survival. The invention provides a rational, conceptual basis for development of novel classes of antimicrobial agents that inhibit Apicomplexan parasites, unique diagnostic reagents, and attenuated vaccines. The inhibitors provide lead compounds for the 15 development of antimicrobial agents. Conserved enzyme active sites or parts of the molecules or genes that encode the protein which are targeted by the inhibitors provide the basis for development of new but related ways to target the enzymes, such as related protein inhibitors, intracellular antibodies, antisense DNA. and ribozymes. Inhibitors are effective against more than one parasite (e.g. 7. gondii. P. 20 fialciparm and C. piaruvmn) and enzymes in these pathways also are present in other bacterial and I'ngIal pathogens such as PneumocY.sus carinii, Aficohacterian IIherculosis).%l .\S'tl;hYlococcus ciueis, and HIenphiu s influenc:a. but not animal s Thus, inhibitors of these pathways al'ect susceptible microorganllsms which - 37 - WO 00/66154 PCT/US00/11478 -41 concurrently infect a host. Because enzymes are utilized ditfferentially in different parasite life-cycle stages, stage-specific inhibitors are within the. scope of the invention. Genes encoding the enzymes in Apicomplexans are identifiable. The genes encoding the enzymes are effectively knocked out in these parasites by conventional techniques. 5 "Knockout" mutants and reconstitution of the missing genes of the parasite demon strate the importance ofgene products to the varying life-cycle stages of the parasite which are identified using antibodies to proteins and ability to form cysts in vivo. which define the life cycle stages. The parasites in which a gene is knocked out are a useful basis for an attenuated vaccine- The genes encoding the enzymes or parts o('.them 10 (e.g., a novel targeting sequence) or the proteins themselves alone or with adjuvants comprise a useful basis for a vaccine. The pathways and enzymes of the invention are useful to design related antimicrobial agents. The sequences and definition ofthe active sites of these enzymes, and pathways, and organelle (e.g., plastid) targeting sequences provide even more specific novel and unique targets for rational design of antimicrobial 15 agents effective against Apicomplexan parasites. For example, proteins which interact with the enzyme and interfere with the function of the enzyme's active site, or are competitive substrates or products or intracellular antibodies (i.e., with a gene encoding the Fab portion of an antibody that targets the protein the antibody recognizes), or antisense nucleic acid or targeted ribozvmes that function as inhibitors are useful, novel 20 antimicrobial agents. Enzymes ofthe invention are a novel basis for unique diagnostic tests. Because some of these pathways are important in dormant parasites, or in selecting the dormant or active life cycle states. they are especially important as antimicrobial agent targets for life cycle stages of the parasite for which no effective WO 00/66154 PCT/US00/1 1478 - 42 antimicrobial agents are known or as diagnostic reagents which ascertain the duration of infection. Identification of the pathways in Apicomplexan parasites provides additional enzyme targets present in these pathways which are not present in or are differentially 5 expressed in animal cells. Identification of the interrelatedness of these pathways with each other provides the basis for the development and demonstration of combinations of inhibitors which together have an effect which is.greater than the expected additive effect (i.e., synergistic). The meaning of synergism is that compound A has effect A', compound B has effect B', compounds A+ B have an effect greater. than A'",B'. 10 Synergism is characteristic of inhibitors of these pathways because an initial pathway affected by an inhibitor often provides a product used as a substrate for another pathway so the inhibition of the first enzyme is amplified. These pathways or their products are interrelated. Therefore, the enzymes or DNA which encodes them are targeted by using two or more inhibitors leading to an additive or synergistic effect. 15 Examples include the additive effect of gabaculine and sulfadiazine and the synergistic effects of NTPMG and sulfadiazine and NPMG and pyrimethamine. One or more of the inhibitors preferentially affect one of the life cycle stages of Apicomplexan parasites. Some enzymes are preferentially used by specific stages of the parasites. Detection of an enzyme of this type or a nucleic acid encoding it offers a novel 20 diagnostic test not only for presence of a parasite, but also for identification of the stage of the parasite Genes encoding enzymes in path..ays ol'f the present invention arc "knocked out using t,:::iques known in the art .- \ parasite with a gene knocked out is said to WO 00/66154 PCT/US00/11478 -43 be attenuated either because the gene expression of the enzyme is stage specific so the parasite cannot become latent, or because the knocked out enzyme is essential for parasite survival. The importance of an enzyme's functions in various life-cycle stages is determined using a mutant-knockout-complementation system. In the former case, 5 the attenuated parasite is useful as a vaccine because the "knocked out" gene is critical for the parasite to establish latency. Its administration to livestock animals results in immunity without persistence of latent organisms. Mutants with the gene "knocked out" also can be selected because when the parasites are grown in vitro they are grown in the presence of product of the enzymatic reaction to allow their survival. However, 10 such attenuated parasites do not persist in vivo in the absence of thile product and, consequently they are useful as vaccines, for example, in livestock animals. The genes that encode the protein also are used in DNA constructs to produce proteins themselves or the proteins or peptides are used in immunized animals. These constructs are used to elicit an immune response and are used for vaccines alone or with 15 adjuvants. Specific examples are incorporation of the gene for alternative oxidase or chorismate synthase in a construct which has a CMV promoter and expresses tilhe protein following intramuscular injection (i.e., a DNA vaccine). This type of construct. but with genes not identified or described as plant-like, has been used as in a vaccines that protect against bacterial and protozoal infections. 20 Plant-like pathways in Apicomplexans were inhibited in vitro. An Apicomplexan GSAT enzyme that is part of a heme synthesis pathway was targeted with inhibitors A Lene with homolo-y to ALA svnthase was identified by analysis of the 7 ,,ndi ESTs (W ashinigton University 7 omodii gene Sequencing project).

WO 00/66154 PCT/US00/11478 - 44 indicating that 7 gomdii has alternative methods for synthesis of ALA. An Apicomplexan glyoxylate cycle was analyzed to determine the sensitivity of tachyzoites and bradyzoites to glyoxylate cycle inhibitors. Specifically, Apicomplexans have isocitrate lyase and malate synthase which present a unique pathway for lipid 5 metabolism that is targeted by inhibitors. Apicomplexan alternative oxidase is targeted, as evidenced by effects of inhibitors of alternative oxidase on this pathway and its expression and immunolocalization in tachyzoites and bradyzoites; Apicomplexan parasites have a metabolically active EPSP synthase enzyme involved in conversion of shikimate to chorismate. These four metabolic pathways, i.e., heme synthesis, 10 shikimate pathway, alternative generation of energy, and the glyoxylate cycle are all exemplified in T. gondii. To show that inhibition was specific for key enzymes in these pathways that are generally absent or used only rarely in mammalian cells, product inhibition studies were used in viitro. For example, growth of 7 gondii is sensitive to NPMG that inhibits the synthesis of folic acid via the shikimate pathway. Because 15 mammalian hosts lack the entire shikimate pathway, it is unlikely that the parasites can obtain either PABA or its precursor chorismate from the host cells so provision of PABA circumvents the need for the substrate pathway for folate synthesis and rescues the EPSP synthase inhibition by NPMG. Further proof of the presence of the plant-like pathways arises from biochemical 20 assays for an enzyme in analogous plant pathways arid isolation of encoding genes Genes are identified by search of available expressed sequence tags (ESTs. i.e.. short, single pass cDNA sequences generated trom randomly selected librar,' clones), by\ PCR aliplil cat loll tusim i primer sequences dcr:, ed f'ron published cnlserved sequences of WO 00/66154 PCTUS00/11478 -45 plant genes with parasite genomic DNA or parasite DNA libraries (Chaudhuri et al., 1996), by the screening of Apicomplexan DNA expression libraries with antibodies to previously isolated homologous proteins or the DNA which encodes them and by complementation of E. coli or yeast mutants deficient in an enzyme. Genes isolated by 5 these techniques are sequenced which permits identification of homologies between plant and Apicomplexan genes using sequence databases such as Genbank. These assays confirm that an enzyme and the gene encoding it are present in Apicomplexan parasites. E. coil mutants and yeast deficient in the enzyme are complemented with plasmid DNA from T. gondii cDNA expression libraries or the isolated gene.or a 10 modification (e.g., removing a transit sequence) of the isolated gene which allows the production of a functional protein in the E. coli or yeast, demonstrating that the gene encoding the enzyme is functional. Homologous genes in T. gondii, P. malaria, Cr)yptosporidia, ANeospora, and Eimeria are identified when relevant plant or T. godii genes are used as probes to DNA obtained from these organisms and the genes are 15 identified either by cloning and sequencing the DNA recognized by the probe or by using the probe to screen the relevant parasite libraries. Genomic DNA is sequenced and identifies unique promoters which are targeted. Unique parts of the genes were identified in the sequences and provide additional antimicrobial agent targets, diagnostic reagents and vaccine components or bases for vaccines. Clade and bootstrap analyses 20 (Kohler et al., 1997) establish the phyvlogenetic origin of novel, sequenced, parasite scenes and this indicates other related antimicrobial agent targets based on components. molecules, and pathways of phylogenetically related organisms. Gene products are expressed and utilized for enzyme assays and olbr screening novel inhibitors. bor making= ' WO 00/66154 PCTIUS00/11478 -46 antibodies for isolation of native protein, for x-ray crystallography which resolves enzyme structures and thus establishes structure-function relationships and enzyme active sites which are useful for the design of novel inhibitors. Immunoelectronmicroscopy using antibodies to enzymes such as chorismate 5 synthase, alternative oxidase, malate synthase or isocitrate lyase immunolocalizes the enzymes within the parasite and determines their location, in particular whether they are in plant-like organelles. Apicomplexan transit peptides are identified by their homology to known transit peptides in other species. Attachment of reporter proteins to the wild type transit peptide, or deletion or mutations of the transit peptide or portionr.of the 10 peptide or gene encoding it, and then characterization of targeting of these constructs alone or in association with reporter constructs establishes that the amino acid sequences of the transit peptide determine intracellular localization and site of function of proteins with this sequence. Stage specificity of these enzymes is determined in vitro by using antibodies to stage-specific antigens in inhibitor-treated cultures, by Western 15 or Northern analyses (detection), by enzyme assays using selected parasite life cycle stages, by using RT PCR (Kirisits, et al. 1996) and a DNA competitor as an internal standard to quantitate the amount of m.NA in parasite samples, by ELISA (quantitation) and by determining whether a parasite with the gene knocked out can develop a bradvzoite phenotype in vito in the appropriate bradyzoite inducing culture 20 conditions. Stage specificity in vivo is determined by observing effects of the inhibitors on diterent te cycle stages in acutely, vs chronically infected mice and by deternimng whether a p::.-ite with the gene knocked. out can form cysts in vivo. Useful techniLICques WO 00/66154 PCTIUS00/11478 - 47 to develop diagnostic reagents for detection of these proteins or nucleic acids include ELISAs, Western blots, and specific nucleotides used as probes.

WO 00/66154 PCT/US00/11478 -48 EXAMPLES Example 1: Novel In Vitro Assay Systems to Assess Antimicrobial EfTects onil T. gondii New in viitro and in vivo assay systems were developed to determine whether 5 plant metabolic pathways are present in Apicomplexans. New elements include use of longer culture times (e.g., extending the duration of the assay to > 6 days is also a unique and useful aspect of this invention, because it allows demonstration of antimicrobial effect for compounds which have to accumulate prior to exerting their effect), use of Me49 PTg and RS strains in vitro, employing synergistic combinations of 10 NPMG and low dosage pyrimethamine in vivo, and assays of parasitemia in vivo using competitive PCR. Improvements were developed in the assays reported by Mack et al. (1984) and Holfels et al. (1994) to measure T gondii replication in tissue culture. The improvements are based on microscopic visual inspection of infected and inhibitor 15 treated cultures, and on quantitation of nucleic acid synthesis of the parasite by measuring uptake of 3 H uracil into the parasite's nucleic acid. Uracil is not utilized by mammalian cells. Parasites present as tachyzoites (RH, Ptg. a clone derived from the Me49 strain), bradyzoites (Mle49), and R5 mutants (mixed tachyzoite/bradyzoites of the Me49 strain that can be stage switched by culture conditions) (Bohne et al., 1993. 20 Soete et dl., 1994, Tomovo and Boothroyd, 1995; Weiss et a/.. 1992) are suitable for assay systems used to study etfiects of inhibitors. Only the RH strain tachyzoites. cultured t0r up to 72 hours, ha, been used in previously reported assays. The use ofi WO 00/66154 PCT/US00/11478 - 49 vc49, Ptg, and RS mutant are unique aspects of the methods used in these assays in this invention. Results using the assay systems are shown in FIGS. 4, 6-8. In these assays toxicity of a candidate inhibitor was assessed by its ability to prevent growth of human 5 foreskin fibroblasts (HFF) after 4 days and after 8 days as measured by tritiated thymidine uptake and microscopic evaluation. Confluent monolayers of HFF were infected with tachyzoites or bradyzoites. Inhibitor was added one hour later. Non toxic doses were used in parasite growth inhibition assays. Parasite growth was measured by ability to incorporate tritiated uracil during the last 18 hours of .culture. 10 Example 2: Detection of Plant-like Pathways in Apicomplexans Using assays disclosed herein, some of which were novel, Apicomplexan parasites were found to contain at least four metabolic pathways previously thought to be unique to plants, algae, bacteria. dinoflagellates, and fungi. Specifically, the presence of a unique hemne.synthesis pathway, an alternative oxidase pathway, a 15 glyoxylate cycle and a pathway necessary for the biosynthesis of chorismate and its metabolites were explored. Growth of the parasite, T. gondii, depends upon these pathways. To examine T. gondii for the presence of plant-like and algal metabolic pathways, certain inhibitors of metabolic pathways are suitable to apply because of their ability to prevent growth of the parasite in tissue culture. 20 Pathwxays which are present in Apicomplexans were analyzed as follows: First. 7 ! o /i tachvzoites were tested to see if they were sensitive in iviro to inhibition by specific inhibitors of target pathw'ays Then bradyzoites arc tested Positive results l'or each pathway provided presumptive evidence that the inhibitor targets were present and WO 00/66154 PCTUS00/11478 - 50 that their activities are important for parasite sur-vival and growth. The inhibitors effective in vitro were screened for activity in vivo in mice. An.example of an effective combination in vivo is NPMG and low dosage pyrimethamine. The presence of an enzyme was further confirmed by product rescue in vitro, in 5 which the product abrogates the need for its synthesis by the enzyme. An example was rescue by PABA for the reaction catalyzed by EPSP synthase. Other methods to demonstrate the presence of an enzyme and thus the pathway include functional enzyme assays, complementation of mutant E. coli strains, PCR, screening of a T. gondii expression library with antibodies or DNA probes, and immunostaining of Western 10 blots. For some enzymes, identification of a partial sequence of a gene in an EST library in the gene database led to cloning and sequencing the full length gene. Demonstration of the enzymes also is diagnostic for presence of the parasites. Examples are demonstration of T. gondii and C. parin' GSAT and T. gondii alternative oxidase and 7 gondii isocitrate lyase and malate synthase by Western 15 analysis and cloning and sequencing of the T. gondii and P. falciparum chorismate synthase gene. Reagents (gene probes and antibodies) obtained during characterization of genes from 7 gondii are used to detect homologous enzymes and pathways in other Apicomplexan parasites. Examples were using the T gondii chorismate synthase gene to probe 1. fulcip)lrui., Eiimeria boris and (Crip i)osporidiumn par'vtm genomic DNA. 20 Other examples are using heterologous plant DNA to detect Apicomplexan GSAT, isocitrate lyase. malate syvnthase, and alternative oxidase genes Such genes are used as )NA probes to screen libraries to clone and sequence the genes to identity PCR prodLIucts WO 00/66154 PCT/US00/11478 -51 Example 3: Effects of Inhibl)itors in Vitro on T. gondii Using the assays described in Example I, five compounds that restrict the growth of T. gondii in vitro were identified: (i) Gabaculine 5 (ii) NPA (iii) SHAM (Salicylhydroxamic Acid); (iv) 8-hydroxyquinoline (v) NPMG Specifically these inhibitors act as follows: 10 i. The Effect of Gabaculine, An Inhibitor Of The 5-Carbon Heine Synthesis Pathway, On the Growth of T. gondii FIG. IA compares heme biosynthesis in plants, algae and bacteria with heme biosynthesis in mammals. In higher plants and algae, ALA is produced in the plastid by the action of GSA aminotransferase on glutamate l-semialdehlyde. In mammals, ALA 15 is formed through the condensation of glycine and succinyl CoA. ALA is subsequently converted to heme. In one dinoflagellate and T. gonidii both pathways are present. Inhibitors of plant heme synthesis pathway restrict the growth of Toxopl)asma gondi in vitro. As shown in FIG. IA, the synthesis of 5-amninolevulinic acid (ALA), the common precursor for hemne biosynthesis. occurs in the plastid of plants, algae and 20 Apicomplexan parasites by the 5-carbon pathway and ALA synthesis occurs by a different pathway in animals The pathway in animals involves the condensation of glvcine and succinvl CoA. The data in FIG. I B-C and a \VWestern blot utilizing- an antibody to the homologous soybean, and barley, and svnechococcus GSATs, WO 00/66154 PCT/US00/11478 - 52 demonstrate that Toxol)asma gondii utilizes the 5-carbon pathway for ALA synthesis and therefore herne biosynthesis. 3-amino 2,3-dihydroxybenzoi.c acid (gabaculine) inhibits GSA in the heme synthesis pathway. First the toxicity of gabaculine was assessed by its ability to prevent growth of 5 human foreskin fibroblasts (-HFF) as measured by 3 H-thymidine uptake and microscopic evaluation. Non-toxic doses were used in parasite growth inhibition assays. In vitro parasite growth inhibition assays included confluent. monolayers of HFF infected with tachyzoites (RH) or mutant Me49 (RS). Gabaculine was added 1 hour later. Parasite growth was measured by the ability to incorporate 3 H-uracil during the last 1.8 hours of 10 culture. In addition, parasite growth was evaluated microscopically in Giemsa stained slides. Toxoplasma organisms were grown in human foreskin fibroblasts alone and in thile presence of different concentrations of gabaculine (3-amino-2,3-dihydrobenzoic acid). Growth was measured by the ability of T. gondii to incorporate tritiated uracil. 15 This compound was effective at inhibiting the growth of T. giodii at thile 20mM concentration. FIG. IB demonstrates the ability of gabaculine (a specific inhibitor of GSA aminotransferase) to restrict the growth of 7 gondii in an in vitro assay over a 4 day period. T7. godmlii growth is measured by ability of the parasites to incorporate tritiated uracil and is expressed as counts/minute (CPM) on the Y-axis. The X-axis 20 describes how the T gondii cultures were treated. Cultures that were grown in medium (medium) produced a CPM ofaround 45,000. Itf no 7 gondii were added to the cultures (nO RHI). a CP 'M ofaround 2,000 was observed Pyrimethanine (0. I ltin/nil) and stuip hadiazine (12 5 tg/mnil) added to cultures resulted in a marked WO 00/66154 PCT/US00/1 1478 - 53 reduction in CPM compared with untreated cultures. At a dose of 5 mM gabaculine restricted around 50%' of CPM and at a dose of 20 mM it almost completely inhibited parasite growth, with counts of about 5,000 CPM. FIG. IC demonstrates the ability of gabaculine to inhibit the growth of 5 T. gondli over 8 days in culture. T. gondii growth is measured by ability ofthe parasites to incorporate tritiated uracil and is expressed as counts/minute (CPM) on the Y-axis. The X-axis represents days post infection.. Parasite growth was evident in the cultures where no drug was added (medium) over the entire time course. Parasite growth was restricted in cultures with 20 mM gabaculine (gabaculine) over the 8 day 10 time course. Similarly, parasite growth was restricted in cultures with pyrimethamine and sulphadiazine (P/S) over the 8 day time course. Similar concentrations showed no toxicity to the foreskin fibroblasts indicating the specificity of this compound for T. gondli. Parallel cultures, fixed and stained with Giemsa and examined by microscopy. clearly demonstrated that T gondii growth was substantially inhibited in 15 the presence of 3-amino-2,3-dihydrobenzoic acid. The results in FIGS. I B and IC indicate that T. gondii utilizes the 5-carbon ALA synthesis pathway. FIG. 7 demonstrates the ability of gabaculine to inhibit the growth of the mutant R5 strain of 7 gondii over 8 days in culture. This mutant strain is atovaquone resistant and possesses certain characteristics of the tachyzoite stage and certain 20 characteristics of the bradyzoite stage. 7: gondii growth is measured by their ability to incorporate tr.:iated uracil and is expressed as counts/minute (CPM) on the Y-axis The X-axis r-. resents days post infection Parasite growth was evident in the cultures where no dru .. as added (medium) over the entire time course. Parasite growth was WO 00/66154 PCT/US00/11478 - 54 restricted in cultures with 20mnNl gabaculine (gabaculine) over the first 6 days of culture, after which a marked increase in parasite growth was detected. Furthermore groups of proliferating organisms which resembled tissue cysts were observed in similarly treated cultures. Parasite growth was restricted in cultures with 5 pyrimethamine and sulphadiazine (P/S) over the entire S day time course. Residual R5 organisms in treated cultures at 8 days begin to incorporate uracil again and some of them appeared cyst-like. Therefore, T. gondii cyst-like structures are selected by gabaculine treatment of cultures. Specific immunostaining of such cultures treated with gabaculine for tachyzoite and bradyzoite specific antigens demonstrates that.gabaculine 10 selects bradvzoites. Table 2 is a schematic representation of experiments designed to test the hypothesis that tachyzoites utilize both conventional oxidase and alternative oxidases, but bradyzoites only use alternative oxidases, therefore interfering with generation of iron sulfated proteins by gabaculine treatment will select bradyzoites. The design and predicted results of stage specific immunostaining (Kasper el al., 1983) 15 if the hypothesis were correct are shown in Table 2 and confirm the hypothesis. These results suggest that T. gondii has stage-specific utilization of alternative oxidases which are utilized when cell cultures are treated with gabaculine because it depletes hemie and thus depletes iron sulfated proteins used in conventional respiration. In summniar, 3-amino-2,3-dihydrobenzoic acid (gabaculine) is an inhibitor of the 2) 5 carbon heme synthesis pathway present in Apicomplexan parasites. H-leine synthesis occurs by a different l)ath\ay in mammalian cells and is therefore unaffected by 3 amino-2.3-di!:. dobcnzoic acid WO 00/66154 PCTIUS00/11478 - 55 Table 2. Gabacule treatment of cultures selects bradyzoites. Antibody Treatment Tachy- Brady- IFA result on culture day used for of zoite zoite 5 IFA culture Control Control 0 2 6 a SAGI media 0 o 0 0 0 0 0 o (expressed O (f (0 on4=0 OD01 Stachy-O o 0 00 0 o 0 zoltes - 0 ( 0 o only) Gabaculine 0 0 0 oo NA 0 C)C 10 o o a a o 00 8 00 00 10 - a BSAG Media 0 0 0 (expressed 00 0 0 n brady- 0 0 0 zoites one C day after 0 00 "O 00 0 a stage Gaculine 0 00 switch) o 00 a0 0 O 15 " oooa eg-Io 00% 0 a BAGS M.edia 00 * 0 0 0R (expressed a 0 A* Z) 00 on brady- C zoites by five day Ga-taculine 0 0 C after stage 0 e 0 0 0 CD switch in 0 A 0 o 00 culture) .-. - --- 20 IFA is immunof:: c scent assay. SAGt is surface antigen 1. BSAG is bradyzoite surface antigen 1. BAG5 is bradyzoite antigen 5. A. Hypothesis. B. Design and predicted results of stage specific irr-..-..ostaining If hypothesis were to be correct. OC) Indicates no specific fluoescence of tine crganism; indicates specific surface fluorescence of the organism due to presence of tLhe antigen recognized by the antibody (e.g., SAGI or aBSAG) 0 Indicates specific Internal f.:rescence in the organism due to presence of the antigen within the parasite recognized by tr- antibody (e.g., aBAG5).

WO 00/66154 PCT/US00/11478 - 56 ii. An inhibitor of the glyoxylate cycle restricts the growth of T. gondii in vitro. 3-Nitropropionic acid is an inhibitor of isocitrate lyase in the degradation of lipid to C4 and inhibits replication of T. gondii in vitro. FIG. 2A illustrates how the 5 glyoxylate cycle manufactures C4 acids. Acetyl CoA, a byproduct of lipid breakdown combines with oxaloacetate to form citrate. By the sequential action of a series of enzymes including isocitrate lyase, succinate is formed. Glyoxalate, the byproduct of this reaction is combined with a further molecule of acetyl CoA by the action.of malate synthase. Malate is then converted to oxaloacetate, thus completing the cycle. 3-NPA 10 and itaconic acid are inhibitors of this pathway. FIG. 2B demonstrates the ability of 3-NPA (an inhibitor of isocitrate lyase) to restrict the growth of T. gondii in an in vitro assay over a 4 day period. This result indicates it is likely that T. gondii degrades lipids using isocitrate lyase. 7 gondii growth is measured by their ability to incorporate tritiated uracil and is expressed as counts/minute (CPM) on the Y-axis. The X-axis 15 described how the 7 gondii cultures were treated. Cultures that were grown in medium (medium) produced a CPM of about 30.000. If no T. gondii were added to the cultures (no RH), a CPM of about 2,000 was observed. Pvrimethamine (0. I pig/ml) and sulphadiazine (12.5 tg/mnl) added to cultures resulted in a marked reduction in CPM compared with untreated cultures. A dose of 0.006 mg ml 3-NPA (3-NPA) 2' restricted arOund 60% of CPMI 3-NPA inhibits the glyoxylate cycle (isocitrate lyase) and/or succinte dehydrogenase in Apicomplexan parasites.

WO 00/66154 PCT/US00/11478 - 57 iii. and iv. Effect of SIIAll and 8-hydroxyquinolilne on alternative oxidase in T. gondii There is a metabolic pathway found in most plants and algae and in Apicomplexans, but absent in most multicellular animals. FIG. 3A describes the 5 electron transport respiratory chain that normally occurs on the inner membrane of mitochondria. In animals, NADH and succinate produced by the action of the citric acid cycle diffuse to the electron transport chain. By a series of oxidation reactions mediated in part through the cytochromes, free energy is released. This free energy yields the potential for the phosphorylation of ADP to ATP. In plants, in addition to 10 the conventional electron transport chain complexes, there is an alternative pathway of respiration. Alternative pathway respiration branches from the conventional pathway at ubiquinone and donates released electrons directly to water in a single four electron step. An important feature of this pathway is that it does not contribute to transmembrane potential and thus free energy available for the phosphorylation of ADP 1I to ATP. The pathway provides a source of energy and is preferred for conditions with relatively low ATP demands. A key enzyme in this pathway is an alternative oxidase that is cyanide insensitive and does not require heme. Toxoplasma gondii utilizes the alternative oxidase for respiration. FIG. 313 demonstrates the ability of SHAM (a specific inhibitor of alternative -0 oxidase) to restrict the growth of ' goniidii in an in vitro assay over a 4 day period. The ability c:'these compounds to inhibit the growth of ' 'goodi was examined by the assay described in Example I. .goid growth is measured by their ability to incorporate "tiated uracil and is expressed as counts/minute (CPM) on the Y-axis.

WO 00/66154 PCT/US00/11478 - 58 The X-axis describes how the 7: gondii cultures were treated. Cultures that were grown in medium (medium) produced a CPM of around 54,00Q. If no T gondii were added to the cultures (no RH), a CPM of around 1,000 was observed. Pyrimethamine (0.1 pg/ml) and sulphadiazine (12.5 p.g/ml) added to cultures resulted in a marked 5 reduction in CPM compared with untreated cultures. A dose of 0.19 pg/ml SHAM (0.19) restricted around 50% of CPM and at a dose of 0.78 ptg/ml it essentially inhibited parasite growth, with counts of about 8,000 CPM. Salicylhydroxamic acid (SHAM) and 8-hydroxyquinoline are inhibitors of the alternative oxidase and are also effective against T. gondii, presumably by inhibiting the 10 alternative pathway of respiration. Salicylhydroxamic acid and 8-hydroxyquinoline inhibit the alternative oxidase of T. gontdii tachyzoites. Since alternative oxidative respiration does not occur in mammals, this makes antimicrobial compounds targeting this pathway therapeutic candidates. v. Effect of NPMG 15 The shikimate pathway is common to plants, fungi and certain other microorganisms and Apicomplexan parasites, but it is not present in mammalian cells. FIG. 4A details the events that result in the production of tetrahydrofolate, aromatic amino acids and ubiquinone in plants, algae, bacteria and fungi. In this pathway, chorismate is formed through the sequential action of a number of enzymes including 20 EPSP-synthase and chorismate svnthase. EPSP-svnthase is inhibited by NPMG. Chorismate is thrther processed to yield tetrahlwdrofolate or ubiquinone by a Ifurther series of tenZ'V2tic reactions This pathway has not been described in mniiiimalls w hic h arc dependent ,n dict or Iolate and therelbre fbr tetrahydrofolbate production. This WO 00/66154 PCT/US00/11478 -59 pathway is required for the synthesis of certain aromatic amino acids and aromatic precursors of folic acid and ubiquinone. It is likely that Toxoplasma gondii utilizes the shikimate pathway for synthesis of folic acid, ubiquinone and aromatic amino acids. N-(phosphonomethyl) glycine, an inhibitor of 3-phospho-5 5 enolpyruvylshikimate (EPSP) synthase and thus an inhibitor of shikimate to chorismate conversion, affects the pathway (Table 1). the ability of this compound to inhibit the growth of T. gondii was examined by the assay described in Example 1. FIG. 4B demonstrates the ability ofNPMG (a specific inhibitor of 10 EPSP-synthase) to restrict the growth of T. gondii in an in vitro assay over a 4 day period. T gondii growth is measured by their ability to incorporate tritiated uracil and is expressed as counts/minute (CPM) on the Y-axis. The X-axis describes how the T. gondii cultures were treated. Cultures that were grown in medium (medium) produced a CPM of around 72,000. If no T. gondii were added to the cultures (no RH), a CPM of 15 around 2,000 was observed. Pyrimethamine (0.1 gg/ml) and sulphadiazine (12.5 pg/ml) added to cultures resulted in a marked reduction in CPM compared with untreated cultures. At a dose of 3.12 mM NPMG (3.12) restricted around 60% of CPM and at a dose of 4.5 mM it inhibited parasite growth by around 80%, with counts of about 12,000 CPM. 20 In FIG. 4C the ordinate shows uptake of tritiated uracil into T. gondii nucleic acids, inhibitory effects of NPMG on nucleic acid synthesis is shown; where PABA at increasing concentrations is added to such cultures, PABA abrogates the inhibitory effects of NPMG on EPSPS synthase restoring nucleic acid synthesis.

WO 00/66154 PCTIUS00/11478 - 60 vi. Branclihed Chain Amino Acid Synthesis Imidazolinones and sulfonylureas inhibit acetohydroxy acid synthase in Apicomplexan parasites. vii. Starch (anmylopectin) Synthesis and Degradation 5 UDP glucose starch glycosyl transferase is inhibited by substrate competition in Apicomplexan parasites. viii. Transit Sequences Antisense, ribozymes, catalytic antibodies, (Pace et al., 1992; Cate et al., 1996; Charbonnier 1997; Askari et.al., 1996) conjugation with toxic compounds allow 10 targeting of parasite molecules using transit sequences. Identification of transit sequences in Apicomplexans provides many means for disruption of metabolic pathways. Antisense or ribozymes prevent the production of the transit peptide and associated protein. Alternatively production of transit peptide sequences, and the conjugation to toxic molecules, allow disruption of organellar 15 function. Catalytic antibodies also are designed to destroy the transit sequence. These antisense compounds or ribozymes or toxic molecules targeted to transit sequences with intracellular antibodies are used as medicines to inhibit the parasite. Example 4: Plant-like Pathways and Enzymes in Apicon plexan Parasites Plasmodittn falcipartmn and Cr'ptrsporidia paranivti 20 Based on the effects of inhibitors of plant-like pathways, abrogation of inhibitor effects, and detection of specific enzymes and/or genes, Apicomplexans. in general, have plant-IiL-uk pai hvays. Results shown in this example broaden the observations of WO 00/66154 PCT/US00/11478 -61 the presence of plant-like pathways in Apicomplexans beyond the representative parasite T7 gondii. i. Heme Synthesis Gabaculine inhibited the heme synthesis pathway (GSAT) in Apicomplexan 5 parasites (FIGS. IB and IC, T. gondii; FIG. 6, Cryptosporidia) but with modest or no affect on P. falciparum (Table 3, Malaria). FIG. 6 demonstrates the effect ofNPMG, gabaculine, SHAM and 8-hydroxyquinoline and 3-NPA on Cryptosporidia in vitro. C. parvuma oocysts at 50,000/well were incubated at 370 C (8% CO 2 ) on confluent MDBKFSD2 cell 10 monolayers in 96 well microtiter plates with the following concentrations of each drug. The concentrations used were: SHAM (0.2% ETOH was added) 100, 10, 1, 0.1 pg/ml; 8-hydroxyquinoline 100, 10. 1, 0.1 pig/ml; NPMG 4.5, 0.45, 0.045 pg/ml; gabaculine 20. 2. 0.2 pg/ml. The level of infection of each well was determined and analyzed by an immunofluorescence assay at 48 hours using an antibody to C. parvitn 15 sporozoites made in rabbits at a concentration of 0.1%. Fluorescein-conjugated goat anti-rabbit antibody was used at a concentration of 1%. 95% Cl count was the mean parasite count per field when 16 fields counted at 10x magnification ± s.d. of the mean. The approximate 95% Cl counts were as follows: media and ethanol - 1200; paromomycin (PRM\) and ethanol - 100: SHAM 100 pg/ml - 400; SHAM 20 10 pg/mil I 100: SHAM 1 p(mil - I100. SHAM 0.1 pg/ml - 1200: media alone I S00 tpg/il: PRM -200, 8-OH-quinoline 100 pug/ml: -300, 8-OH--quinoline 10 pug/nl: - 900, 8-01 1-quinoline I pg/mln I 100. 8-01-1-quinolineC 0. I pg/mil - 1300: NPMG WO 00/66154 PCT/US00/11478 - 62 4.5 pg/ml - 900; NPMG 0.45 pg/ml - 1200; NPMG 0.045 - 1200; gabaculine 20 pg/ml - 200; gabaculine 2 pg/ml -600; and gabaculine 0.2 pg/ml - 1300. Thus each of these compounds are promising lead compounds as antimicrobial agents effective against Cryptosporidia. 5 ii. Glyoxylate cycle 3-NPA inhibited the glyoxylate cycle (isocitrate lyase) and/or succinate dehydrogenase in Apicomplexan parasites (FIG. 2B13, T. gondii) and also inhibited P. falciparumn and C. parvuni. To determine whether there is an Apicomplexan glyoxylate cycle, to analyze the 10 sensitivity of T. gondii tachyzoites and bradyzoites to glyoxylate cycle inhibitors and to determine whether Apicomplexan parasites have isocitrate lyase which presents a unique pathway for lipid metabolism that can be targeted with inhibitors, the following methods are suitable. The inhibitor of isocitrate lyase is 3-nitropropionic acid (concentration ranging 15 from 0.005 to 5mg_/mI in vitro, and 5 to 50 mg/kg/day in vivo). Mutants [Yale Stock Center] used for complementation are as follows: E. coli strains; DV 21A01 (aceA which lacks isocitrate lyase) and DV21 A05 (aceB which lacks malate synthase). Plant gene sequences suitable for comparison are those described by KahIn et cl. (1977), Maloy et al. (19S0); and Maloy et al. (1982). A biochemical assay for isocitrate lyase 20 activity is thle method of Kahn et ul. (1977). The polyclonal antibodies to cotton mialatc synthasc and cotton isocitrate Ivase which hybridize to I goldii proteins of WO 00/66154 PCT/US00/11478 - 63 approximately 60 kd are used to identify these enzymes in other Apicomplexan parasites.

WO 00/66154 PCT/US00/11478 - 64 iii. Alternative Oxidase SHAM and 8-hydroxyquinoline inhibited the alternative pathway of respiration, i.e., the alternative oxidase in Apicomplexan parasites [FIG. 3, T. gondli; FIG. 6, Cryptosporidia parvun; Table 3, Plasmodiumfalciparumn (clones W2, D6), 5 pyrimethamine resistant or sensitive clones. Because Cryptosporidia appear to lack mitochondria, the plastid is a likely site for the alternative pathway of respiration. Table 3. Effect of NPMG, SHAM, 8-OH quinoline, 3NPA and gabaculine on the D6 and W2 clones of Plasmodium falciparum_ Inhibitor Parasite Clone Conc ng/ml) IC 50 IC 90 NPMG 06 823 2510 W2 1716 3396 SHAM 06 6210 25066 W2 5705 42758 8-OH-quinoline D6 1204 1883 W2 1631 4521 *Assays were performed in accordance with Milhous et at., 1985; Odula et al., 1988. Concentrations (ng/ml) of other compounds that inhibited these clones in this assay were as follows for the W2 and 06 clones: Pyrimethamine (82.10, 0.05), Chloroquin (40.86, 2.88), Quinine (38.65, 4.41), HAL (0.33, 0.51). Atovaquovone (0.13, 0.12). 3NPA also inhibited P. falciparum with IC 50=3304, 2817: IC 90=4606, 2817 but with a very small or no significant effect of qabaculine (IC 50 1 45,000). Effect of SHAM on wild type malaria in viltro had been described earlier (Fry and 10 Beesley, 1991). However, this observation was presented without knowledge that SHIAM affected alternative oxidase function. iv. Shikimn:ite/Chorismate NP.\IG inhibited the shikimnate pathway in Apicomplexan parasites (FIG. 411. I' .,'oidil " Table 4. A aIt/lri a: FIG. 6, (ln)to./)r)idic) WO 00/66154 PCTUS00/11478 - 65 Presence of a product of the enzymatic reaction in the pathways of the present invention abrogates the effect of the inhibitor on a specific enzyme because the product no longer has to be made by enzyme catalysis ofa substrate. Thus, addition of the product proves the specificity of the effect of the inhibitor on the enzyme. The addition 5 of PABA abrogates the exogenous effect of NPMG which is an inhibitor of EPSP synthase (FIG. 4B13, T gondii). Because PABA ablates the effect of the inhibitor NPMG on EPSP synthase, the presence of the shikimate pathway in Apicomplexan parasites is demonstrated. Other specific methods to determine whether Apicomplexan parasites have a 10 metabolically active EPSP synthase enzyme involved in conversion of shikimate to chorismate and further characterize this metabolic pathway in T. gondii are as follows: Use of the inhibitor N-(phosphonomethyl) glycine (concentrations of 3.125mM in vitro and 100 mg/kg/day in v'ivo). The product rescue assays are performed with PABA. The mutants for complementation are as follows: E. coli, AroA; E. coli, AroC: 15 and yeast, AR. [Yale Stock Center] Plant gene sequences for comparison are outlined by Klee et al. (1987). A biochemical assay for EPSP synthase activity in cellular lysates is as described by Mousdale and Coggins (1985). Other enzymes in this pathway also are characterized (Nichols and Green, 1992). The full length nucleotide sequence of chorismate svnthase was obtained following restriction digestion and primer-based "0 sequencing o'f the Tg EST zyllc05.r I clone obtained from the "Toxoplasma EST Project at \Vashington University" and of I'.faciparun EST czap PFD d2. I clone obtained from the "malaria lEST project. D) Chakrabarti, Florida. The 7T.-voplV ia ,ldId sequence has substantial homology wxvith tomato and several other chorismate WO 00/66154 PCTUS00/11478 - 66 synthases and a region of the T. gondii protein has 30% identity and 45% homology with the transit sequence ofZea mays sweett corn). Other inhibitors of EPSP synthase are Inhibitors 4 and 5, sulfosate (Marzabadi et al., 1996). Other inhibitors of enzymes in this pathway also have been developed by others and provide a paradigm for the 5 rational synthesis of competitive substrate inhibitors of Apicomplexan parasites. v. Branched Chain Amino Acid and Other Essential Amino Acid Synthesis Acetohydroxy acid synthase is an enzyme present in plants but not animals and 10 is inhibited by imindazolinones and sulfonylureas in Apicomplexan parasites. Inhibitors of histidine synthesis restrict growth of Apicomplexan parasites. vi. Starch (Amylose/Amylopectin) Synthesis and Degradation UDP glucose starch glycosyl transferase, starch synthetase and Q (branching) enzymes are inhibited by substrate competitors in Apicomplexan parasites. 15 vii. Lipid Synthesis The plant-like acetyl coA decarboxylase is inhibited by a number of inhibitors shown in Table I B. Linoleic acid and linolencic acid synthases are inhibited by newly designed competitive substrates. viii. Auxins and Giberellins 1 The known auxin mimics and Giberellin synthesis and Giberellin inhibitors inhibit .Apicceuplexa1 parasites' growth WO 00/66154 PCT/US00/11478 - 67 ix. Glutamine/Glutanmate Synthesis Glufosinate inhibits Apicomplexan glutamine/glutamate.synthesis because the critical enzyme is plant-like. x. Transit Sequence 5 The transit sequence is conjugated with toxic molecules such as ricins and used to disrupt plastid function in Apicomplexans. Other strategies, such as antisense, ribozymes or the use of catalytic antibodies prevent translation of DNA to protein or catalyze the destruction of mature protein. This interferes with functioning of the molecule and thus the parasite's growth and survival. 10 Example 5: The Combined Effects of Inhibitors of Apicomplexan Parasites The effect of enzymes in pathways "in parallel" are additive and in "series" are more than the additive effect of either inhibitor used alone (i.e., synergistic). FIG. 5 demonstrates the inter-relationship of the shikimate pathway and heme synthesis with the electron transport chain. The shikimate pathway produces 3,4-dihydroxybenzoate 15 which is converted to ubiquinone, an essential component of the electron transport chain. Thus. NPMG, an inhibitor of EPSP-synthase, indirectly affects ubiquinone production and, thus, the electron transport chain. Similarly, hemne is required for the production of cytochromes in the electron transport chain. Thus, inhibition of heme production by gabaculine also indirectly affects the conventional electron transport 20 chain. This scheme allows synergistic combinations of drugs. Thus, NPMG and sulphadiazine (a competitive PABA analogue) which act at different points of the folatc snthesis pa:hway are predicted to be synergistic, whereas the elects ot gabaculine and sulphadizinc l a cnpCtitive PA13A analogue) which act on different pathways, are WO 00/66154 PCT/US00/11478 - 68 predicted to be additive. Similarly, gabaculinc and SHAM are a predicted synergistic combination of inhibitors. Table 4 demonstrates the additive inhibitory effect of sulphadiazine and gabaculine on the growth of T. gondii over 4 days in culture. 7: gondli growth is measured by their ability to incorporate tritiated uracil and is 5 expressed as counts/minute (CPM). Cultures that were grown in medium (medium) produced a CPM of about 36,000. If no T. gondli were added to the cultures (no RH). a CPM of about 2,000 was observed. Pyrimethamine (0.1 .tg/ml) and sulphadiazine (12.5 pg/ml) added to cultures resulted in a marked reduction in CPM compared with untreated cultures. The growth of T. gondlii was inhibited by about 60% in cultures 10 treated with 5 nuM gabaculine (gabaculine). The growth of T. gondii in cultures treated with sulphadiazine (1.56 lig/ml) was reduced by approximately 60%. When this dose of sulphadiazine was used in combination with 5 mM gabaculine, as expected, the combined effect of gabaculine plus sulfadiazine is additive because the pathways are in parallel. In contrast, NPMG and sulfadiazine combine in a synergistic manner. 15 Because hemne is needed for conventional mitochondrial respiration, it is expected that if both the hemie synthesis and alternative oxidase pathways are present, then 3-amino 2,3-dihydrobenzoic acid and SHAM will demonstrate synergy. Similarly, ubiquinone or end products of the shikimate pathway are needed for mitochondrial respiration and NPMG plus SHA.M therefore demonstrate synergy. Table 4 also shows that; the 20 (effects of abaculine and SHAM are not synergistic as would be predicted by this simple model The likely reason for this is that ALA svnthase is present in T gondii and provides , dcfault pathway fOr the synthesis of 6-aminoleuliniic acid Thus. the effects of ual'aculine plus SI IA:\M are lnot svncrgisli ycloguanil which affects the WO 00/66154 PCT/US00/11478 - 69 plant-like DH-FR-TS of 7. gonfii (McAuley et al, 1994) also is synergistic with NPMG and other inhibitors of enzymes in the shikimate pathway which provides an improved, novel method to treat this infection. Use of synergistic combinations provide an improved strategy for the development of new medicines for the treatment of disease 5 and eradication of the parasite.

WO 00/66154 PCTUSOO/1 1478 -70 0 cu (U' r- LO x c M z;. N y qr <a <) a) C) (n c 00 Q~0) U (D 00- (D (U( LO ~ 0 o0)0 0 - 0 a + a) C)U- ( ) Al 4-00 C7 N'- a) 0:)) -. 0 (U ().2 Q-H n D l o) coI 0< CO U-) C:a)" 0 LO CV) c C co(D0100) 5>.C6 0 ( D

-

_c: -H -H 71 z) (0r- CY) <~ ol-0 T- It 0)C _ L o C C C40~ a)i a)Z C-) LO a)) a0 C14'~~ C0I 6E < 0) a))3 a)~ ~ ~ C Y e ) C c -Q C C > -. +1 +1+1 0) < 0 f < 2 m 0) ) .!Q En0 CL0 00 aJ C) a) Z a)) O C~ (n ~. C: c 0 0 0 ) 1 1 1 ) 5 < a m) <) m m .!2, 'I I -~ ] a) H~ c zz'z 000 0( WO 00/66154 PCT/US00/11478 -71 Example 6: Effects of Inhibitors In Viv' Candidate inhibitors are administered to animals by daily intraperitoneal injection or by addition to the drinking water. To inhibit EPSP synthase, in vivo, NPMG is administered at a dose of 100mg/kg/day. 5 a) Survival: Five hundred tachyzoites of the RH strain are administered intraperitoneally to BALB/c mice. Cumulative mortality is followed in groups of mice given inhibitor compared to untreated controls. b) Formation of Cysts: C3H/HeJ mice that have been infected perorally with the Me49 strain of T. gondii for 30 days are treated with the inhibitor for 30 days 10 Cyst burden and pathology in the brains of inhibitor-treated and control mice are compared using methods described previously (Roberts, Cruickshank and Alexander, 1995; Brown et al., 1995; McLeod, Cohen, Estes, 1984; McLeod et al., 19SS). Cyst numbers present in a suspension of brain are enumerated, or cyst numbers in formialin fixed paraffin embedded sections are quantitated. 15 c) Persistence ofCvsts: C3H/HeJ mice are infected orally with 100 cysts of T. gondii (Me49 strain). Inhibitors are administered to groups of mice from day 30 post infection to day 50 post infection. Cyst burden, mortality and pathology are compared in treated and control mice on days 30 and 50 post infection and in mice that receive antibody to gamma interferon which leads to recrudescence of disease 20 d) Svnergy: If marked synergistic effect is demonstrated in vitro by showing that the subinhibitorv concentrations used together exert an effect greater than WO 00/66154 PCT/US00/11478 - 72 the additive effects of each used separately, for any combinations, their effect alone and together in ivo is compared. e). New Assays Which Determine the Effects of Antimicrobial Agents on T. gondii hit Vivo 5 Previously reported assay systems measure protection against death following intraperitoneal infection if an animal is infected with the virulent RH strain of T. gondii. Novel aspects of the assay systems in the present invention are using the Me49 (AIDS repository) strain of T. gondii to determinethe effect on brain.cyst number following acute peroral infection by an Apicomplexan parasite, the effect on the established 10 number of brain cysts during subacute/chronic infection, and use of the Me49 and RH strains to demonstrate synergy of inhibitors of plant-like pathways of the present invention which are "in series," and a novel system to demonstrate reduction of parasitemia which is quantitated using a competitive PCR technique. In this competitive PCR method the 7 gondli B I gene is amplified by PCR in the presence of I5 a construct which produces a product slightly smaller than the wild type B I gene. The amount of construct can be quantitated to semiquantitate the amount of the competing wild type gene. For example, presence of a greater amount of the wild type gene will result in lesser use of the competitor. f). Effect of Antimicrobial Agents on Apicomplexanl Parasites hit io 20 A demonstration of the effect of inhibitors of plant-like metabolic pathways in vivo is the synergistic effect of1 NPMG and low dosage pyrimethamiine NPNIG is anl inhibitor of in action and promotes sumrival of mice infected %k ith the virulent RI I strain WO 00/66154 PCT/US00/11478 - 73 of T gooidii when utilized in conjunction with a low dose of pyrimethamine, whereas neither low dosage pyrimethamine nor NPMG alone are protective. Sulfadiazine reduced manifestations of infection in vivo. SHAM affects parasitemia and number of brain cysts. 5 FIG. 8 demonstrates the ability of NPMG and pyrimethamine administered in combination to protect mice from an otherwise lethal challenge with the virulent RH strain of T. gondii. Mice were infected intraperitoneally with 500 tachyzoites and left untreated (control) or treated by the addition of pyrimethamine (PYR),. NPMG (NPMG) or both pyrimethamine and NPMG (PYRNPMG) to their drinking water. 10 Percent survival is marked on the Y-axis and days post infection on the X-axis. Untreated mice and those treated with either pyrimethamine or NPMG died between day 7 and 9 post infection. In contrast 66 percent of mice treated with pyrimethamine and NPMG survived until day 9 post infection and 33 percent survived until the conclusion of the treatment (day 30 post infection). After the withdrawal of treatment. 15 all of these mice survived until the conclusion of the experiment (day 60 post infection). Example 7: Presence of an Enzyme in a Specific Life Cycle Stae Predicts Efficacy of Inhibitors of the Enzyme on this Stage of the Parasite The effect of candidate inhibitors on different life cycle stages and their effect on stage conversion is of considerable interest and clinical importance. The bradyzoite 20 form of T. indii was studied by electron microscopy and was found to have a plastid Intralparasite immunolocalization of the enzymes is also performed. Gabaculine treated cultures arc stained with antibodies to tachyzoites and bradyzoites. Tachyzoites of tlh.

WO 00/66154 PCT/US00/11478 - 74 RH strain are grown in the peritoneum of ND4 mice for 3 days. Tachyzoites are harvested in saline (0.9%) from the peritoneal cavity of euthanized mice and purified by filtration through a 3pim filter. Bradyzoites are isolated as described herein in the Material arid Methods. The tachyzoites are pelleted by centrifugation and the pellet is 5 fixed in 2.5% glutaraldehyde. Cysts and bradyzoites are purified from the brains of CS7BLIO/ScSn mice as described herein in the Materials and Methods and thenfixed in 2.5% glutaraldehyde. Immunoelectronmicroscopy is as described by Sibley and Krahenbuhl (1988) using gold particles of different sizes with antibodies to the enzymes to identify the 10 enzyme localization in different organelles which are identified morphologically. Immunoelectronmicroscopy localization is accomplished with Amersham Immunogold kit and cryosectioning using standard techniques in the electronmicroscopy facility at the University of Chicago or at Oxford University, Oxford, England. Extracellular organisms are studied as well as tachyzoites and bradyzoites at intervals after invasion. 15 Morphology of the parasites, their ultrastructure and the localization of the intracellular gold particles conjugated to the antibodies is characterized. Invasion is synchronized by placing tachyzoites and bradyzoites with PS 15 cells at 4 0 C, then placing cultures at 37 0 C. Intervals to be studied are before 1, 5. arid 10 minutes and 4 hours after invasion. 20 immunostaining and immunoelectronmiicroscopy using an antibody to soybean. or synechococcus. or barley GSAT indicate whether the enzyme is present or absent in WO 00/66154 PCTUS00/11478 - 75 both the tachlvzoite and bradyzoite life cycle stages and localizes the enzyme in the parasite. a) Immunostaining for tachyzoites and bradyzoites Immunostaining of tachyzoites and bradyzoites is evaluated with fluorescent 5 microscopy. This is performed on cultures of fibroblasts in Labtech slides infected with tachyzoites (RH strain) or bradyzoites and permeabilized using triton, or saponin or methanol, as described by Weiss et al., 1992; Dubremete and Soete, 1996; Bohne et al. (1996). Slides are stained. 1, 2, 4, 6, and 8 days post infection with anti-BAG (Weiss et al., 1992) and anti-SAGI (Mineo etaL., 1993; McLeod et al., 1991; Roberts and 10 McLeod, 1996). b) Antibodies Antibodies to the bradyzoite antigens (Weiss et aL., 1992; and Bohne et al., 1993) and monoclonal and polyclonal antibodies to SAGI (Kasper et al. 1983) as a marker for tachyzoite stage specific antigens are used for immunostaining of parasites 15 to establish stage of the parasite. Transgenic parasites with bradyzoite genes with reporter genes are also useful for such studies. c) Inhibitors and Stane Switchinm The etYffect of inhibitors of conventional (KCN, Rotenone, Antimycin A or Mvxothiazol ) respiration and alternative respiration on inhibition of growth of 20 tachvzoites and bradyzoites are compared using standard inhibition experiments i n conjunction v.ith immunostaining techniques Taclhyzoites uIse conventional and altcrnativ\C plt.hways of rCespirationl whereas the bradyzoite stage relies on alternative WO 00/66154 PCT/US00/11478 -76 respiration. Inhibitors of conventional respiration favor tachyzoite to bradyzoite switching whereas inhibitors of alternative respiration inhibit tachyzoite and bradyzoite stages. d) Svnergy studies, gabaculine treatment 5 Synergy studies with gabaculine are of particular interest because heme is used in the conventional oxidase pathway. If there is synergy, iron influences stage switch ing. For alternative oxidase, immunostaining for bradyzoites and tachyzoite antigens is performed using gabaculine treated and control cultures. This is especially informative concerning whether bradyzoites utilize alternative oxidases exclusively, because 10 gabaculine treatment of cultures would limit use of conventional oxidases and thereby select bradyzoites. e) Western Blot Analysis, and ELISAs to determine stage specific expression of enzymes Bradyzoites and tachyzoites also are compared directly for the relative amounts 15 of alternative oxidase, using northern blot analyses, enzyme assays of parasites, isolation of mRNA and RT-PCR, using a competitor construct as an internal standard, and by Western blotting and ELISAs using antibodies to the enzymes (e.g., alternative oxidase). UDP-glucose-starch glyvcosyl transferase. chorismate synthase, isocitrate lyase, GSAT also are studied in a similar manner. 20 E x:almple S: Prohitg Aicomplev Iat DNA with lomtolo~ouis lPl:nitt-like Genes or Polnc ti:allv I loi lontous Genes rolnt Other P:trasites WO 00/66154 PCT/US00/11478 - 77 The presence of the gsa genes, alternative oxidase genes, EPSP synthase genes. chorismate synthase genes, isocitrate lyase genes, and malate synthase genes are identified by probing, and then sequenced. For example, the cDNA clone of soybean gsa is labeled for chemiluminescent detection (ECL) or 32P detection to identify 5 homologous gsa sequences in T. gonidii. Probes are used on a membrane containing the genomic DNA of T. gondii and soybean (positive control). When T. gondii genes are isolated, they are used to probe other Apicomplexan DNA. Thus, the gsa genes of Cryptosporidia, Eimeria, and Malaria are detected in the same manner as the T. gondii gsa. 10 In addition, DNA probes complementary to Trypanosome alternative oxidase DNA are used to probe the Apicomplexan DNA. The gene for T. gondii alternative oxidase is identified by screening T. gondii cDNA expression libraries using the 7D3 monoclonal antibody or the tobacco alternative oxidase gene used as a probe and thus detecting the gene expressing the relevant protein. This gene is used to detect the 15 alternative oxidase genes of other Apicomplexan parasites by Southern analysis and screening other Apicomplexan cDNA libraries. A nucleotide sequence generated from random sequencing of a 7 gondi tachyzoite cDNA library and placed in the Genbank database was found to encode a protein with homology to tomato chorismate synthase. The EST was obtained, clone, 20 and the full length sequence of the i gondii chorisnlate synthase gene and deduced amino acid sequences were obtained (FIGS. 9 and 10) This provides evidence for these plant-like pathways and information useful in preparing a probe to isolate and WO 00/66154 PCTIUS00/11478 -78 sequence this full gene from other Apicomplexan parasites as well. This gene was used as a probe and identified a chorismate synthase in Eimeria bovis DNA and Cyptosporidium parvum DNA. A P. falciparni EST has also been cloned and sequenced. Probes for gsa (soybean) alternative oxidase (soybean and tobacco), 5 isocitrate lyase (cotton), UJDP glucose starch glycosyl transferase (sweet corn), and acetohydroxy acid synthase (sweet corn) also are used to screen for clone, and sequence Apicomplexan genes. Large numbers of T. gondii genes from tachyzoite and bradyzoite cDNA libraries are being sequenced and deposited.in Genbank. Putative homologous genes encoding plant enzymes are used to compare with these sequences 10 to determine whether they are identified in the libraries and if so to determine whether the enzymes are encoded in thile nucleus or plastid. Example 9: Identification of Genes Encoding Enzymes of the Plant-Like Biochemical Pathiways in Apicontplexan Genes are isolated from a cDNA library by hybridization using specific probes 15 to genes known to encode enzymes in metabolic pathways of plants. (see Example 9) Genes are cloned by complementation from a T. gondli cDNA expression library using a series ofE. colt mutants that lack these enzymes and thus depend on the addition of exogenous additives for their optimal growth. Transformed bacteria are used to isolate and sequence plasmid DNA and from those sequences, probes are generated to 20 determine whether other Apicomplexans have ,__enes homologous to those in T guodul 1) eDNA libraries A cDNA library v..as constructed by Stratagene romn mRN :\ isolated from / mtoi;n tachiyzoites ol'f the N1e49) strain olf 7 gondi using the WO 00/66154 PCT/US00/11478 - 79 Uni-ZAP XR cDNA library system. The titer of the amplified library is 1-2 X 10to/ml. Other cDNA libraries also are utilized. The phagemids were excised with R408 or VCS-M13 helper phage and transduced into XL I-Blue Cells. The plasmid DNA was purified using the Qiagen 5 maxiprep system. Other libraries, e.g., early Me49 bradyzoite, in vivo Me49 bradyzoite, and Me49 tachyzoite libraries also are suitable, as are other tachyzoite and bradyzoite libraries prepared by Stratagene.. 2) Screening of library for genes. This is done in a standard manner using monoclonal or polyclonal antibodies or a radiolabeled gene probe. 10 3) cDNA expression libraries are probed with DNA from the genomes of: a) Toxoplasma gondii; b) Plasmodium mnalariae: c) C7yp)tos)poridint parvum: d) Eimeria. 15 The existence of plant-like pathways is confirmed in members of the Apicomplexa by demonstrating the existence of genes encoding the enzymes required for the pathways. Genomic DNA is examined by Southern blot analysis for the presence of the sequences encoding enzymes required for specific algal or plant metabolic pathways. Genomic DNA is extracted from Apicomplexan parasites by 20 proteinase K digestion and phenol extraction DNA(5-10g) is digested with restriction enzymes, electrophorcsed through Io Agarose and transferred to a nylon membrane lThe ECL (Amersham) random prime system is used for labeling of DNA WO 00/66154 PCT/US00/11478 - 80 probes, hybridization and chemiluminescence detection. Alternatively, the Boebringer Mannheim Random Prime DNA labeling kit is used to label the DNA with J P with unincorporated nucleotides removed using G-50 Sephadex Spin columns. Hybridiza tion with the 32"P-labeled probe is carried out in [IM NaCI, 20 mM NaH 2 PO4 pH 7.0, 5 1% SDS, 40% formamide, 10% dextran sulfate, 5 mg/ml dry milk, 100 pg/ml salmon sperm DNA] at 37 0 C. Washes are optimized for maximum signal and minimum background. Probes are prepared from T gondii cDNA clones obtained and characterized as described in Example 9. If lack of overall sequence.conservation limits ability to detect homology, highly conserved regions are useful. For example, two 10 highly conserved regions of the gsa gene are useful to generate oligonucleotide probes (Matters el al., 1995). 4) PCR: An alternative approach for identifying genes encoding enzymes ofthe present invention is by using PCR with primers selected on the basis of homologies already demonstrated between plant protein sequences for the relevant 15 gene. For example, for the gsa gene, polymerase chain reaction technology is used to amplify homologous sequences from a T gondii cDNA library or T. gondii genomic DNA using primers generated from two highly conserved regions of GSAT. The JVetrospora1 crssa alternative oxidase gene has been isolated using degenerate primers designed from conserved regions in alternative oxidase sequences from plant species 20 (Li et l., 1996). These primers are used to detect and clone the alternative oxidase gene from I. gomdii. Candidate PCR products are cloned using the Invitrogen TA cloning kit WO 00/66154 PCT/US00/11478 -81 5) Sequencin : DNA from candidate cDNA clones is extracted using the Promega Wizard Miniprep System. Clones of interest are purified in large scale using the Maxiprep Protocol (Qiagen) and are sequenced by modified Sanger method with an automated sequencer (ABI Automated Sequencer) by the University of Chicago Cancer 5 Research Center DNA Sequencing Facility. 6) Homology Search: to determine whether there is homology of isolated genes with other genes, e.g. gsas, sequences are compared against those in Genbank using the BLASTN (DNA -- DNA) and BLASTX (DNA -> Protein) programs. T. goniidii sequence data is available in Genbank. Sequences for plasmodia also are 10 available as are some isolated sequences for the other Apicomplexan parasites. T. gondii sequences are searched for homologies to the known plant genes gsa, glutamyl-tRNA reductase, isocitrate lyase, malate synthase, alternative oxidase, EPSP synthase, and chorismate lyase using the BLASTN (DNA-4 DNA) and TBLASTN (Protein -+ Conceptual Translation of DNA Sequence) programs. The conserved plant 15 gene sequences for the shikimate pathway are those described by Kahn el al. (1977) and Maloy et al. (1980; 1982). Conserved plant gene sequences for comparison of homologies are outlined by Klee et al. (19S7). Similar libraries and sequence data for Plasmodia also are compared for homologies in the same manner. 7) Complementation: To isolate T. gondii genes or to demonstrate that a 20 gene encodes a functional enzyme product. plasmids from the cDNA library detailed above, or modified constructs, arc used to com lemnent /. co// mutant strains GEl 376 or GE-1377 tem/.) and RP523 (hemul)' from the Yale I-. coh genetic stock center and WO 00/66154 PCT/US00/11478 - 82 SASX4 I B (hemnA) from D. Soil. This strategy has been successful for cloning g.vsa genes from plants and algae (Avissar and Beale, 1990: Elliott et al., 1990; Grimm, 1990; Sangwan and O'Brian, 1993). The hemnA gene encodes glutamate-tRNA reductase, an enzyme important in the CS-pathway for heme synthesis. The henB gene 5 encodes ALA dehydratase, an enzyme common to both heme biosynthesis pathways that should be common to all organisms and is included as a positive control. Mutant bacteria are made competent to take up DNA with CaCI 2 treatment and are transformed with plasmids from the cDNA library. Briefly, chilled bacteria (O.D. 550nm -0.4-0.5) are centrifuged to a pellet and resuspended in ice-cold 10 0. 1 M CaCl 2 and incubated for 30 minutes on ice. Following further centrifugation, the cells are resuspended in 0. 1 M CaCl 2 , 15% glycerol and frozen at -80 0 C in transformation-ready aliquots. 0.2ml ice-thawed competent bacteria are incubated on ice for 30 minutes with approximately 50ng plasmid DNA. Cells are placed at 43 0 C for 2.5 minutes and cooled on ice for 2 minutes. Following the addition of0.8ml Luria 15 Broth, cells are incubated at 37 0 C for I hour and 0. I ml is plated onto M9 minimal media plates. The M9 (Ausubel et al., 1987) medium contains 0.2% glycerol as the carbon source, I mM MgSO 4 .0.1m\ CaCI 2 , I mrM IPTG, 0.2 mg/ml Ampicillin, and 40 p0ml threonine. leucine, and thiamine. Nonselective medium contains 25 pg/ml 5 aminolevulinic acid (hemL and hemA.4) or 4 pg/mil hemin (hemB). Alternatively, bacteria 20 can take up DNA by electroporation. Chilled bacteria are prepared by a repetition of centrilituation and resuspension. The cells are washed in an equal volume ol'f cold water, a V volume of cold water, a 5 volume of cold 10% glycerol, and 1lnallv in a WO 00/66154 PCTUS00/11478 - 83 1/500 volume of cold 10% glycerol and frozen in 0.04 ml aliquots at -80 0 C. Cells are thawed at room temperature and chilled on ice. Cells are mixed with the DNA for 0.5-1 minutes and then pulsed at 25pF and 2.5 KV. The cells are rapidly mixed with SOC medium and grown at 37 0 C for I hour. Cells are plated in the same way as for 5 CaCI 2 transformation. Successful complementation of the E. coli mutants with a T. gondii gene is determined by plating the transformed bacteria onto minimal medium which lacks the supplement. required for optimal growth of the E. coli mutant. Growth on the selective medium is compared to growth on nonselective medium, which contains 25 :g/ml 10 6-aminolevulinic acid (hemniL or hemA) or 4 pg/ml hemin (hemB). Clones that complement each E. coli mutant are tested for their ability to complement each of the other mutants. Clones of putative T. gondii gsa and glutamate-tRNA reductase should complement only hemnL and hemA mutants, respectively. Clones that suppress more than one henm mutation are candidates for alternative oxidase gene clones. 15 A cDNA clone containing the entire soybean gsa gene was able to transform the E. co/i hemnL mutant from auxotrophic to prototrophic for 6-aminolevulinic acid (ALA). Thus the system for obtaining 7 gondli genes that complement E. coli mutants is available For the glyoxylate cycle the mutants used for complementation are as follows 20 DV21 AOl i ace.-4 which lacks isocitrate lvase) and DV21 A05 (aceB which lacks malaie s'nthasel WO 00/66154 PCTIUS00/11478 - 84 For the shikimate pathway the mutants for complementation are available and used as follows: E. coli, AroA and yeast AR. The same procedures are used for Plasmnoditnfalciparunt and Plasmodiun 7knowlesii, Criptosporidiumn and Eimeria complementation. When transit sequences 5 lead to production of a protein which does not fold in such a manner that the protein can be expressed in E. coli or yeast constructs that lack these sequences are prepared to use for complementation that lack these sequences. Example 10: Analysis of Alternative Oxidases in T. gondii T. gondii bradyzoites use unique alternative oxidases. Alternative oxidases are 10 necessary and sufficient for bradyzoite survival. MNethods to characterize plant alternative oxidases are as described (Hill, 1976; Kumar and Soll, 1992; Lambers, 1994; Li et al., 1996, McIntosh, 1994). For in vitro studies, cell lines that lack functional mitochondria are used. These cell lines are used to allow the study of inhibitors effective against the conventional or 15 alternative respiratory pathways within the parasite, but independent from their effects on the host cell mitochondria. SHAM, an inhibitor of the alternative respiratory pathway is used at concentrations between 0.25 and 2 pg/ml in vilro, and 200 mg/kg/day orally or parenterally in vivo alone or in conjunction with other inhibitory compounds. Other approaches include complementation of alternative oxidase 20 deficient LE. call mutants to isolate and sequence the alternative oxidase gene, immunostainhIn using antibodies for potentially homologous enzymes, enzymatic assay WO 00/66154 PCT/US00/11478 - 85 and the creation of mutant-knockouts for the alternative oxidase gene and studying stage specific antigens in such knockouts. 1) Cell lines: Two cell lines, a human fibroblast cell line (143B/206) lacking mitochondrial DNA, and the parental strain (143 B) which possess functional 5 mitochondria are used. These cell lines have been demonstrated to support the growth of T. gondii (Tomavo and Boothroyd, 1995). 2) Inhibitor studies: Inhibitor studies are carried out as described herein. SHAM concentrations are 0.25 to 2 mg/ml in vitro and 200 mg/kg/day in vivo: 3) Immunostainine for tachvzoite and bradyzoites: Immunostaining is 10 performed on cultures of fibroblasts in Labtech slides infected with tachyzoites (RH strain) as described herein. Slides are stained 1, 2, 4, 6 and 8 days post infection with anti-BAG and antiSAGl. 4) RT-PCR is as performed using the protocol of Hill (Chaudhuri et al., 1996) with degenerate primers based on consensus sequences. The product is cloned, 15 sequenced and homology with known alternative oxidases documents its presence. 5) Complementation and alternative oxidase aene cloning: Complementation is used to demonstrate function and is an alternative approach to isolate the gene. Proper function of the complementation system is demonstrated by using complementation with a plant alternative oxidase gene. Mutants suitable for use 20 are hemL. hcm.-, henil. The alternative oxidase gene, AOX. is cloned from a 7' goidic cDNA expression library by coniplementation of the E. coh heml, mutant. Henil. mutants of/.. c/I cannot synthesize liene and are therefore deicient in respiration.

WO 00/66154 PCT/US00/11478 -86 This cloning strategy has been successful in isolating AOX genes from Arabidopsis (Kumar and Soll, 1992). The procedure employed for recovering transformants is identical to that used for cloning the T. gondli gsa gene. The distinction between the gsa and AOX genes is that the AOX gene should restore function not only to hemL 5 mutants but also to other hem mutants of E. coli. In addition, respiratory growth of E. coli on the alternative oxidase should be antimycin-insensitive and SHAM-sensitive. Clones recovered are tested for complementation of hemL, hemB and hemnA mutants. Growth is tested for inhibitor sensitivity. Sequences of cDNA clones that provide functional alternative oxidase activity by these tests are compared with known AOX 10 gene sequences (Mcintosh, 1994). The Escherichia coli strain XL I-Blue was prepared for infection with the 7. gondii phage library according to Stratagene manufacturer's protocol. The RH tachyzoite library, in the X-ZAP vector system was titred, and 106 pfu are added to the XL I-Blue preparation. Approximately 6 X 105 plaques are plated on agar onto 15 150 mm 2 petri dishes containing NZY medium, and grown at 42 0 C for 3.5 or 8 hours, depending upon which screening method is employed. If antibodies are used for screening, IPTG-soaked nitrocellulose filters are placed on the plates after the short incubation period, and the growth of the plaques is allowed to proceed for an equivalent period of time. Filters are blocked in BLOTTO overnight. Screening is 2) carried out under the same conditions which had been optimized during westernn blotting with that primary antibody, and the appropriate secondary antibody If DN.\ probes are used I'or scrcc1nin_. the plaques are grown for S hours post-itllection. Mand WO 00/66154 PCT/US00/11478 - 87 placed at 45CC for 2 hours to overnight. Nitrocellulose filters are placed on the plates, and all subsequent steps for lysis and fixing of the DNA are as specified in the Stratagene protocol. Filters are placed into a pre-hybridization solution containing Denhardts, SSC, SDS, and denatured salmon sperm DNA, as directed in Ausubel et al. 5 (1987). Blots are hybridized to 32 P-labeled probe overnight. Low stringency washes, containing 5X SSC and 0.1% SDS are performed twice at room temperature, and high stringency washes with 0.2X SSC and 0.1% SDS are performed at a temperature dependent upon the degree of homology between the probe and the T. gondii DNA. 6) Assays for the presence of genes: Evidence for the presence of the 10 genes which encode the novel enzymes is obtained by demonstrating enzyme activity and/or Western blot analysis of Apicomplexan whole cell lysates and/or polymerase chain reaction and/or probing the genomic DNA of the parasite with the homologous DNA. Identification of the genes is accomplished by screening an Apicomplexan cDNA library with the antibody to homologous enzymes from plants or other 15 microorganisms or probes which recognize the genes which encode them and/or complementation of mutant bacteria lacking the enzyme with Apicomplexan DNA. 7) Mutant-Knockouts: The alternative mitochondrial oxidase pathway is the preferred oxidative pathway for bradyvzoites and is likely to be important for their survival. The genetic system used to examine the function of the gene via targeted 20 gene knock-outs and allelic replacements essentially as described (Donald & Roos. 1993. 1)94. 1995) The alternative oxida;e is not absolutely required for growth w\vhen cvtochronie eo:idase can be active and l::ants are recoverable. The AOX-null strains WO 00/66154 PCTIUS00/11478 - 88 may be hypersensitive to GSAT inhibitors, both in vilro and in vivo. The ability of the AOX-null strains to switch stages, both in vitro and i vivo is determined. The AOX null strains are examined for stage specific antigens. Virulence and ability to form cysts are assessed in vivo in C3H/HeJ mice as described herein. 5 Knockouts with a bradyzoite antigen reporter gene are produced and these constructs and organisms with the genes knocked out are cultured under conditions that would ordinarily yield a bradyzoite phenotype. These are used to determine. whether expression of the "knocked out" gene is critical for bradyzoite antigen expression and the bradyzoite phenotype. 10 8) Similar "knockouts" of EPSP synthase or chorismate synthase are produced. 9) Similar procedures are used for other Apicomplexan parasites. For example, a similar genetic system is available for P.falciparunm. Example 11: Production. Testing. and Use of Vaccines against Apicomplexa 15 "Knock out" organisms (e.g., lacking GSAT, or alternative oxidase or EPSP synthase or chorismate synthase or UDP-glucose starch glycosyl transferase) are produced as described herein. The knock-out vaccine strain in some cases is cultivated in tissue culture because components which are deficient are provided by a single product or a plurality of products. DNA constructs and proteins are produced and 20 tested as described herein (see Materials and Methods) using unique genes and sequences ar: assay systems and methods which are known to those of skill in the art and disclosed >erein. Briellv. they are used to immunize C3H- mice. and tissues of WO 00/66154 PCT/US00/I 1478 -89 immunized and control mice are subsequently examined for persistence of parasites. These immunized mice and controls are challenged perorally with 100 cysts of Me49 strain or intraperitoneally with 500 RH strain tachyzoites. Effect of immunizations on survival, and tissue parasite burden are determined (McLeod et al., 1988). Parasite 5 burden refers to quantitation of numbers of parasites using PCR for the B I T. gonidii gene, quantitating numbers of cysts in brain tissue, quantitating numbers of parasites by inoculating serial dilutions of tissues into uninfected mice when the RH strain of T. gondii is utilized and assessing survival of recipient mice as I parasite of the RH strain of T gonidii is lethal. Ability to prevent congenital transmission and to treat 10 congenital infections is also a measure of vaccine efficacy. Vaccines are useful to prevent infections of livestock animals and humans. Standard methods of vaccine development are used when substantial prevention of infection is achieved in murine models. Example 12: Nucleotide and Deduced Amino Acid Sequence of T. :gondii 15 Chorisnimate Svnthase cDNA Animals and most protista (e.g. Leishmania) rely exclusively on exogenous folates. Previous studies which demonstrate the efficacy of anti-folates for the treatment of toxoplasmosis have implied that T gondii has the enzymes necessary to synthesize folates. For this purpose. T. gondii uses PABA. The biochemical events "I that lead to PABA production in T gondii or any other Apicomplexan have not been previously characterized In algae. plants. certain bacteria and tungi. the shikimate patlihway facilitates the conversion of shikimate to chorismate, a three step reaction WO 00/66154 PCTIUS00/11478 - 90 catalyzed by three enzymes, shikimate kinase, 3-phospho-5-enolpyruvyl shikimate synthase (EPSP synthase) and chorismate synthase. Chorismate is then used as a substrate for the synthesis of PABA. In plants, EPSP-synthase and chorismate synthase are encoded in the nucleus. In plants, algae and bacteria, chorismate is not only an 5 essential substrate for the synthesis of folate, but it is required for the synthesis of ubiquinone and certain aromatic amino acids. The shikimate pathway may occur both inside and outside of the plastid: For example, EPSP synthase exists in.two forms in Euglentia, one associated with the plastid of those grown in the light and the other found in the cytosol of those grown in the dark. 10 Apicomplexan parasites utilize the shikimate pathway for folate synthesis. An inhibitor of the EPSP synthase, an essential enzyme in this pathway, restricts the growth of 7 gondii. P. falciparum and C. parvim in vitro. This inhibitor, NPMG, synergizes with pyrimelhamine and sulfadiazine to prevent T. gondli multiplication. NPMG also synergizes with pyrimethamine to protect mice against challenge with the virulent RH 15 strain of T. gondii. The sequence of a T. gondii gene that encodes a putative chorismate synthase, that has considerable homology with chorismate synthases from other organisms, provides information useful in developing novel antimicrobial agents. A partial cDNA sequence of approximately 250 bases was identified from the "Toxoplasma EST Project at Washington University." This sequence, when translated. 20 had approximately 30% homology with chorismate synthase from a number of organisms Both strands of the corresponding clone were sequenced and found to be 2312 bases i; length (FIG. 9). Analysis revealed a large open reading fame of 160S WO 00/66154 PCT/US00/11478 -91 base pairs which would encode a 536 amino acid protein. Homology was determined by the use of CLUSTAL X, a computer program that provides a new window base user interface to the CLUSTAL W multiple alignment program. (Thompson, 1994). The deduced amino acid sequence has considerable identity (44.5 to 51.4%) with 5 chorismate synthases ofdiverse species (FIG. 10). The putative T. gonidii protein differs from other known chorismate synthases in length. Chorismate synthases from other organisms range in length from 357-432 amino acids. The larger size of the T. gondii protein is due to an internal region that has no counterpart in other known. chorismate synthases and is novel. The function of this region remains to be 10 determined. The T. gondii chorismate synthase sequence was used in a search with the BLAST program. An EST from a Plasmodiumnfalciparunm cDNA library was located that has considerable homology with the T. gondii sequence. Chorismate synthase is also present in M.ycobacterium tuberculosis. The nucleotide sequence of the cDNA which encodes a putative T. gonidii 15 chorismate synthase and the amino acid sequence deduced from it is shown in FIG. 9. The deduced amino acid sequence of putative T. gondii chorismate synthase has substantial homologies with chorismate svnthases from diverse organisms including SolICTunn Itco I)ersicumln (tomato), S enechocysli-s S)ecies, Hetnophilus influetnza. Saccharomnces cerevis iae, and ANeurospora crassa. (FIG. 10). 20 The Apicomplexan data base in Genbank was searched for homologies to the 7' vodhi chorismiate synthase gene A homnologous P. /alcil.'arum EST (FIG. 11) was WO 00/66154 PCT/US00/11478 - 92 identified. It was sequenced. This provided additional evidence that at least a component of the shikimate pathway also was present in P. falciparum. Sequencing Method Characterizationi of Insert and Design of Sequencing Strategy. 5 Clone TgESTzyl lc05.rl was obtained from the Toxoplasma project at Washington University and supplied in the Bluescript SK vector as a phage stock. Phagemid DNA was excised by simultaneously infecting XLI-Blue cells with the phage stock and VCS-M13 helper phage.. Purified phagemids.were used to infect XLl-blue cells. Infected XL I-Blue cells were grown in LBmediaand plasmid DNA purified 10 using Qiagen maxi-prep kits. The cDNA insert was excised using EcoR I and Xho I restriction enzymes and found to be approximately 2.4KB. Initial sequencing of the 5 prime end of the insert's plus strand and its translation, revealed 30% homology with previously described chorismate synthases from other organisms. However, sequencing of the 5 prime end of the minus strand yielded a sequence that when translated had little 15 apparent homology with any known protein. A series of restriction digestion experiments were performed to establish a restriction map of the insert. Restriction fragments were electrophoresed through a 1% agarose gel and fragments visualized by ethidium bromide staining and ultra-violet illumination. Due to the lack of available restriction enzyme sites within the insert, sequencing with the conventional technique of 2," using sub-cloned overlapping restriction fragments as templates would prove to be laborious and time consumin,-. To circumvent this potential problem and facilitate rapid seque::cing. a strategy was designed that used both conventional sub-cloned WO 00/66154 PCT/US00/1 1478 - 93 overlapping restriction fragments with standard vector annealing primers and the fill length clone with custom designed primers. Thus, sequencing was first carried out by using sub-cloned restriction fragments and the information obtained used to custom design unique sequencing primers. These primers allowed efficient sequencing of the 5 internal regions and the external 3 prime end of each strand. The customized primers were: CUSTOMIZED PRIMERS: CSI 5' TGTCCAAGATGTTCAGCC.T3' CS2 5' AGG CTG ATC ATC TTG GAC A 3' 10 CS2 5' TCG GGT CTG GTT GAT TTT 3' CS4 5' GAGAGAGCGTCGTGTTCA T 3' CS5 5' ATG AAC ACG ACG CTC TCT C 3' CS6 5' CAT GTC GAG AAG TTG TTC 3' CS7 5' GAA CAA CTT CTC GAC ATG 3' 15 CS8 5' ACTTGTGCATACGGGGTA C 3' CS9 5' GTA CCC CGT ATG CAC AAG T 3' CSIO 5'TGAATGCAACTGAACTGC3' CSI I 5' GCA GTT CAG TTG CAT TCA 3' CSI2 5' AGC CGT TGG GTG TAT AAT C 3' 20 CSI3 5'CTACGGCACCAGCTTCAC3' CSI4 5 CGT CCT TCC TCA ACA CAG TG 3' CS15 5' GI( AAG CTG GTG CCG TAG 3 WO 00/66154 PCT/US00/11478 - 94 CSI6 5' CGC CTC TGA TTT GGA AGT G 3' CS17 5'TCTGCCGCATTCCACTAG3' CS18 5' G.AAk-kGCCAAGCAGTTCAGT T 3' 5 Sub-cloning Sub-clones were made from restriction fragments isolated by agarose gel electrophoresis and purified using the Qiaex gel extraction kit.Qiagen, Chatsworth CA. Double digestions of the plasmid.with Hinc II and Pst I resulted in 4 fragments of 500, 800, 300 and 4000 base pairs. The 800 bp fragment, corresponding to the base pairs 10 800-1600 was ligated into the bluescript KS vector. The 1600-2400 base pair portion of the insert was obtained in a similar manner using Pst I and Xho I restriction enzymes and ligated into the bluescript KS vector. Ligations were performed for 12 hours at 18 degrees centicgrade on a PTC 100, programmable thermal cycler, MJ Research Inc. Watertown. Massachusetts. Plasmids containing the restriction fragments were used to 15 transform DHSe competent cells. Plasmid DNA was purified using Qiagen maxi-prep kits. Primer Sequetce Desigi Prime-s were designed based on the sequencing information obtained from restriction enzyme fragments. To facilitate sequencing of a region on the same strand 20 and 5 prime t.: an already sequenced portion of insert, primers were designed from an area approxi::::ely 200-300 nucleotides 5 prime into the last obtained sequence. For sequenicing t: .:e complementary strand, primers were designed to be the complement WO 00/66154 PCT/US00/11478 - 95 and reverse of the same region. Primers were designed to be 18-25 nucleotides in length and have a Tm of 55-60 degrees centigrade. G plus C content was 45-55 percent. Primers were designed to have minimal self annealing and to have a low propensity for primer to primer annealing. Primers with the ability to form stable 5 secondary structures were not designed. These criteria for the design of primers were based on theoretical considerations and results of other experiments which found that primers which had Tins of much less than 55 degrees centigrade failed to work or performed poorly, producing ambiguous sequences of low quality. Sequencing and Assembly of Sequence Information. 10 All sequencing was performed using a Perkin Elmer automated sequencer. The three purified plasmids containing the entire cDNA or a restriction fragment were used as templates for sequencing reactions with the standard M 13 and reverse primers. The sequences obtained were used to design primers which allowed sequencing of the internal regions of the inserts. This process was repeated until both strands of the 15 entire clone were sequenced. Chromatograms were critically edited and controlled for quality using Sequencher software. Edited chromatograms of excellent quality were assembled with the same software package and a consensus sequence obtained. The consensus sequence was analyzed for open reading frames using Macvector software package. Kodak International Biotechnology, Inc., New Haven, CT. 20 Ex:umple 13: Tr:ansit Sequence of T. ,omlii Chorismiate Synthlse Homology with other peptides was sought using the Genbank database and the unique sequence in the T godii chorismlate synthase (amino acids 284 to 435, WO 00/66154 PCT/US00/1 1478 -96 Figure 1 1). There was thirty percent identity and forty-five percent homology, with a number of conserved motifs, between this unique sequence of T. gondli chorismate synthase and the amyloplast/chloroplast transit (translocation) sequence of the Waxy protein (UDP-glucose starch glycosyl transferase) of Zea mays (sweet corn). The 5 same methods whereby the Zea miays transit sequence was analyzed (Klosgen and Well, 1991), i.e., construction of the transit sequence with a reporter protein, immunolocalization of the protein, creation of the construct with deletions or mutations of the transit sequence.and.subcellular immunolocalization using immunoelectronmicroscopy are useful for proving that this is a transit sequence in the 10 7: gondii chorismate synthase. A useful reporter protein for a chimeric construct is 0 glucoronidase of E. coli, expressed under the control of the 355 promoter of cauliflower mosaic virus. The P3 glucoronidase alone is expressed, in parallel. The transit peptide chimeric construct is found in the plastid. The control 3 glucoronidase is found in the cytoplasm. Another useful reporter system is green fluorescent protein 15 (gfp). Antibodies to the chorismate synthase protein are also used to detect the presence of the product of the gene (with the transit sequence) in the plastid and the product of a construct in which the transit sequence is not present in the cytoplasm only. This is used to immunolocalize proteins in different life-cycle stages. Further mutations and deletions are made which identify the minimal transit sequence using the same techniques as described above for the entire peptide. Antisense, ribozyme or intracellular antibodies directed against the transit sequence nucleic acid or translated protein are .uetul as medicines. The amino acid or nucleic acid which encodes the WO 00/66154 PCT/US00/11478 - 97 transit sequence are the bases for diagnostic reagents and vaccine development. This transit sequence is useful for the construction of ribozyme, antisense nucleic acids, intracellular antibodies which target a key parasite protein, and creation of constructs with accompanying molecules which are lethal to the parasites (Roush, 1997; Mahal 5 et al., 1997). This transit sequence also is useful because it provides a general extension of the concept of transit and targeting sequences in Apicomplexan parasites that enable targeting of other parasite.organelles in addition to plastids. The transit sequence.of Zea mays. and: T.. gondii. are.shown in Figure : 1. Example 14. Nucleotide and Deduced Amino Acid Sequences of P. falcipartum 10 Chorismate Svnthase EST. Sequencing of P. falciparutim chorismate synthase EST followed the same pattern as described above for sequencing the T. gondii chorismate synthase gene with the following exceptions: There was difficulty in obtaining sequence from the 3' region of the cDNA due to an unstable polyA tail. This made it necessary to do all sequencing 15 approaching from the 5' end using gene walking techniques and subcloning of restriction fragments. The AT richness ofP.falciparum genes increased the complexity of design of the customized primers. The customized primers utilized were: PFCSI AGC TAT TGG GTG GATC PFCS2 TCC ATG TCC TGG TCT AGG 2 PFCS3 ATA A.-AA ACA CAT TGA CTA TTC CTT C PFCS4 GGG GAT TTT TAT TTT CCA ATT CTT TG PFCS5 TT( AVI CGT TGA ATG ATA AGA C WO 00/66154 PCTIUS00/11478 -98 PFCS6 TTT TAG ATC AGC AAT CAA ACC PFCS7 AAC TTT TTA TCT CCA TAC TTT G PFCS8 GAA GGA ATA GTC AAT GTG TTT TTA T PCFS9 GTA TTT TAC CAA GAT TAC CAC CC 5 PFCS 10 CCC CCA ACA CTA TGT CG PFCSI I CAG TGG GCA AAA TAA AGA PFCSI2 CCA GTG GGC AAA ATA A PFCS 13 GGA AGA GAA ACA GCC AC PFCS14 TGC TGC TGG GGC GTG 10 The gene and deduced amino acid sequences are in Figure 12. Example 15: Southern Blotting Demonstrates Presence of Chorismate Synthase (and by Inference nil of the Shikimate Pathway) in Apicomplexan Parasites 15 Southern blotting using the T. gondli chorismate synthase gene as a nP labeled probe demonstrated homology at moderate stringency (e.g. 0.2 x SSC, 0.1% SDS at 42oC) [more stringent conditions define greatest relatedness of genes] with Eimeria bovis and Cr1pto./)oridimtn /)p rvlltl DNA. This 7. gondii cDNA also comprises a probe for screening cDNA libraries of all 2) other Apicomplexa to identify their chorismate synthase genes The same principles are applicable to all the other enzymes in Table I WO 00/66154 PCT/US00/11478 - 99 Example 16: Genc Expression. Recombinant Protein. Production of Antibody and Solvine the T. conii and P. fialcipartium Crystal Structures of chorismate svnthase to establish their active site and secondary structure. 5 These are done using standard techniques. The gene construct is placed within a competent E. coli. Recombinant enzyme is identified by homologous antibody reactivity and purified using affinity chromatography. Fusion proteins are useful for. isolation of recombinant protein. Protein.is injected into rabbits and antibody specific to the protein is obtained and utilized to purify larger amounts of native protein for a 10 crystal structure. The crystal structure provides information about enzyme active site and facilitates rational drug design (Craig and Eakin, 1997). Recombinant proteins are used for high through put screens to identify new antimicrobial agents. Example 17: Other Uses (e.g. in diagnostic reagents and vaccines) of the Chorismnate Svnthase Gene as a Representative Example of Uses of 15 Each of the Genes and Enzymes in These Pathways That are not Present or Rarely Present in Animals. These uses include T. gondii genes and proteins used as diagnostic reagents and as a vaccine to protect against congenital infection. Recombinant protein (all or part of the enzyme) is produced and is used to elicit monoclonal antibodies in mice and 2 polyclonal antibodies in rabbits These antibodies and recombinant protein (c ,g . to -~,'nohli chorism.ate synthasc) are used in ELISA (e.g. antibody to human IgG or 1lN. or igA or I-- attached to ELISA plate seirum to be tested + antibody conjugated to WO 00/66154 PCT/US00/11478 - 100 enzyme + enzyme substrate). The recombinant proteins, pooled human sera from known uninfected individuals (5 individual sera pooled) and infected individuals (5 individuals with acute infection sera pooled, 5 individuals with chronic infection sera pooled) are the controls. This test is useful with serum or serum on filter paper. 5 Another example of a diagnostic reagent are primers to amplify the target transit sequence or another portion of the chorismate synthase sequence unique to T. gondii. PCR with these primers is used with whole blood to detect presence of the parasite. Such assays have proven to be useful using the T. gondii B Igene(Kirisits, Mui, Mack, McLeod, 1996). 10 Another example of a diagnostic reagent is useful.in outpatient settings such as an obstetrician's office or in underdeveloped areas of the world where malaria is prevalent. FABs of monoclonal antibodies (which agglutinate human red cells when ligated) (Kemp. 1988) are conjugated to antibodies to the target sequence or selected enzyme. Antigen conjugated anti-red cell Fab also is used to detect antibody to the 15 component. A positive test occurs when the enzyme or antibody is circulating in the patient blood and is defined by agglutination of red cells (in peripheral blood from the patient) mixed with the conjugated antibodies. Controls are the same as those specified for the ELISA Examples of vaccines are protein. peptides. DNA encoding peptides or proteins 20 These are administered alone or in conjunction with adjuvants. such as ISCOMS. These vaccine preparations are tested lirst in mice then primates then in clinical trials Endpoints aic induction otf protectiv'e immune responses, protection measured as WO 00/66154 PCTUS00/11478 - 101 enhanced survival, reduced parasite burden, and absent or substantial reduction in incidence of congenital infection (McLeod et al., 1988). Example 18: T. gondii Chorismate Synthase Genomic Sequence Genomic clones are isolated from commercially available genomic libraries 5 (AIDS repository) using the identified cDNA clones as probes in the screening process. The genomic library, as X phage, is isolated onto NZY agar plates using XL 1-Blue E. coli as the host, resulting in plaques following a 37 0 C incubation. The cDNA sequence is radiolabeled with 32P and hybridized to nylon membranes to which DNA from the plaques has been covalently bound. Plasmids from candidates are excised and their 10 restriction enzyme-digested inserts sequenced. Experimental details are as described in Ausubel et al. (1987). Example 19: P. falciparumn Chorismate Synthase Genomic Sequence. This is done with a gene specific subgenomic library as described in Example 18 (see example 41). 15 Other examples of enzymes and the genes which encode them and which are characterized as outlined above include: glutamyl-tRNA-synthetase; glutamyl-tRNA reductase; prephenate dehydrogenase aromatic acid aminotransferase (aromatic transaminase); cyclohexadienyl dehydrogenase tryptophan synthase alpha subunit; tryptophan synthase beta subunit; tryptophan synthase beta subunit; indole-3-glycerol 20 phosphate synthase (anthranilateisomerase), (indoleglycerol phosphate synthase), anthranilate posphoribosyltransferase; anthranilate synthase component I; phosphobiosyl anthranilate isomerase anthranilate synthase component II; prephenate dehdryatase WO 00/66154 PCT/US00/11478 - 102 (phenol 2-monooxvgenase) catechol 1,2-deoxygenase (phenol hydroxylase); cyclohexadienyl dehydratase; 4-hydroxybenzoate octaprenyltransferase; 3-oxtaprenyl 4-hydroxybenzoate carboxylyase dehydroquinate synthase (5-dehydroquinate hydrolase); chorismate synthase (5-enolpyruvylshikimate 3-phosphate phosph-lyase); 5 dehydroquinate dehydratase; shikimate dehydrogenase; 3-deoxy-d-arabino heptuloonate 7 phosphate synthase; chorismate mutase (7-phospho-2-dehydro-3 deoxy-arabino-heptulate aldolase); 3-deoxy-d-arabino-heptuloonate.7 phosphate synthase; shikimate 3-phosphotransferase (shikimate kinase); UDP glucose starch glycosyl transferase; Q enzymes; acetohydroxy acid synthase; chorismate synthase 10 malate synthase, isocitrate lyase; 3-enolpyruvylshikimate phosphate synthase (3 phosphoshikimate- I carboxyvinyltransferase). Example 20: T. gomlii Chorismate Svnthase. EPSP Svnthase. and Shikimate Kinase Activities were Demonstrated Assay for chorismate synthase, EPSP synthase and shikimate kinase in T. 15 gondii were performed and demonstrated such activity. Example 21: T. oirdii Dehvdroquinate Dehvdratase Activity is Demonstrated An assay tfor dehydroquinate dehydratase in T. gondli was performed and demonstrated such activity. Example 22: GSAT activity is demonstrated in T. ,,mondii tachvzoite Ivsates -0 An e:nzvmatic assay (Sangwan and O'Brian. 1993) demonstrates GSAT activity in T .- odi vsates. The buffer contains 0.1 I NIOPS (3-[f N iiiorliholinoji-rolancsullonic acid), p-i 6 8.0.3M glycerol. 15 niNI N gCI, I iNmM WO 00/66154 PCT/US00/11478 - 103 dithiothreitol, 20 p.l pyridoxal phosphate, I mM PMSF (phlenylmethylsulfonyl fluoride) The MOPS, glycerol and MgCI 2 are combined and then pH'd. This is important because the glycerol alters the pH, so it must be added first. This is filter sterilized and has a long shelf life. When the buffer is needed, DTT, pyridoxal 5 phosphate and PMSF are added immediately prior to use: The protein extract stock should be -10 mg/ml if possible. The principle of the assay is conversion of substrate which produces a.change in color due to the reactant. Example-23:. Isocitrate lvseactivitv is demonstrated in TLgondii taclivzoite.. Ivsates 10 An enzymatic assay demonstrates isocitrate lyase activity in 7 gondii isolates prepared by disruption of the parasite membranes using french press or a lysis buffer. Demonstration that the lysis buffer does not alter enzyme activity is carried out by performing the assay with known substrate and enzyme in the lysis buffer and documenting presence of enzyme activity. 15 Example 24: Alternative oxidase activity is demonstrated in T. condii preparations. 7: gondli tachyzoites and bradyzoites are assayed for alternative oxidase activity and such activity is found to be present in greater amounts in bradyzoites. Example 25: Novel Substrate Competitors and Trnsition State Anlogues of 20 Enzymes Inhibit Apicomplexan Enzymnies Some inhibitors are competitive substrates or transition state analogues and they are utihized in the enzyme assay. ti vitro with tachvzoite and bradyzoite WO 00/66154 PCT/US00/11478 - 104 preparations and with native enzyme, tissues culture assays and in in vivo models as described above. These provide a model paradigm for designing inhibitors of any of the enzymes specified above. Briefly, inhibitors are produced as follows: Competitive substrates are produced by designing and synthesizing compounds similar to known 5 compounds but modified very slightly. For example, inhibitors related to glyphosate are known. The structures of glyphosate, sulfosate and the precursor for EPSP have similarities (please see below). Inhibitors are designed by modifying substrates in such a manner that the modification interferes with the enzyme active site. This can be performed using molecular modeling software. Similarly, halogenated substrates for 10 other enzymes have functioned effectively as nontoxic inhibitors. The principles are applicable to the design of inhibitors for any of the unique enzymes with well characterized substrates and active sites. The approaches to rational design of inhibitors include those standard in the art (Craig and Eakin, 1997; Ott et al., 1996). These methods use information about 15 substrate preference and three-dimensional structure of the target enzyme (e.g., chorismate synthase or EPSP synthase). In one approach, the structure of the target is modeled using the three dimensional coordinates for amino acids in a related enzyme. An example of this is that the crystal structure of GSAT from a plant has been solved and its active site is 20 known. In another part of this approach. expression ot'high levels of recombinant enzy'me is produced using cDNA (e.g.. the chorismate synthlase of i gwndui or I'.

WO 00/66154 PCT/US00/11478 - 105 falciparuwn) and quantities of protein adequate tor structural analysis, via either NMR or X-ray crystallography are obtained. Drug resistant mutants are produced in vitro following mutation with nitrosoguanidine and culture with the inhibitor. The surviving organisms have acquired 5 resistance to the inhibitor. This process is carried out either with the Apicomplexan parasite or with bacteria or yeast complemented with the gene encoding the enzyme or part of the gene (e.g., without the transit sequence). PCR amplifies the relevant cDNA and this cDNA encoding the resistant enzyme is cloned.and sequenced. The sequence is comparedwith that of the enzyme that is not resistant. With the information about 10 the inhibitor target and three-dimensional structure, the point mutations which cause resistance are analyzed with computer graphic display. This information provides the mechanism for altered binding of the drug. and the inhibitory compound is then modified to produce second generation medicines designed to treat resistant pathogens prior to their development in nature. 15 An example of the use of toxic analogues to kill parasites used by others provides a means whereby there is production of analogues toxic to parasites. Specifically, the purine analogue prodrugs, 6 sulfanylpurinol. 6 thioguanine, 6 thioxanthine and allopurinol interact with hypoxanthine phosph~oribosyltransferase which is responsible for salvage of purines used to produce .kIP and GMP. Such 20 toxic analogues are effective against the plant-like enzyvmes in. the pathways (see Table I ) in Apicomplexans.

WO 00/66154 PCT/US00/11478 - 106 Transit state analogues bind with extraordinarily high efficiency to the enzyme active site and are predicted from the three-dimensional structure and kinetic information. Analogues that mimic the structural properties and electrostatic surface potentials for the transition state are designed and synthesized. Empirical testing using 5 recombinant enzyme demonstrates that these transition state analogues are good leads with high affinity for the active site of the target enzyme. Multisubstrate analogues are useful becatzse they markedly enhance the binding affinity to the enzyme. Similarly, if enzymes in a cascade are linked in such a manner that the substrate for one reaction provides the substrate for the next reaction, 10 multisubstrate analogues are more useful. Selective inhibitor design and lead refinement: Co-crystallization of inhibitors with target enzymes of host and pathogen enable three-dimensional analysis of molecular constructs and atomic interactions between inhibitors and enzymes and redesign of inhibitors (leads) to enhance their affinity for the pathogen enzyme. 15 Iterative crystallography, lead redesign and inhibitor testing in vitro and in vivo enable design and development of potent selective inhibitors of the target of thile pathogen enzyme. Recombinant methods for screening large numbers of analogues for those that bind selectively to the enzymes of specific parasites provide justification for inclusion of the analogues which bind best in the design of transition-state or 20 multisubstrate analogues. Additional examples (included to illustrate principles employed) but already patented by others include Inhibitor of EPSiP sy nthse have been designed based oni tlhe WO 00/66154 PCT/US00/11478 - 107 similarities of the inhibitor to the substrate. Based on molecular modeling algorithms additional inhibitors are designed. Phosphoenolpyruvate = CH 2 O I III C-O-P 0 5oC 0 0 10 glyphosate = HO-C-CH 2

N-CH

2 -P-OH H OH Inhibitors that effect components of these pathways are halogenated substrates or 15 analogues which are effective competitors. Inhibitors of Ubiquinone: Modifications (substitutions) ofbenzhydroxamic acids produce CoQ (ubiquinone) analogues such as esters of 2, 3 and 3,4 dihydroxybenzoic acid and structurally related compounds. Inhibitort of Isoleucine/valine biosynthetic pathway: These are noncompetitive 20 inhibitors as is shown by the lack of relatedness of the inhibitors (e.g., imidazolinones, sulfonylureas) to the target enzymes. Inhibitors of GSAT The following acids (5 amino-1.3 cyclohendienyl carboxylic acid, 4 amino 5 hexynoic acid ( acetylenic, GABA), 4 amino 5 hexonoic acid ( vinyl GABA) 2 amino 3 5 butanoic acid (vinyl glycine), 2 amino 4 methoxy-tranls- 3 butenoic acid. 4 amino > hloropentanoic .,cid alter catalysis dependent formation of a stable covalent aildducI WO 00/66154 PCT/US00/1 1478 - 108 Inhibitors of lvsine biosyntlihetic pathway: There are noncompetetive inhibitors of lysine synthesis that target enzymes in this patway (e.g., azi DAP, 3, 4 didehydro DAP, 4 methylene DAP4, 4 methylene DAP6) and inhibitors of other plant-like enzymes as in the Table IA and B. 5 Example 26: Modifications of Inhibitory Compounds to Improve Oral e ution especiallyy to brain and eye). Tissue distribution is characterized using radiolabeled inhibitor administered to mice with its disposition to tissues measured by quantitation of radiolabel in tissues. 10 Compounds are modified to improve oral absorption and tissue distribution by standard methods. Example 27. Ecac of Antimicrobia CmundsAione. Toetherand In Conioin~t Infctions in Murine Models: Inhibitors of plant-like pathways are effective against the Apicomplexan 15 infection alone, together with the bacterial and/or fungal infections and also treat the bacterial and fungal infections alone. Presence of inhibitory activity of new antimicrobial compounds is tested using Apicomplexans, bacteria and fungi in enzymatic assays. in vitro, and in vivo assays as described above and known to those of skill in the art. 20 Infections are established in murine models and the influence of an inhibitor or combination of inhibitors on outcomes are determined as follows: WO 00/66154 PCT/US00/11478 - 109 Infections: Infections with Toxop lasma gondlii, Pneumnoc"slis carinii, AMycobacteriuan tuberculosis, Aycobacterium avium intracellular and Cryptosjporidiumni parvuin are established alone and together using an immunosuppressed rodent model. Endpoints in these infections are: 5 Survival: Ability of an inhibitor to protect the infected animal is measured as prolonged survival relative to the survival of untreated animals. Parasitemia: Is a measure using isolation of mRNA and RT-PCR. A 10 competitive inhibitor is used for quantitation. Tissue Parasite Burden: Is determined by quantitating brain and eye. cyst numbers. Inflammatory Response: This is noted in histopathologic preparations. Representative combinations of inhibitors are NPMG and sulfadiazine, SHAMNI 15 and atovaquone, NPMG and pyrimethamine, NPMG and SHAM. Example 28. Establishing Efficacy. Safety. Phiarmakokinetics. and Theraputic/Toxic Index: 20 The testing in murine models includes standard Thompson tests. Testing of antimicrobial agents for efficacy and safety in primate models for malaria is performed Dosages are selected based on safety information available from data bases of information concerning herbicides and the literature. Measurements of serium and tissue levels of antimicrobial compounds are performed using assays which detect 25 inhibitor concentrations and concentrations of their metabolites RepresentatlVe assays are high performance liquid chromatography. and assaying tissues for percentage 1" WO 00/66154 PCT/US00/1 1478 - 110 radiolabeled compounds administered, using liquid scintillation, and other assays also are used. Example 29. Determining whether there is Carcinoienicitv and Teratogenicity: Standard assays to evaluate carcinogenicity and teratogenicity include 5 administration of medicines as described above to rodents and observation of offspring for teratogenic effects and carcinogenicity (i.e. development of malignancies).. Observation includes general physical examination,.autopsy and histopathologic studies which detect any teratogenic or carcinogenic effects of medicines. Example 30. Constructs to Mesure Prasitemia: 10 Portions of genes are deleted and the shorter gene is used as an internal standard in RT PCR assays to measure amount of parasites present (Kirisits, Mui, McLeod, 1996). Ex:amuplc 31. Vaccine Constructs and Proteins and their Administration: These are prepared, as described. They include DNA constructs (Ulmer, 15 Donnelly and Liu, 1996) with the appropriate gene or portions of the gene alone or together, with adjuvants. Representative adjuvants include ISCOMS, nonionicsurfactant, vesicles, cytokine genes in the constructs and other commonly used adjuvants. Native and recombinant proteins also are used in studies of vaccines. Protection is measured using immunologic mn vitro assays, and assessing enhanced 20 survival, reduction of parasitemia tissue and parasite burden and prevention of congenital infection [McLeod et al.. 198S] WO 00/66154 PCT/US00/11478 -Ill Example 32: Staze-Specific Expression of Proteins This is evaluated by enzyme assays, northern or western analysis, ELISA, semi-quantitation of mRNA using RT-PCR with a competitor as internal standard in gene-knockout organisms using culture conditions (e.g. alkaline pH, increased 5 temperature, nitric oxide exposure) which ordinarily elicit a bradyzoite phenotype, or engineering a reporter construct and characterizing presence of the reporter in stage specific expression of antigens. Ability to change between life cycle stages or to persist in a particular life cycle stage is affected by presence or absence of particular plant-like genes and by treatment of inhibitors with plant-like processes. Suitable 10 examples of plant-like enzymes which make parasites less able to switch from or persist in a specific life cycle stage include: alternative oxidase, enzymes critical for amylopectin synthesis such as starch synthases, UJDP glucose-glucosyl starch transferase and branching (Q) enzymes. Example 33. Preparation of Diagnostic Test Reaents and Dianostic Tests: 15 These assays are as described (Boyer and McLeod, 1996). Sensitivity and specificity are established as is standard in the field. Tests and reagents include ELISAs in which antibodies to the proteins or peptides and recombinant proteins of this invention such as chorismate synthase (Aroc) are used and PCR methodology in which primers to amplify DNA which encodes the enzymes, or parts of this DNA, are 20 used. A test usetiLl in an outpatient setting is based on conjugation of a monoclonal antibody to human red blood cells with antibody to plant-like peptides or proteins based on an assay described by Kemp et al. (Kemp eti al., 1988). The red cells are WO 00/66154 PCT/US00/11478 - 112 cross linked via the monoclonal antibody moietyv. resulting in agglutination of the red blood cells in thile blood sample if the antigen or antibody to the parasite component is present in the blood sample. ELISA and PCR can be utilized with samples collected on filter paper as is standard in Newborn Screening Programs and also facilitates 5 outpatient and field use. Example 34. Develoment and use of Antisense Olisonucleotides in Desizn and Use of Medicines to Protect Against Apicomplexans: Antisense oligonucleotides directed against the nucleic acids which encode the enzymes of the essential parasite metabolic process described herein are effective 10 medicines to treat these infections. Antisense oligonucleotides also are directed against transit sequences in the genes. Antisense oligonucleotides are short synthetic stretches of DNA and RNA designed to block the action of the specific genes described above, for example, chorismate svnthase of T. gondti or P. falciparun, by binding to their RNA transcript. They turn off the genes by binding to stretches of 15 their messenger RNA so that there is breakdown of the mRNA and no translation into protein. When possible, antisense do not contain cytosine nucleotides. Antisense reagents have been found to be active against neoplasms, inflammatory disease of the bowel (Crohn's Disease) and HIV in early trials Antisense will not contain cytosine nucleotides followed by guanines as this generates extreme in:mune responses (Roush. 20 1997) Antisense oligonucleotides with sequence for thytiidine kinase also is used for reulatable g,ne therapy E'x:aniiiplc 35. Rihozvinm es id Other 'lToxic Compo iuids :Is Antimnicrohli:. A\vents: WO 00/66154 PCT/US00/11478 -113 Ribozymes are RNA enzymes (Mack, McLeod, 1996) and they and toxic compounds such as ricins (Mahal et al. 1997) are conjugated to antisense oligonucleotides, or intracellular antibodies, and these constructs destroy the enzyme or other molecules. 5 Example 36. Intracellular Antibodies to Target Essential Enzymes Proteins and Organelles: Intracellular antibodies are the Fab portions of monoclonal antibodies directed against the enzymes of this invention or portions of them (e.g., anti-transit sequence antibodies) which can be delivered either as proteins or as DNA constructs, a's I 0 described under vaccines. Example 37. Development of New Antimicrobial Compounds Based on Lead Compounds: The herbicide inhibitors comprise lead compounds and are modified as is standard. Examples are where side chain modifications or substitutions of groups are 15 made to make more active inhibitors (Table 1). Their mode of action and structure as well as the enzyme and substrate structures are useful in designing related compounds which better abrogate the function of the enzymes Examples of such substrate or active site targeting are listed in Table I. Native or recombinant protein used in enzymatic assays and in vitro assays 20 described above are used to test activity of the designed newly synthesized compounds Subsequently, they are tested in animals WO 00/66154 PCT/US00/11478 -114 Example 3S. Trials to Deionstrate Efficacv of NovelAntimicrobbialAents for Human Disease: Trials to demonstrate efficacy for human disease are performed when in vitro and murine and primate studies indicate highly likely efficacy and safety. They are 5 standard Phase I (Safety), Phase II (small efficacy) and Phase III (larger efficacy with outcomes data) trials. For medicines effective against T. gondii tachyzoites, resolution of intracerebral Toxoplasmnia brain lesions in individuals with HIV infection with no other therapeutic options available due to major intolerance to available medicines is the initial strategy for Phase 11 trials. Endpoints for trials of medications effective 10 against T gondli bradyzoites include absence of development of toxoplasmic encephalitis in individuals with HIV. HIV infected patients who also are seropositive for T. goindii infection are evaluated. Evaluation is following a one-month treatment with the novel anti 71 gonidii medicines. Observation is during a subsequent 2 year period when the patients peripheral blood CD4 counts are low. Effective medicines 15 demonstrate efficacy measured as absence of T. gondii encephalitis in all patients. Otherwise, 500/o of such individuals develop toxoplasmic encephalitis. When medications efficacious against bradyzoites and recrudescent toxoplasmic encephalitis in patients with AIDS are discovered and found to be safe, similar trials of efficacy and safety for individuals with recurrent toxoplasmic chorioretinitis are performed. All 20 such trials are performed with informed consent, consistent with Institutional NIH. and Helsinki guideliness applicable to treatment trials involving humans WO 00/66154 PCT/US00/11478 -115 Example 39. Vaccine Trials for Hlumans After vaccine efficacy in rodent models to prevent congenital and latent Toxoplasma infection are established, for component vaccines only, trials to establish safety and efficacy in prevention of congenital and latent infection are performed. 5 They follow standard procedures for phase 1, 11 and III trials as outlined above and as is standard for vaccine development. Endpoints for vaccine effect and efficacy are development of antibody and cell mediated immunity to T. gondii (effect) and most importantly, prevention of T. gondii congenital infections. After establishing in phase I trials that the vaccine is entirely 10 safe, nonpregnant women of childbearing age will be vaccinated with recombinant vaccine. Assay for efficacy is via a serologic screening program to detect newborn congenital toxoplasmosis (described in Boyer and McLeod, 1996) with usual testing to document whether seropositive infants are infected (described in Boyer and McLeod, 1996). 15 Example 40. Vaccine Efficacy and Safety for Livestock Animals The efficacy of candidate vaccines is tested in sheep as previously described (Buxton et al.. 1993). Vaccines are live attenuated, genetic constructs or recombinant protein. The most eflicious routes and frequency of inoculation is assessed in a serious of experiments as described below. Intra-muscular, sub-cutaneous and oral are the o20 preferred routes, although intravenous. intraperitoneal and intradermal routes may also be used Scottish blackface or!and swaledale ewes. four to six years old are tested for luG antibodies to T'oo.vl1a.snt g'm/ usinLIg an EL ISA\ assay Only scro-negative WO 00/66154 PCTIUS00/11478 -116 animals are used for the study. Three groups of 10-15 ewes are used for each experiment. Groups I are vaccinated, while group 2 and 3 are not. Three months later all ewes are synchronized for estrous and mated. At 90 days gestation the ewes in groups I and 2 are given 2000 sporulated oocyst of T. gondii. 5 The outcome of pregnancy is monitored in all groups. Aborted lambs or those dying soon after birth are examined histologically and by PCR for the B 1 gene or sub inoculation into mice or tissue culture, for the presence of T. gondii. All placentas are examined histologically and as above for parasites. Lambs are weighed at birth. Pre colostral serum is taken from each lamb. Congenital transmission is assessed by 10 performing ELISA assays on the serum for IgG or IgM. Protection is measured as a decrease in congenital transmission, a decrease in the incidence or severity of congenital disease, or a decrease in abortion.

WO 00/66154 PCT/US00/11478 -117 Example 41: T. condiiChorismate Svnthase Genomic Sequence is Used to Produce "Knockouts" (Attenuated Vaccine Strain). The genomic sequence of chorismate synthase is in FIG. 13. As 5 with other genomic sequences herein, it provides an example of a gene which is "knocked out" to produce an attenuated vaccine and also can be utilized as described in other parts of this document. A chorismate synthase knock out parasite was produced as follows: The genomic T. gondii chorismate synthase sequence consists of 9 exons. 10 To prepare the knockout construct, this sequence was digested with EcoN I to remove a 1.8 kb fragment that included exons 2, 3, and 4. The EcoNI digested ends were blunt ended followed by dephosphorylation. A 1.9 kb piece bearing HXGPRT flanked by the 5' promoter region and 3' untranslated region of dhfr (called dhfr HXGPRT) was isolated by 15 digestion of a construct, obtained from J. Boothroyd, and Xbal and hoL After blunt ending, the 1.9 kb fragment was cloned into the chorismate synthase construct so that dhfr HXGPRT replaced chorismate synthase exons 2, 3 and 4. This construct was used for knockout of the wild type chorismate synthase gene. 20 The sequence of the construct was verified by PCR. Following transfection into T. gondii (deficient in HXGPRT) and selection in medium containing 25 pg/ml mycophenolic acid and 50 pg/ml Xanthine, successful transfection was confirmed by PCR of the chorismate synthase/dhfr HXGPRT junction and sequence the product. Parasites were 25 cloned by limiting dilution and clones were cultured in the presence or absence of folate and other aromatic products in this medium with replica cultures. Aromatic compound deficient medium with 10% AlbuMax® as a serum substitute was prepared. Final concentrations of aromatic compounds in the supplemented medium are 0.1M phenylalanine, 30 tyrosine, tryptophan, PABA, 2,3 dihydroxybenzoate and p hydoxybenzoate. DNA was extracted from those replicate cultures of parasite clones that grew only in the presence of aromatic compound -.-- tar-r- cuc-r Itr (Il II 1= Q1) WO 00/66154 PCT/US00/11478 -118 supplementation. PCR primers were designed to confirm presence of the knockout construct and demonstrated that homologous recombination occurred resulting in replacement of exons 2-4 with the dhfr HXGPRT sequence. The knockout parasite was passaged in aromatic compound 5 supplemented medium. Whether this selection clearly demonstrates inability of the knockout parasite to grow in aromatic compound deficient medium, but ability to grow in aromatic compound sufficient medium using a uracil assay. Such aro deficient strains of bacteria have been used as vaccines precisely because they are nonpersistent. Complementation 10 with aroC in an episomal vector to prove that the phenotype of the chorismate synthase knockout organisms is due to deletion of the chorismate synthase gene, was also done. This complementation system also allows characterization of the effects of mutations in chorismate synthase or its promoter region on transcription or on enzyme function, 15 importance of the pathway for parasite viability, stage switch and subcellular localization. An episomal vector was obtained from John Boothroyd. Chorismate synthase was cloned within this plasmid under control of a constitutive promoter (e.g., the promoter for tubulin or DHFR). The resulting construct was transfected into the chorismate 20 synthase knockout parasite described above. Proof that the construct produces mRNA for chorismate synthase is with northern and western blotting. The lack of ability of the knockout and the ability of the complemented parasite to grow in folate and other aromatic compound deficient medium indicates a functional construct. This knockout 25 organism is suitable for use as an attenuated vaccine strain. Example 42: T. gondil Chorismate Synthase cDNA Sequence in a DNA Vaccine Vector Elicits Antibodies. T. gondii chorismate synthase cDNA sequence placed in a DNA vaccine vector with a CMV promoter (Vical, San Diego) and 30 administered intramuscularly to mice elicits serum antibodies to chorismate synthase (FIG. 14 A and B). Antibody production is detected on Western blot and in other immunoassay systems. This is an example of Mr-CTIlFl=I SHEET (RULE 91) WO 00/66154 PCT/US00/1 1478 -119 a recombinant vaccine and a system to produce antibody reagents useful min diagnostic tests without the need to produce recombinant protein. Example 43: T. gondii Chorismate Svnthase-green Fluorescent Protein Construct is Made and Used in Parasite 5 Survival Assays to Test Antimicrobial Agents. A T. gondii chorismate synthase-green fluorescent protein DNA construct elicits a fusion (reporter) protein detectable with conventional immunofluorescence microscopy and deconvolution microscopy (FIG. 15) and other techniques known in the art to detect fluorescence. This 10 construct accelerates the growth rate of the parasite and is useful for measuring effects of antimicrobial agents on the parasite by detecting relative amounts of the green fluorescent reporter protein. This is useful for testing antimicrobial agents. Example 44: Chorismate Svnthase and Life Cycle. 15 Chorismate synthase is differentially located and expressed in different life cycle stages indicating that it can be an antimicrobial agent target in, and reagent to detect, specific stages of the parasite. ImmunostaininE This is performed as is standard in the art with tachyzoites, converting organisms, intestinal life cycle stages using 20 specimens produced in vivo and in vitro. In some tachyzoites, chorismate synthase was concentrated in a small area contiguous to the nucleus in the area of the plastid (FIG. 16A). In other life cycle stages it was distributed diffusely throughout the cytoplasm (FIG. 16B, C). It was most abundant in bradyzoites and 25 macrogametes. A C-terminal green fluorescent protein reporter alters its localization in tachyzoites (FIG. 15). Unique stage-associated expression and subcellular localization of T. gondii chorismate synthase is identified in tachyzoites, bradyzoites and in the stages of the parasite in the cat intestine including macrogametes, microgametes but not schizonts. 30 Stage-associated expression of T. gondii chorismate synthase (FIG. 16A-C) is an example of the expression and differential subcellular localization of this protein. This stage-associated expression demonstrates m.ar- wr -sr)-m cet tar-l" 1 II !" Oil WO 00/66154 PCT/US00/1 1478 -120 that this protein is present in tachyzoites (A), bradyzoites (B) and microgametes (C) and macrogametes (C). This is an antimicrobial agent target, useful diagnostic reagent and vaccine constituent for infections with these life cycle stages. The differential stage associated subcellular 5 localization demonstrates that organelle targeting is another way to target these enzymes. Example 45: Recombinant Chorismate Svnthase is Useful for Antibody Production and in Enzyme Assays for High Throughput Screens. 10 Recombinant chorismate synthase was produced and is useful for high throughput screens, development of diagnostic reagents and a vaccine. Overexpression of Chorismate Svnthase Chorismate synthase was expressed in E. coli using a pGEX expression system (Pharmacia). 15 Briefly, PCR was used to amplify the coding region and to introduce BamHl and EcoRl sites to the 5' and 3' ends respectively. Following removal of the 3'adenosine overhangs, the PCR product was first cloned into pUC 8 using the Sureclone Ligation Kit (Pharmacia Biotech, Herts, UK). The pUC18 plasmid containing the insert was digested with EcoRl 20 and BarnmH I and following purification by electrophoresis, the insert was eluted from an agarose gel and then cloned into pGEX-2T. DNA sequencing confirmed that the nucleotide sequence was in frame and that no PCR errors had been introduced. Following transformation the protein was expressed in BL21. To optimize expression and to test protein for 25 enzymatic activity, expression is increased using BL21 Codon Plus (Stratagene). This strain of E. coli has been engineered to contain extra copies of tRNAs for codons in E. coli that are rarely used (argU, ileY, leuW and proL). In some cases the presence of an N-terminal tag can interfere with the ability of a protein to function and that although a GST 30 tag can be removed with thrombin this treatment itself can be too harsh to retain the activity of some proteins. Thus as an alternative approach is to employ the Protein C Epitope Tagging system (Roche). This system .-..-- ,-,- tr-vr-% eUJr-Ir- ftl II 1 C14l WO 00/66154 PCT/US00/11478 -121 allows the production of recombinant proteins which have either C terminal or N-terminal protein C tags. The protein C tag is used to purify protein using an antibody that binds the protein C tag only in the presence of Ca 2 . Calcium chelation then provides a gentle means of eluting the 5 purified protein from the antibody. The Pichia Expression System (Invitrogen) is also used. This system offers advantages of bacterial systems such as high-level expression and ability to use large scale cultures. In addition, it offers certain advantages of eukaryotic expression systems that facilitate protein 10 processing, folding and post-translational modifications. The system makes use of the powerful alcohol oxidase promoter (AOXI) to aid high expression levels. Tranformants are selected by Zeocin resistance and inframe C-terminal His tag allows purification by metal-chelating resins and detection through an anti-myc antibody. This produces additional 15 recombinant chorismate synthase protein, in order to produce polyclonal antisera to chorismate synthase. Antisera is employed to determine subcellular localization of T. gondii chorismate synthase. Recombinant protein also is used for later crystallography studies and for high throughput screens. 20 Production of anti-chorismate synthase antibody To produce polyclonal antiserum to the entire protein, mice with 10 ug of recombinant protein emulsified with TiterMax initially and then again 2 weeks later. A commercial source for immunization of rabbits is also suitable. Preimmune sera and sera containing polyclonal antibody, is obtained 7 25 days after the second immunization. To produce monospecific antibody, anti-peptide antibodies to specific regions of the protein also is produced in rabbits by a commercial laboratory (Alpha Diagnostic, San Antonio, TX). Analysis for B cell epitopes indicates that amino acids 342 to 363, KHERDGCSAATLSRER ASDGRT, and amino acids 35 to 55, 30 SVEDVQPQLNRRRPGQGPLST are peptides that should elicit monospecific antibodies. The advantage of polyclonal antibodies is that . . .. ,-nt-,,r- e r'-r''*r trI II C- C4\ WO 00/66154 PCT/US00/11478 - 22 they recognize native folded protein, and of the anti-peptide antibodies is that when they recognize native protein. peptide epitopes are defined. Development of enzyme assay for high throughput assays To measure chorismate synthesis, a phosphate release assay is performed using a 5 malachite green dye and the product is detected spectrophotometrically with a plate reader. This is adapted for large scale screening for high throughput screens. This assay is performed anaerobically (i.e., in a nitrogen environment) using polyethylene bags. Substrate EPSP will be synthesized as described previously. 10 Example 46: Antibody to Recombinant Chorismate Svnthase is Useful in Diagnostic Assays. Antibody to recombinant chorismate synthase was produced in mice and is useful as an immuno-diagnostic test kit reagent. Example 47: Isocitrate Lvase. 15 T. gondii isocitrate lyase activity was demonstrated and has the same uses as chorismate synthase activity, and other enzymes, e.g., it is useful for high throughput screens of T. gondii, Isocitrate lyase enzyme activity (FIG. 17C, D) and its inhibition by 3 nitropropionic acid (3NPA) (FIG. 17D) was identified. This exemplifies the presence of a key enzyme 20 in the glyoxylate cycle, and provides a method useful for both screens of available libraries of compounds and rational development of combinatorial libraries of compounds based on lead compounds and their interactions with the enzme and analysis of enzyme structure. Use of a knockout microorganism complemented with the parasite ICL gene is 25 another example of a method useful for high throughput screens to idnetify an inhibitor of ICL. sequences Antisense gene sequences to interfere with parasite growth or survival. This is a representative example of inhibition of this enzyme in this pathway. This enzyme is potentially useful in development of antimicrobial agents, diagnostic reagents or vaccines. ,-u-r.Ir-,-r r-r--- (DZI II O1\ WO 00/66154 PCT/US00/11478 -123 Example 48: The T. ontdii Isocitrate Lvase Binding Pocket and Active Site Form a Basis for Rational Antimicrobial Agent Development. The T. gondii isocitrate lyase cDNA sequence (FIG. 18), amino 5 acid sequence (FIG. 19), and isocitrate lyase binding pocket and active site (FIG. 20, box) were identified and have absolute homologies with all other isocitrate lyases and not with other partially homologous enzymes such as CPEP mutase. A yeast with a mutation in a base encoding a lysine (K) only in this area produced an inactive isocitrate lyase. This observation is 10 useful for development of antimicrobial agents as described for other sequences herein. Example 49: T. gondii Isocitrate Lvase Genomic Sequence is Useful for Vaccine Development A genomic ICL sequence is in FIG. 21 and is useful for vaccine 15 development as described for thoer genomic sequences. Example 50: Demonstration of T. gondii Isocitrate Lyase Stage Associated Protein and mRNA. T. gondii isocitrate lyase stage associated protein is present in bradyzoites and is useful as described herein for producing diagnostic 20 reagents, identifying anti-microbial agents and for vaccines. T. gondii, isocitrate lyase stage-associated protein is present in bradyzoites (FIG. 22) and there is stage associated mRNA expression and protein (FIG. 23). This observation is useful in the same manner as other examples of mRNA and protein described herein in for diagnostic reagents, antimicrobial agent 25 and vaccines. Example 51: Additional Inhibitors of Apicomplexan Isocitrate Lvase are Based on Compounds that Inhibit Isocitrate LYases of Other Organisms. Additional inhibitors of apicomplexan isocitrate lyases are 30 identified and designed. They are used as lead compounds for designing new inhibitors as described herein and this is useful for development of e- - r-- cU cr" tol II 1"' Oil1 WO 00/66154 PCT/US00/11478 -124 diagnostic reagents, antimicrobial agents and vaccines as described for other enzymes herein. 5 Example 52: Genetic, Enzymatic and Functional Evidence and Active Inhibitors of Apicomplexan Acetvl coA Carboxvlases 10 Such as Clodinafop Provide a Basis for Development of Novel Antimicrobial Agents, Diagnostic Reagents and Vaccines. FIG. 24 presents enzymatic, genetic and functional evidence of a wheat-like T. gondii acetyl coA carboxylases. Partial gene sequences were 15 identified for T. gondii, Plasmodia and Cryptosporidia acetyl coA carboxylases. Inhibitors of T. gondii acetyl coA carboxylase inhibited parasite survival in vitro. This is useful for diagnostic reagents, antimicrobial agents and vaccines as described for other sequences herein. Example 53: Synergism of Antimicrobial Agents that Inhibit 20 Apicomplexan Lipid Synthesis. Other examples of synergistic effects on lipid synthesis pathway are the synergistic effects of clodinofop, thialactomycin, and cerulin. Example 54: Growth of Toxoplasma gondii is Inhibited by Aryloxyphenoxy-propionate Herbicides Targeting 25 Acetyl-CoA Carboxylase. The recently discovered plastid-like organeldies in apicomplexan parasites provide new targets for antimicrobial agents. Aryloxyphenoxypropionates, known inhibitors of the plastid Acetyl-CoA Carboxylase (ACC) of grasses, inhibit Toxoplasma gondii ACC by 50% at 30 a concentration of 20 pM Clodinafop, the most effective of the herbicides m,--rEsen CWulmT (RI11 M1 WO 00/66154 PCT/US00/11478 -125 tested, inhibits growth of T. gondii in human fibroblasts by 70% at 10 gM and is not toxic to the host cell even at much higher concentrations. Infected fibroblasts treated with Clodinafop for two days show a substantial reduction in the number of T. gondii cells at 10 gM and almost complete removal of 5 parasites at 100 gM. Longer treatments are even more effective. Fragments of genes encoding biotin carboxylase domain of multi-domain ACCs were cloned. One ACC from T. gondii (ACC1) clusters with the putative Cyclotella cryptica chloroplast ACC and Plasmodium ACC, while another (ACC2) clusters with Cryptosporidium ACC, probably the cytoplasmic form. 10 In plants, genes encoding enzymes for fatty acid synthesis, including various subunits of ACC except one, are present in the nuclear genome and their protein products are imported and function in plastids. ACC, catalyzing the first committed step of de novo fatty acid biosynthesis, is a known selective target of aryloxyphenoxypropionate ("fops") and cyclohexanedione 15 ("dims") herbicides in sensitive species. The molecular mechanism of inhibition/resistance of the enzyme is not know but there is a strong correlation between the enzyme structure and its origin. The high molecular weight multi-domain ACC that is localized in plastids of grasses is extremely sensitive to these herbicides. All of the multi-subunit chloroplast enzymes of 20 dicot plants and bacteria as well as other multi-domain cytosolic ACCs, such as those from man, chicken, rat and yeast, are resistant. ACC activity is conveniently measured in vitro by the incorporation of the carboxyl group from bicarbonate into an acid-stable form using crude protein extracts after Sephadex G50 filtration. Substantial, acetyl-CoA dependent activity was 25 observed in extracts from tachyzoites of the RH strain of T. gondii isolated from infected mice, and no ACC activity could be detected in a control extract of macrophages from uninfected mice, the usual minor contaminant of the parasite preparation. Two biotin-containing proteins were revealed with streptavidin following electrophoresis of the extract proteins. One band at WO 00/66154 PCT/US00/11478 -126 240 kDa corresponded to the expected size for a subunit of ACC, while another at 130 kDA corresponded to the size expected for pyruvate carboxylase (PC). Structures of fops and dims were tested on the ACC-containing protein 5 extracts of T gondii described above. Three of the four fops were striking inhibitors of the activity, while none of the dims had any effect against the enzyme. There was 50% inhibition at 20 iM and 90% inhibition at 100 gM by Clodinafop, Quizalofop, and Haloxyfop. Effects of the herbicides on uninfected fibroblasts and on T. gondii growth and replication were tested as 10 previously described by Roberts et al., 1998 using incorporation of radiolabeled thymidine by growing fibroblasts to assess toxicity and incorporation of radiolabeled uracil to measure T gondii growth and persistence. Anti-parasite activity and toxicity for four fops and one representative dim were determined. Pyrimethamine and sulfadiazine, 15 antimicrobial agents which are known inhibitors of folate synthesis, were included as positive control. The combination of candidates inhibited uracil incorporation by T. gondii by more than 95% without toxicity for fibroblasts. Consistent with the data for ACC activity in vitro, the inhibitory activity of the fops and the dim on T. gondii growth in fibroblasts was in the same 20 concentration range. Clodinafop was even more active in this assay than in the enzyme assay, giving 70% inhibition at 10 gM. With regard to toxicity, fops are mildly toxic at the highest concentration, 400 giM. In separate experiments, the effect of Clodinafop on T. gondii was assessed by light microscopy. Micrographs showed infected fibroblasts treated with 25 Clodinafop at 10 and 100 tM compared with control infected cells without herbicide and uninfected cells. There is substantial reduction of the number of Toxoplasma tachyzoites at 10 gM and almost complete removal at 100 jM. The effectiveness of Clodinafop at 10 jM is greatly enhanced by a 4-day treatment with one change of medium and inhibitor after 2 days. In this WO 00/66154 PCT/US00/11478 -127 experiment, cultures were incubated for 2 more days without the inhibitor. No parasite cells were found in infected fibroblasts treated in this way. The active form of fops used as herbicides in the field are esters, which are converted to free acids by plant esterases. The true inhibitor of ACC is the 5 free acid. Two esters of Halosyfop, two esters of Quialofop and one ester of Clodinafop (Topik) have no effort on T. gondii ACC activity in crude extracts and were relatively inactive in the uracil incorporation assay except for Topik that was as active as the free acid, suggesting significant level of hydrolysis of this ester. In general, in this assay fop esters are not more effective than free 10 acids. Single stranded cDNA prepared from total RNA extracted from T. gondii tachyzoites was used as a template for the PCR amplification of a 440 bp fragment encoding the biotin carboxylase (BC) domain of ACC, using primers and conditions described for wheat ACC. Several independent PCRs 15 yielded five different products. Two of them appeared to encode eukaryotic type multi-subunit ACCs. Genomic clones encoding the entire BC domain were then isolated from a genomic library using the PCR-cloned fragments as probes and these were sequenced. Similarly, sequences of a fragment of the BC domain of ACCs of P. knowlesii, P. falciparum and C parvum were 20 determined from PCR-cloned gene fragments. A phylogenetic analysis was performed based on amino acid sequence comparisons of the two candidate ACCs from T. gondii with those of other BC domains. Three apicomplexan sequences (T. gondii, P. knowlesii, and P. falciparum) cluster together with Cyclotella cryptica ACC, an enzyme thought to be in the diatom chloroplast. 25 This isozyme, called ACC1 in T. gondii, is likely the plastid form. This assignment awaits cloning and sequencing of the 5'-terminal portion of the cDNA, where a sequence encoding a signal/transit peptide ought to be found. The other ACC, called ACC2 in T. gondii, clusters with the ACC of C parvum. These two are probably cytosolic forms. The partial genomic WO 00/66154 PCT/US00/11478 -128 sequences revealed differences in intron number and location before ACC1 and ACC2 of T. gondii, and the three ACC genes from the other apicomplexa. One of the other PCR products encoded a BC domain similar to that of pyruvate carboxylases. Deduced amino acid sequences encoded by the 5 remaining two PCR products were similar to the BC domains of rat ACC and prokaryotic-type biotin-dependent carboyxlases, respectively. These fragments were assumed to encode the host mouse ACC and a carboxylase from a bacterial comaminat. Protein gels blotted with streptavidin revealed pyruvate caroxylases (130 kDa) in addition to ACC (240 kDa), but no 10 bacterial-type biotin carboxyl carrier protein (20 kDa) or biotinylated subunit of propionyl-CoA carboxylase (70 kDa). There is a very strong correlation between the pattern of sensitivity/resistance of the ACC activity and Toxoplasma growth inhibition by the twelve different compounds tested. This result provides important 15 evidence linking the Toxoplasma growth phenotype to the effect of the compounds on the enzyme activity. The basis for the sensitivity of some of the multi-domain ACCs to fops and dims is not known, nor is it known why some, like the T. gondii ACC activity reported here, are sensitive to fops but resistant to dims. Compounds in the fop family differ in their properties as 20 well, with a clear correlation between activity and structure, e.g. relatively low inhibitory activity of Fluazifop. The target for sensitivity (herbicide binding site) is likely in to a region encompassing the 3 domain of carboxytransferase, based on experiments using yeast gene replacement strains, in which chimeric genes encoding wheat 25 ACCs replace the yeast ACC1 gene. Such strains are herbicide-sensitive if they contain a gene encoding sensitive ACC. Availability of the genes encoding T. gondii ACCs may clarify which of the isozymes is targeted to the plastid and whether one or both of them are sensitive to fops (the majority of the activity in the protein extracts was inhibited).

WO 00/66154 PCT/US00/11478 -129 Inhibition of T. gondii growth in infected fibroblasts by herbicides targeting ACC suggests, based on earlier studies of herbicide action on plants and yeast gene-replacement strains, that inhibition of ACC activity in sensitive species leads to metabolite depletion to a level at which the organism 5 cannot support its needs. This reflects an essential contribution of ACC to the pathway of de novo fatty acid synthesis and is the basis for the use of the ACC inhibitors as herbicides in agriculture and their potential future use in medicines. Example 55: An Apicomplexan Glvoxvlate Cycle. 10 To determine whether there are additional plant-like metabolic pathways as potential targets for novel chemotherapeutic agents, because they are not present in animals or differ substantially from those of animals, evidence was sought that the glyoxylate cycle might be operational in apicomplexan parasites, and play an essential role in certain stages of the life 15 cycle of these organisms. Evidence was sought for the presence of isocitrate lyase and malate synthase, key enzymes unique to the glyoxylate cycle. Enzymes of the glyoxylate cycle were detected in protein extracts of T. gondii. Polyclonal antibodies to cotton malate syntase and isocitrase lyase were used to detect 20 heterologous apicomplexan proteins by western blot analysis. A protein band of approximately 64 kD was detected using antibodies to cotton isocitrate lyase and malate synthase in lysates of T gondii tachyzoites. Isocitrate lyase was also sought, and found in western blots of T. gondii bradyzoites. Antibody to cotton isocitrate lyase also was used for immunohistochemistry to 25 study bradyzoites within cysts in brain tissue. Isocitrate lyase was identified in bradyzoites. Whether there was stage related expression of isocitrate lyase in T. gondii was studied by using smaller number of parasites in semiquantitative western blots. There was greater expression of isocitrate lyase in parasites undergoing stage conversion in vitro on the first and second WO 00/66154 PCT/US00/11478 -130 days of culture following pH shock, with loss of detectable isocitrate lyase protein on the third and seventh day with concomitant appearance of increasing levels of the bradyzoite marker BAG 1 as the bradyzoites matured when relatively small numbers of parasites were used. Stage specific 5 expression of the gene was analyzed by RT PCR using mRNA obtained from Me49 strain T. gondii tachyzoites differentiating in bradyzoites in vitro. Tachyzoites had demonstrable ICL mRNA whereas bradyzoites did not. These results suggest that expression of isocitrate lyase may be developmentally regulated. In other microorganisms, isocitrate lyase is 10 regulated at a number of different steps. For example, in E. coli there is an ace operon comprised of ace B, A, and K encoding malate synthase, isocitrate lyase and isocitrate dehydrogenase kinase phosphatase, respectively. Expression of the ace operon is under the transcriptional control of two genes, the iclR gene andfadR. The fadR is also involved in the regulation of fatty 15 acid degradation. It has been suggested that these genes encode repressor proteins, which act independently or in concert, to repress the ace operon. Moreover, functionally related isoenzymes with distinct roles in the metabolic pathways needed for growth under different minimal conditions also have been described. In addition, different isoforms of the isocitrate lyase enzyme 20 related to the age of the organism have been identified. Interestingly, in germinating seeds, isocitrate lyase plays a time-limited role with decline in isocitrate lyase activity in the senescent endosperms. Next, evidence for the presence of a functional glyoxylate cycle enzyme and its inhibition in apicomplexan parasites was obtained isocitrate 25 lyase enzyme activity and its inhibition by 3 Nitropropionic acid (NPA) was detected in lysates of T. gondii tachyzoites. Functional evidence for the glyoxylate cycle was sought by examining the effects of inhibitors of isocitrate lyase on growth and survival of apcomplexan parasites in vitro. Uracil incorporation by T. gondii in the presence and absence of inhibitor was WO 00/66154 PCT/US00/11478 -131 measured in tachyzoites. 3 NPA inhibited parasite growth. Similarly, 3NPA inhibited growth of P. falciparum. Then, genetic evidence for the presence of isocitrate lyase was obtained in T. gondii. First the primary structure of isocitrate lyases from varied organisms 5 (bacteria to higher plants) were compared, and absolutely conserved amino acid sequences were identified across species. A partial complementary DNA sequence was next identified from the WashU-Stanford-PAMF-NIH Toxoplasma EST project (EST TgESTzz53cO8.rl; GenBank accession number AA520237; Steve Parmly, PAMF, www.ncbi.nln.nih.gov/Malaria/plasmodiumbl.html). Both 10 strands of the corresponding clone were sequenced. This sequence when translated had an open reading frame (ORE) of 857 base pairs, had over 30% homology with isocitrate lyases from varied organisms (range: 29 - 53% identities; 43 - 67% positives). A T gondii RH strain genomic Lambda DASH II library (Stratagene) was then screened using TgESTzz53c08.rl as a probe, and a 15 genomic clone was obtained and sequenced (GenBank accession number to be assigned). The binding pocket and catalytic site that are absolutely conserved among other isocitrate lyases was identified in the T. gondii gene. The deduced amino acid sequence also showed partial homology with putative carboxyphosphonoenolpyruvate phosphonomutase from E. coli and Salmonella 20 species. Two regions of isocitrate lyase have been implicated as part of the active site. The motif KKCGHM(L) is conserved in all isocitrate lyases, and it is proposed that the cysteine is a critical active site residue. The absolute identity of the T. gondii sequence in the region of the active site, the binding pocket and other conserved regions to that of all isocitrate lyases, not demonstrated by any 25 carboxyphosphonoenolpyruvate phosphonomutase, makes it highly likely that the gene cloned is an isocitrate lyase gene. Also, interestingly, a single WO 00/66154 PCT/US00/11478 - 132 mutation of a K to R at the second lysine in the KKCGHM(L) motif (a substitution noted in a number of carboxyphosphonoenolpyruvate phosphonomutase genes) in a yeast and E. coli isocitrate lyase rendered it inactive (Figure 4B)

"

'. The putative T. gondii isocitrate lyase gene sequenced thus far has predicted 4 exons. These studies provide protein, enzymatic, functional and genetic evidence for the presence of a glyoxylate cycle in apicomplexan parasites. The presence of the glyoxylate cycle pathway enzymes, but not expression of its mRNA appears to be more abundant in certain life cycle stages of T. gondii in which lipids may be utilized in preference to carbohydrates as an energy source. This pathway provides a novel antimicrobial agent target and an inhibitor of an enzyme in this pathway has been identified. MATERIALS AND METHODS T. gondii Swiss Webster mice (12-15 mice per assay) were infected intraperitoneally with T. gondii tachyzoites (Rh strain, 2x10 7 per mouse) 2 days prior to assay. Tachyzoites were extracted with a peritoneal lavage using 5 ml of sterile saline per mouse. Alternatively, the PTg strain of T. gondii was cultured as tachyzoites or tachyzoites induced to become bradyzoites, as described. Antibodies Rabbit control preimmune serum was obtained and then antibodies to cotton malate synthase or isocitrate lyase were produced in rabbits SDS PAGE and Western blots T. gondii tachyzoites or bradyzoites were obtained at indicated time points from host cells by scraping the monolayer, passing the infected cells through a syringe with a 25g needle twice to disrupt them, and then organisms were counted and centrifuged at 2000 rpm for 10 minutes at 4*C to pellet the parasites. The supernatant was discarded and the pellet was suspended in SDS PAGE loading buffer (with 2 mercaptoethanol) at a WO 00/66154 PCT/US00/11478 - 133 concentration of lx 10 5 parasites per p.l and boiled for 10 minutes. Unless otherwise indicated, material from 2x 106 parasites was utilized per lane. This was electrophoresed in a 12% polyacrylamide gel under reducing conditions and transferred onto nitrocellulose membranes blocked with 5% milk in PBS tween (0.05%), and probed with rabbit preimmune serum or polyclonal antibody to cotton isocitrate'lyase or malate synthase, or mouse monoclonal antibody to BAGI antigen, followed by HRP conjugated anti rabbit or anti mouse secondary antibodies'as appropriate. Bands were visualized using ECL. PCR and Northern blots Messenger RNA, isolated on oligo dT solid phase matrix columns and reverse transcribed using a random priming method, was used for semi-quantitative PCR analysis of tachyzoite surface antigen (SAG)1, bradyzoite cytosolic antigen (BAG) 1-5, and isocitrate lyase (ICL), relative to beta tubulin (TUB). The primer sets were as follows: SAGI (5'-CGG' TTG TAT GTC GGT TTC GCT-3' and 5'-TGT TGG GTG AGT ACG CAA GAG TGG-3'), BAG 1-5 (5'-CCC ATC GAC GAT ATG TTC GAG-3' and 5'-CGT AGA ACG CCG TTG TCC ATT G-3'), ICL (5'-TTG CCG TTC TGG AAA GCT AGT AAG A-3' and 5' GCA AAC GCT GGT CCT CAA TGT-3'), and TUB (5'-GTT TCC AGA TCA CCC ACA GTC TTG G-3' and 5'-GAG CAA ACC CAA TGA GGA AGA AGT G-3'), yielding PCR product sizes of, 346, 225, 574, and 420bp, respectively. The BAGI-5 primers flank an intron serving as a control for genomic DNA contamination, yielding a PCR product of 784 bp. cDNA from T. gondii tachyzoites of the RH strain and induced bradyzoites from the Me49 strain were used as templates. Immunohistochemistrv Immunoperoxidase staining was performed' as previously described using control preimmune or immune rabbit antisera. Enzyme assays Parasite lysates were obtained from tachyzoites, suspended in elution buffer (100 mM KCI. 20% glycerol, 7 mM 2-mercaptoethanol, 20 mM Tris-HCI, pH 7.5, and WO 00/66154 PCT/US00/11478 - 134 complete TM protease inhibitor cocktail [Boehringer Mannheim, I tablet per 50 ml buffer], sonicated 3 times for 3 seconds at 30 sec intervals, and centrifuged at 12,000 g for 15 min. The supematant collected was applied to a Sephadexo G 100 column (25 ml, Pharmacia) equilibrated with elution buffer, eluted with 15 ml of elution buffer, and = 1.5 ml fractions were collected. Fraction(s) with the peak protein concentrations (protein analysis performed on a spectrophotometer at 280 nm) were selected and used in enzyme assays. A discontinuous method described by Ko and McFadden1 7 was employed with minor modifications to measure the ability of isocitrate lyase to convert isocitrate to succinate and glyoxylate. This method utilizes the colorimetric reaction between the phenylhydrozone of glyoxylate and ferricyanide. Reaction mixtures (92 mM MOPS,.5 mM MgCIl,, I mM DTT, 1% phenylhydrazine, 4.4 mM isocitrate, in 0.5 ml with fractionated parasite lysate) were incubated in a 37 "C water bath for a determined amount of time. After incubation, enzymatic reactions were stopped with concentrated HCI, mixed with 25% (w/v) potassium ferricyanide, and then measured in a spectrophotometer at 520 nm. Culture of parasites in vitro with inhibitors Parasites were cultured with host cells and inhibitors and the effects analyzed as described'. Identification of T. gondii isocitrate Iyase genes: Library screening, phage DNA purification, Southern blot (cloning and sequencing), host strains and vectors XL I Blue MRA and pBluescript KS" DH5 a were used. Lambda Dash II (Stratagene) was the vector for the genomic library. A 550 bp ECORI-XhoI fragment of the cDNA EST clone TgZZ13 CO8 r 1 was labeled with a ( 5 2 P) dCTP and used for initial screening of the library. For subsequent secondary and tertiary screening to obtain pure phage, a biotinylated, non-radioactive, labeled probe of the entire 857 bp EST clone was prepared and used. The genomic library was screened (Stratagene), phage purified to >99% homogeneity, the clone amplified and DNA extracted (Current Protocols in Molecular Biology).

WO 00/66154 PCT/US00/11478 - 135 Southern blot The purified phage DNA was digested with NotI; xhol or EcoRI enzymes, run on a lx agarose gel, transferred onto a nylon membrane probed with the biotinylated probe (above). A -4kb band which was identified with the probe and was cloned into pBluescript KS' and sequenced. DNA sequencing and sequence analysis DNA sequencing was performed using an automated DNA sequencer. This sequence was compared to peptide sequence databases at The National Center for Biotechnology Information (NCBI) using the program TBlastX or BlastP (for derived open reading frames). Gene construction using the sequence obtained was also performed utilizing the Baylor College of Medicine program. Primers for sequencing were made at Integrated DNA Technology. Sequence analysis was carried out by software programs MacVector, ClustalX and MACH Box.

WO 00/66154 PCT/US00/11478 - 136 MATERIALS AND METHODS A. MIethods to Assay Candidate Inhibitors 1. Inhibitors of Toxoplasma gondii a) Cell lines: Fibroblasts. Human foreskin fibroblasts (HFF) are 5 grown in tissue culture flasks in Iscoves' Modified Dulbecoes Medium (IMDM), containing 10% fetal bovine serum, L-glutamine and penicillin/streptomycin at 37 0 C in 100% humidity and a 5% CO 2 environment. Confluent cells are removed by trypsinization and washed in IMD)M. They are used in a growth phase for toxicity assays or when 100% confluent for parasite inhibition assays. 10 b) Tachyzoites: Tachyzoites of the RH and pTg strains of 7 gondii are passaged and used for in vitro studies (McLeod et al., 1992). The R5 mixed tachyzoite/bradyzoite mutant was derived from mutagenesis with nitrosoguanidine in the present of 5 hydroxynapthoquinone. These organisms are used for in vitro experiments at a concentration of 2 x 10', 2 x 10', or 2 x 10: organisms per ml, 15 dependent upon the planned duration of the experiment (i.e., larger inoculations for shorter duration experiments). c) Bradyzoites: Bradyzoites are obtained as described by Denton et al. (1996b). Specifically, C57BLI0"ScSn mice are infected intraperitoneally with 20 cysts of the Me49 strain of 7T gondii. Their brains are removed 30 days later 20 and homogenized in PBS by repeated passage through a 21 gauge needle. Aliquots containing the equivalents of 3-4 brains are diluted in PBS and 6 5 mils of 90% percoll added to the mixture which is allowed to settle for 30 nins :mls of 90% Percoll is WO 00/66154 PCT/US00/11478 -137 then added as a bottom layer and the mixture centrifuged for 30 mins at 2500xg. The cysts are recovered from the bottom layer and a small portion of the layer above. After the removal of Percoll by centrifugation, the contaminating red blood cells are removed by lysis with water followed by the addition of I ml of 10xPBS per 9 ml brain 5 suspension in water. Bradyzoites are released from the purified cysts by digestion in a 1% pepsin solution for 5 minutes at 37 0 C. This method routinely permits recovery of greater than. 90% of the cysts present which yields approximately 100 bradyzoites per cyst. Bradyzoites are used at concentrations of2xl0', 2x10 4 and 2x10 5 per ml in parasite growth inhibition assays. pH shock is also used to retain organisms in 10 bradyzoite stage when such pH does not interfere with inhibitor activity. d) Inhibitors: Inhibitor compounds are tested over a range of concentrations for toxicity against mammalian cells by assessing their ability to prevent cell growth as measured by tritiated thymidine uptake and inspection of the monolayer using microscopic evaluation. A range of concentrations that are non-toxic in this 15 assay are tested for their ability to prevent the growth of T gondli and also other Apicomplexans within these cells. i.) Heme Synthesis: The inhibitor of the heme synthesis pathway, gabaculine (Grimm. 1990. Elliot et al.. 1990; Howe et al., 1995; Mets and Thiel. 1989; Sangiwan and O'Brian 1993: Matters and 20 Beale, 1995) is used at a concentration of 20 m.11 [which has been demonstrated to be effective against tachvzoites of the RH and R5 -trains] Other inhibitors ofthis pathway include 4 amino-5-hexynoic WO 00/66154 PCTIUS00/11478 - 138 acid and 4-aminofluoropentanoic acid which provide additional corroborative evidence that this pathway is present. ii) Glyoxylate Cycle: The inhibitor of isocitrate lyase is 3 nitropropionic acid (ranging from 0.005 to 5 mg/ml in vitro). 5 iii) Alternative Oxidase T. gondli bradyzoites use unique alternative oxidases. Alternative oxidase is necessary and sufficient for bradyzoite survival. Methods to characterize plant alternative oxidases are described (Hill, 1976; Kumar and Soil, 1992; Lambers, 1994; Li et al., 1996, Mclntosh, 1994). 10 For the in vitro studies, cell lines that lack functional mitochondria are used. These cell lines are used to allow the study of inhibitors effective against the conventional or alternative respiratory pathways within the parasite, but independent of their effects onil the host cell mitochondria. Two cell lines, a human fibroblast cell line (143B/206) lacking mitochondrial DNA. and the parental strain (143B) which poses 15 functional mitochondria are used. These cell lines have been demonstrated to support the growth of T gondii (Tomavo S and Boothroyd JC, 1995). SHAM, an inhibitor of the alternative respiratory pathway is used at concentrations between 0.25 and 2 lg/ml ill Viro. iv) Shikimate Pathway. For EPSP synthase, the inhibitor is 2) N-(phosphononiethyl) glycine (concentrations of 3 125mN'I in folate deficient media) e) Culture assay svst:m tor assessin, inhibitor effect: WO 00/66154 PCT/US00/11478 - 139 i) Toxicity assays: Aliquots ofcells (HFF) are grown in 96-well tissue culture plates until 10% confluent. Cells are incubated with various concentrations of drug for 1, 2, 4 and 8 days. Cultures are pulsed with tritiated thymidine (2.5 pCi/well) for the last 18 hours of the culture after which the cells are 5 harvested using an automated cell harvester and thymidine uptake measured by liquid scintillation. ii) In vitro parasite growth inhibition assays: Confluent monolayers of HFF cells, grown in 96-well plates are infected with T. gondii tachyzoites of the RH strain and serial dilutions of anti-microbial compound are applied 1 hour later. 10 7: gondii growth is assessed in these cultures by their ability to incorporate tritiated uracil (2.5 pCi/well) added during the last 18 hours of culture. After harvesting cells with an automatic cell harvester, uracil incorporation is measured by liquid scintillation. Alternatively. confluent HFF cells are grown in the chambers of Labtech slides and parasite growth is assessed microscopically following fixation in aminoacridine and 15 staining in 10% Giemsa (McLeod et al., 1992). f) Product rescue assays to evaluate specificity of the inhibitor: To attempt to demonstrate specificity of the site of action of the inhibitor, growth inhibition assays are performed in the presence of varying concentrations of product, e.g., in the case where gabaculine is thile inhibitor, ALA is added simultaneously to 20 determine whether product rescue occurs. This type of study is only interpretable when rescue is demn..nstrated because it is possible that exogIenous "product" is not WO 00/66154 PCT/US00/I 1478 - 140 transported into the 7. gondii within host cells. For EPSP synthase, product rescue assay is performed with PABA. g) Assays for synergy in vitro. This is an assay in which < 50% inhibitory concentrations of two antimicrobial agents are added alone and together to determine 5 whether there is an additive, synergistic or inhibitory interaction. All other aspects of this assay are as described herein. 2. Inhibitors of Cryptosporidia parvium C. pavaum oocysts at 50,000/well were incubated with each drug (PRM=paromomycin which is the positive control, NPMG, gabaculine, SHAM, 8 10 hydroxyquinoline) at 37 0 C (8% carbon dioxide) on confluent MDBKF5D2 cell monolayers in 96 well microtiter plates. The level of infection of each well was determined and analyzed by an immunofluorescence assay at 48 hours using as an antibody C. parrumn sporozoite rabbit anti-serum (0.1%), and using fluorescein conjugated goat anti-rabbit antibody (1%). Data are expressed as mean parasite 15 count/field when 16 fields counted at 10x magnification "s.d. of the mean. (FIG. 6) 3. Inhibitors of Plasmodiumn falciparuin This assay is performed in folate deficient RPMI 1640 over a 66 hour incubation in plasma as described by Milhous et al. (1985). Both the VW2 clone D-IFR resistant phenctype and the D6 clone are used (Odula et al., 1988) (Table 3). 20 4. Inhibitors of Einmeria tenella S usce:ibility of lineria enella in vitrio is analyzed by a method similar to that described byv NcLeod e't a/., 19 )92 or for( 'rrputoridiun as disclosed herein.

WO 00/66154 PCT/US00/11478 - 141 5. In vivo stud(lies, measurement of parasitemnia of Toxoplasna gondii A method to measure the amount of parasitemia in mouse peripheral blood has been developed. Briefly, the target for PCR amplification is the 35 fold repetitive B I gene of T. gondli and the amplification was performed using primers previously 5 reported. In order to semiquantitate the PCR product and to avoid false negative results, a competitive internal standard is generated using a linker primer and the original B 1 primers. Competitive PCR was performed by spiking individual reactions (containing equal amounts of genomic DNA) with a dilution of the internal standard. Since this internal control contains the same primer template sequences, it competes 10 with the B I gene of T. gondii for primer binding and amplification. The sensitivity of the PCR reaction in each sample can be monitored. Following competitive PCR, the PCR products are distinguished by size and the amount of products generated by the target and internal standard can be compared on a gel. The amount of competitor DNA yielding equal amounts of products gives the initial amount of target gene. I 5 6. Interpretation of DataStatistical Analisis of Data: I vitro studies are performed with triplicate samples for each treatment group and a mean ± sd determined as shown in the FIGs. All in vivo studies utilize at least 6 mice per group Statistical analysis performed by Students' t-test or the Mann-Whitney U-test. A p value of < 0.05, is considered statistically significant. 120 B. Western Blots Demionstrate Plant-Like Enzymes \Wester: analysis for GS.\AT. isocitrate lyase, malate svnthase, alternative oxidase and EPSP is used to demonstrate the presence of plant-like enzymes in many WO 00/66154 PCT/US00/11478 - 142 Apicomplexan parasites, e.g., P'lasmodia, Toxop/asmna, Criptosporidia, ,alaria and Eimneria. Tachvzoites and bradyzoites (McLeod et al. 1984, 1988; Denton et al., 1996a, b), or their mitochondria and plastids are isolated as previously described. Equivalent 5 numbers of tachyzoites and bradyzoites are separately solubilized in 2x sample buffer and boiled for 5 minutes. Samples are electrophoresed through a 10 percent SDS polyacrylimide gel. Proteins are transferred to.a nitrocellulose membrane at 4oC, 32V with 25mM Tris and 192mM glycine, 20% v/v methanol, pH 8.3. Blots are blocked in PBS (pH 7.2) containing 5% powered milk and 0. 1% Tween 20 for 2 hours at 20'C. 10 After washing in PBS (pH 7.2), 0.1% Tween 20, blots are stained with polyclonal or monoclonal antibodies specific for alternative oxidases in PBS (pH 7.2) containing 0.1% Tween 20 for 1 hour at 20 0 C. Following washing in PBS (pH 7.2) containing 0.1% Tween 20, blots are incubated with an appropriate secondary antibody conjugated to HRP at a dilution to be determined by methods known in the art. After further 15 washes, binding is visualized by chemoilluminescence (Amersham). Antibodies to various enzymes, e.g., soybean GSAT, barley GSAT, synechococcus GSAT, plant and/or trypanosome alternative oxidase, cotton isocitrate lyase, cotton malate synthase, soybean malate synthase, petunia EPSP synthase were used to determine whether homologous enzymes are present in 7' gondii tachyzoites, 20 bradyzoites. mitochondrial and plastid enriched preparations. Antibodies used include monoclonal antibodies to Thipinoso nmi hruc'ii and Voo Doo Lily (Chaudhuri et al. 1996) alternative oxidase and polyclonal antibody to 'ti7ano.omua bruceii alternative WO 00/66154 PCT/US00/11478 - 143 oxidase. The hybridizations with antibodies to plant and related protozoan alternative oxidases demonstrated the relatedness of 7 gondii metabolic pathways to those of plants and other non-Apicomplexan protozoans. The products GSAT and alternative oxidase were demonstrated by Western analysis. Both polyclonal and monoclonal 5 antibodies were reacted with alternative oxidase to confirm this observation. C. Probing Other Parasite Genes. The genes isolated from T. gondii as described herein are used to probe genomic DNA of other Apicomplexan parasites including Plasmniodia, Cryptosporodium, and Eimeria. D. Genomic Sequence. Genomic clones are identified and sequencedin the same 10 manner as described above for cDNA except a genomic library is used. Analysis of unique promoter regions also provide novel targets. E. Enzymatic Activity Demonstrates Presence of Plant-Like Enzymes in Metabolic pathways The presence of the enzymes putatively identified by inhibitor studies is 15 confirmed by standard biochemical assays. Enzyme activities ofGSAT, isocitrate lyase. malate synthase, alternative oxidase, and EPSP synthase, chorismate synthase, chorismate lyase, IUDP-glucose starch glycosyl transferase and other enzymes listed herein are identified using published methods. Representative methods are those of Jahn et al., 1991 Weinstein and Beale, 1995; Kahn et al., 1977: Bass et a/., 1990, 20 Mousdale and Coggins (1985) In addition, enzyme activity is used to determine in which of the .chvzoite and bradyzoite life cycle stages each pathway is operative Tachvzoites a:: bradyzoites are purilied as described herein. Tihe parasites are lysed in WO 00/66154 PCT/US00/11478 - 144 . 50mM HEPES (pH7.4) containing 20% glycerol, 0.25% Triton X-100 and proteinase inhibitors (5mnM PMSF, 5FM E64, IFM pepstatin, 0.2mM 1,10-phenanthroline). This method has proven successful for measurement of phosphofructokinase, pyruvate kinase, lactate dehydrogenase, NAD- and NADH-linked isocitrate dehydrogenases and 5 succinic dehydrogenase activity in tachyzoites and bradyzoites of T. gondii (Denton et al., 1996a,b). 1) GSAT: GSAT activity is measured by the method of Jahn et al., (1991), which uses GSA as substrate. GSA is synthesized according to methodsofGough el al. (1989). Heat-inactivated (60'C, 10') lysates are employed as non-enzymatic 10 controls. ALA is quantified following chromatographic separation (Weinstein and Beale, 1985). This approach allows the definitive detection of GSAT activity in crude extracts. 2) ALA Synthase: To determine whether parasites contain ALA synthase, an activity also present in mammalian host cell mitochondria, cell fractions from 15 purified parasites are assayed. (Weinstein and Beale, 1985) ALA produced from added glycine and succinyl CoA is quantified as for GSAT. 3) Isocitrate Lvase: The biochemical assay for isocitrate lyase activity used is the method of Kahn et al. (1977). 4) Alternative Oxidase: activity is measured in parasite lysates or purified 20 mitochondria or plastids by oxygen uptake using an oxygen electrode described by Bass ei al. (1990) Conlirmation of the oxidation being due to alternative oxidase(s) is WO 00/66154 PCT/US00/11478 - 145 achieved by successful inhibition ofoxygen uptake in the presence of 0.5mM SHAM, but not in the presence of KCN. 5) Shikimate Pathway: The biochemical assay for EPSP synthase, chorismate synthase, chorismate lyase; activity in cellular lysates is conducted as 5 described by Mousdale and Coggins (1985) and Nichols and Green (1992). 6) Branched Amino Acids: The biochemical assay for hydroxy acid synthase is as described. 7) Amvlopectin Synthesis: The biochemical assays for starch synthase, Q enzymes, and UDP-glucose starch glycosyl transferase are as described. 10 8) Lipid Synthesis: Assays for lipid synthases are as described. Some of the additional representative enzyme assays are precisely as described by Mousdale and Coggins(1985) and are as follows: 5-Enolpyruvylshikimate 3-phosphate synthase is assayed in forward and reverse directions as described previously (Mousdale and Coggins 1984). 15 Shikimate:NADP oxidoreductase (shikimate dehydrogenase), shikimate kinase, 3-Dehydroquinase (DHQase) are assayed. Assay mixtures contained in a total volume of I ml: 100 mM potassium phosphate (pH 7.0) and 0.8 mM ammonium 3-dehydroquinate. 3-Dehydroquinate synthase is assayed by coupling for forward reaction to the 3-dehydroquinase reaction; assay mixtures contained in a total volume of I ml: 10 mM potassium phosphate (pH 7.0), 50 pil N."\D 0 I mM CoCIl. 0.5 nkat partially-purified EIch'ricihia coll DlIQase WO 00/66154 PCT/US00/1 1478 - 146 and (to initiate assay) 0.4 mM DAHP. The DAHP is prepared from E. coli strain AB2847A and DHQase from E. coli strain ATCC 14948. Assay of DAHP synthase is by a modification of the method of Sprinson et al.. Assay mixtures contained in a total volume of 0.5 ml: 50 mM 1,3-bis 5 [tris(hydroxymethyl)-methylamino] propane-HCI (pH 7.4), 1 mM erythrose 4 phosphate, 2 mM phosphoenolpyruvate and 1 mM COC1 2 . The reaction is initiated by the addition of a 50 to 100 Vl sample containing DAHP synthase and terminated after 10 min at 37 0 C by 100 jil 25% (w/v) trichloroacetic acid. The mixture was chilled for I h and centrifuged to remove precipitated protein. 10 A 200 pl aliquot of the supernatant was mixed with 100 pl 0.2 M NalO 4 in 9 MNI HPO- and incubated at 37 0 C for 10 min; 0.5 ml, 0.8 M NaASO 2 and 0.5 M Na 2

SO

4 in 0. 1 M H 2 SO4 in 0.1 m H 2 SO was then added and the mixture left at 37 0 C for 15 mrin; 3 ml 0.6% (w/v) sodium thiobarbiturate and 0.5 M NaSO 4 in 5 mM NaOH was added and the mixture placed in a boiling-water bath for 10 15 min. After cooling to room temperature the solution was centrifuged (8500 xg. 2 min) and the optical density at 549 nm read immediately. Appropriate controls assayed in triplicate lack substrates, sample or both." Another representative assay is an assay for chorismate lyase which is as described by Nichols and Green. 1992: 20 Chorismate lyase assays are carried out in a volume of0.5 ml containing 50 mM Tris-1-CI (pil1 7 5), 5 mM EDTA. 10 mM 2-mercaptoethanol, 60 pM chorismnate, and 0.2 to 4 U ofchorismate vlyase After incubation at 37 0 C for WO 00/66154 PCT/US00/11478 - 147 30 min, 4-hydroxybenzoate is detected and quantitated by high-pressure liquid chromatography (HPLC). Fifty microliters of each reaction mixture is applied to an HPLC system (Waters 625) equipped with a Nova-Pak Ci 8 column equilibrated in 5% acetic acid and monitored at 240 nM. The height of the 5 4-hydroxybenzoate peak is compared with those of standard curves generated by treating known amounts of 4-hydroxybenzoate in a similar manner. One unit of chorismate lyase activity is defined as the amount of enzyme required to produce I nmol of 4-hydroxybernzoate in 30 min at. 37 0 C. Assays for 4-aminobenzoate and 4-amino-4-deoxychorismate arepeformed as 10 described previously." Enzyme Assays: The 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase assay entailed monitoring the generation of EPSP using HPLC. Reaction components were separated using a Hypersil H3APS2 HPLC column (Hichrom Limited, Reading, UK) and a NaH2PO4 elution gradient (50-400 mM). UV spectra (200-320 nm) of the column eluate were collected to identify eluants. I5 Shikimate-3-phosphate and 5-enolpyruvylshikimate-3-phosphate, synthesized enzymatically and purified to at least 95% purity as described (12), eluted after 3.9 and 6.8 min, respectively; phosphoenolpyruvate did not interfere with the EPSP detection and eluted after 5.3 min. The peaks at 215 nm were integrated; the EPSP produced was quantified using a standard curve of authentic EPSP. Parasite extracts were 2, produced at 4:C by suspension of pure tachyzoites in extraction buffer (50 mM Tris I-ICI, pH 7 5. containing complete TNI protease inhibitor cocktail [Bochringer Mannheim, I tablet per 50 ml bufler]). sonication 3 times for 3 seconds at 30 second WO 00/66154 PCTIUS00/11478 - 148 intervals, and centrifugation at 12000 g for 15 rnin. The resulting supernatant was diluted 6-fold with extraction buffer and loaded onto a ResourceQ column (1 ml, Pharmacia) equilibrated with extraction buffer. The bound protein was eluted in a single step using extraction buffer containing 500 mM KCI. The eluted material was 5 used for enzyme assay. The assay mix contained I mM phosphoenolpyruvate, I mM SP and 50 mM HEPES, pH 7.5. The reaction was started by addition of parasite extract and incubation was at. 30 0 C. Times 10 :1 I aliquots were subjected to HPLC analysis. Protein concentrations of lysates were determined using the Lowry method. (Roberts et al., 1998, In Press) 10 E. Construction and Analy'sis of Gene "Knock-Outs" In order to determine whether a gene, e.g., chorismate synthase or alternative oxidase is essential for growth or survival of the organism, gene knockout organisms are generated by the method of Roos et al., 1996. Specifically, the strategy for creating mutants is with homologous recombination and to generate a targeted gene knock-out 15 a sequential positive/negative selection procedure is used (Roos et al., 1996). In this procedure positive and negative selectable markers are both introduced adjacent to, but not within the cloned and suitably mutated locus. This construct is transfected as a circular plasmid. Positive selection is applied to yield a single-site homologous recombinant that is distinguished from non-homologous recombinants by molecular 20 screening. In the resulting 'pseudodiploid,' mutant and wild-type alleles flank selectable marker and other vector sequences In the next step, parasites are removed from positive selection, which permits recombination between the duplicated loci. This WO 00/66154 PCT/US00/11478 - 149 event appears to occur at a frequency of 2 x 10" per cell generation. These recombinants are isolated with negative selection. Next, they are screened to distinguish those that have recombined in a manner that deletes the mutant locus and yields a wild-type revertant from those that deleted the wild-type gene to leave a 5 perfect allelic replacement. This 'hit-and-run' approach has the disadvantage of being time-consuming. Nonetheless, it offers several distinct advantages over other gene knock-out strategies. First, because gene replacement occurs by two sequential single-cross-overs instead of. one double cross-over which is a very rare event, it is morelikely to be successful. 10 Second, because selectable marker(s) are located outside of the targeted gene itself, experiments are not limited to gene knock-outs. A variety of more subtle point mutations are introduced as allelic replacements. Third, this strategy provides a means of distinguishing essential genes from those which cannot be deleted for purely technical reasons. Specifically, if the hit-and-run mutagenesis procedure yields only 15 wild-type revertants instead of the theoretical 1:1 ratio of wild-type:mnutant, this provides positive evidence that the locus in question is essential. An example is a knockout created for the chorismnate synthase gene. It also can be made more general to include knockout of other genes for attenuated vaccines such as EPSP synthase and alternative oxidase The parasite with the gene of interest to be 20 knocked out is grown ("manufactured") m vitro in presence of product, but when used int " m the needed product is not present The parasite functions as an attenuated vaccine as described below under vaccines A specific example 1followvs: Specifically.

WO 00/66154 PCT/US00/11478 - 150 the strategy of product inhibition discussed above is also useful for growing gene knockout parasites (which lack a key gene for their survival) in vitro by providing the essential product and thus bypassing the need for the gene during inii vitro propagation of the parasite. Such gene knockouts cultivated in vitro in this manner are useful 5 attenuated organisms that are used as attenuated vaccines. The chorismate synthase cDNA clones are used as hybridization probes for recovering genomic clones from a T. gondii genomic cosmid library. Coding regions are mapped onto the genomic clones using the cDNA clones as a guide. Appropriate, sections are sequenced to verify the. gene location. Ultimately. full genomic sequences 10 are obtained. Enough of the genomic clones are sequenced to develop a strategy for generating a putative null allele. Segments that can be deleted at the 5' end of the coding region to generate an allele that is unlikely to generate a functional gene product are identified. A putative neutral allele is generated that can be distinguished from the wild type allele on the basis of an introduced restriction site polymorphism, but that 15 does not differ in encoded protein sequence. These putative chorismate synthase-null and chorismate synthase-neutral alleles are cloned into the pminiHXGPRT transfection vector plasmid. The resulting chorismate synthase -null and chorismate svnthase-neutral plasmids are transfected into HXGPRT-negative strains of 7 gondii (strains 20 RH(EP)'HXGPRT [a ME49 derivative]. Numerous independent clones are selected lbr survival on mnvcophenolic acid to select for insertion o'f the plasmid These strains are screened by Southern analysis designed to detect the presence o both the normal and WO 00/66154 PCT/US00/1 1478 - 151 modified copies of the chorismate synthase gene and for tandem location of the two copies (with the vector HXGPRT gene between). This is the structure expected for insertion of the plasmid by homologous recombination at the AroC genomic locus (tilhe "hit" needed for the hit-and-run gene knock-out strategy). The feasibility of recovering 5 these strains is critically dependent upon the ratio of homologous to non-homologous integration following transfection, which will depend upon the length of homologous, genomic DNA in the clone (Donald and Roos, 1994; Roos et aL., 1996). Eight kb of homology is sufficient to obtain >50% homologous integration (Roos et aL., 1996): HXGPRT clones with verified pseudodiploid structure of thechorismate: 10 synthase alleles are selected for loss of HXGPRT using 6-thioxanthine (the "run" part of the protocol). Numerous clones are selected. If the loss of HXGPRT is based upon random homologous exchange between the two chorismate synthase pseudodiploid alleles, theoretically half of the events should lead to excision of the modified chorismate synthase allele along with the HXGPRT, leaving the original wild type allele 15 in thile chromosome. The other half should excise the wvil type allele, leaving the modified allele in the chromosome. During selection and grow-out of these clones, the medium is supplemented with chorismate at thile concentration determined to best rescue cells from inhibitor toxicity. The purpose of the supplementation is to enhance the chances of recovering chorismate synthase-null strains. Thile genomic structure of 20 the selected clones is examined by Southern analysis to confirm loss of the vector SlXGPRT and of one colpy of the chorismate svnthase and to identify the remaining allele of chorismate synthliasc The ratio 'of muLtant to wild type is tabulated. The WO 00/66154 PCT/US00/11478 - 152 chorismate synthase-neutral allele is intended as a positive control to confirm that either allele (wild type or mutant) can be lost in this procedure. If chorismate synthase neutral strains can be recovered but chorismate synthase-null strains cannot, the conclusion is that the chorismate synthase gene is essential for growth. If it proves 5 possible to recover chorismate synthase-null strains, they are subjected to further phenotypic analysis, first, using immunoblotting of electrophoretically separated cell extracts to confirm absence of chorismate synthase protein, then, determining if these strains show hypersensitivity to inhibitors of the alternative oxidase. or to any of the other potential inhibitors. Sensitivity to chorismate.synthase inhibitors is analyzed to. 10 determine the relative specificity of inhibition. If chorismate synthase is the sole target of the inhibitors, then the null mutants should be insensitive to further inhibition. Sensitivity analysis is conducted in vitro as described herein. Whether strains show alterations in expression of the alternative oxidase or in any stage-specific antigens is of interest. These analyses are conducted by immunoblotting of electrophoretically 15 separated cell extracts. In vivo analysis using a mouse model is conducted to determine ifthese strains are infective and what stages of parasites can be detected following infection. Genetically altered T. gondii organisms are used to infect C3/HU-leJ mice by the intraperitoneal route. Mortality is monitored and brains examined for cysts at 30 days post infection. 20 Knockouts with bradyzoite reporter genes are useful to determine whether these enzymes influence staue switch.

WO 00/66154 PCTUS00/11478 - 153 Stage switch also is characterized by quantitating relative amounts of parasite mRNA present in each stage ofparasite using Northern blotting, isolation of mRNA and RT-PCR using a competitive inhibitor, and enzyme assay. G. Reagents used for construction of "Knock-Outs" 5 Library Me49 genomic libraries are used. Plasmids pminiHXGPRT. Contains T gondli HXGPRT gene under control of DIHFR-TS 5' and 3' flanking sequences. Functions as either a positive or negative selection nriarker 10 (using 6-thioxanthine or mycophenolic acid, respectively) in suitable host strains. Parasite Strains (obtained from AIDS Repository, Bethesda, Md.) RH(EP). Wild-type host strain RH (highly pathogenic in mice). RH(EP)"HXGPRT. HXGPRT knock-out mutant of RH strain. Suitable for positive selection of HXGPRT-containing vectors. 15 P(LK). Wild-type host strain P, (clonal isolate of strain ME49; produces brain cysts in mice). P(LK)HXGPRT-. HXGPRT-deficient mutant of P strain. Suitable for positive selection of HXGPRT-containing vectors.

WO 00/66154 PCT/US00/11478 - 154 I1. Antibodies Antibodies have been raised against homologous plant enzymes by standard techniques for both polyclonal and monoclonal antibodies (Current Protocols in Immunology, 1996). 5 1) Hemne Synthesis Antibody to soybean, barley and synechococcus GSAT are polyclonal antibodies with preimmune sera the control for the barley and synechococcus antibodies. 2) Glvoxvlate Cycle 10 T. gondii contains a glyoxylate cycle that allows growth using lipids as a carbon source, thus the lipid mobilization pathway of T. gondii is similar to the pathway of plants (Tolbert, 1980). A similar approach using polyclonal antibodies to isocitrate lyase and to malate synthase and preimmnune control sera are used. 3) Alterinative Enervgy Genteration 15 MNonoclonal and polyclonal antibodies to alternative oxidases in plants (Mcintosh et aL., 1994) and Trypanosomes (Hill, 1976) are used. 4) Shikimate Pathway To demonstrate that 7 gondii has the same unique enzymes that permit interconversion of shikimate to chorismate as plants do, the antibody to shikimate 20 pathway plant EPSP synthase is used.

WO 00/66154 PCT/US00/11478 - 155 5) Synthesis of Branched Chain Amino Acids Antibodies to acetohydroxy acid synthase are used. 6) Amvlose and Amviopectin Synthesis and Decradation Antibodies to starch synthesis, branching (Q) enzymes and UIDP glucose starch 5 glycosyl transferase are used. I. Complementation of Enzyme Deficient E. coli Demonstrates Functional Product The E. coli AroC mutant which lacks chorismate synthase (AroC) was obtained from the E. co/i genetic stock center. AroC bacteria is made competent to take up 10 DNA by transformation with CaCl 2 treatment. Alternatively, the cells are electroporated to take up DNA. The presence of the plasmid is demonstrated in this system by growth on media which contains ampicillin, as the plasmid contains an ampicillin resistance gene. Complementation is confirmed by demrnonstrating growth on media lacking the product catalyzed by (i.e., chorismate). Thus, this transformation/ 15 complementation is used with the T. gondli cDNA librarY system or a construct which contains some or all of the chorismate synthase gene to transform the AroC mutant. Functional enzyme is then demonstrated. J. Immuniizations Of Mice For Polvclonal Antibody Production: As an alternative approach if complementation studies are unsuccessful and the 20 monoclonal antibodies to a plant protein are not cross reactive, purified plant protein is used to immunize mice to raise polyclonal antibodies to each enzyme. Where necessary, a:ibodies to the pertinent enzymes are generated in mice, ND4 outbred WO 00/66154 PCT/US00/11478 - 156 mice are immunized with 20 pg of enzyme emulsified in Titermax complete adjuvant injected intramuscularly into their gluteal muscle. Two weeks later mice are immunized with a further 20 ug of enzyme emulsified in Titermax. After a further 2 weeks mice receive a further boost of enzyme alone in PBS by the intraperitoneal route. Mice are 5 bled and the serum tested for specificity by the standard Western blotting technique. K. Immniunofluorescence Antibodies used to identify enzymes in the Apicomplexan metabolic pathways disclosed here are used for immunofluorescence studies. Examples are demonstration of alternative oxidase in T gondli by immunofluorescence assay (IFA). T. uodii 10 alternative oxidase is immunolocalized to mitochondria. L. ELISAs ELISAs are used for documenting the presence and quantitating the amounts of alternative oxidase M\1. Reporter Constructs To Demonstrate Organelle Targeting Are Made And 15 Characterized As Described Using (3 Glucoronidase Or Other Chimeric Constructs Importance of the targeting sequence for localization of the enzyme to an organelle is demonstrated with imnmunoelectronmicroscopy. Organelle targeting sequences in proteins expressed in bacteria which lack the organelle cause misfolding of 20 proteins and thereby impair protein function A use'il reporter protein for a chimeric construct is 3 glucoronidase. expressed in . cu under control of the 355 promoter of caulillower mosaic virus The WO 00/66154 PCT/US00/11478 - 157 glucoronidase alone without the transit sequence is expressed in parallel. The transit peptide construct is found in the plastid. The control glucoronidase is found in the cytoplasm. Antibodies to the chorismate synthase protein are also used to detect the presence of the product of the gene (with the transit sequence) in the plastid and the 5 product of a construct (in which the transit sequence is not present) in the cytoplasm only. Further mutations and deletions are made which identify the minimal transit sequence using the same techniques as described above for the entire peptide. Antisense, ribozyme or intracellular antibodies directed against the transit sequence nucleic acid or translated protein are useful as medicines. The amino acid o&nucleic 10 acid which encodes the transit sequences are the bases for development of diagnostic reagents and vaccines. N. Modifications of Inhibitory Compounds to Improve Oral Absorption Tissue Distribution (especially to brain and eve). Tissue distribution is characterized using radiolabeled inhibitor administered to 15 mice with its disposition to tissues measured. Compounds are modified to improve oral absorption and tissue distribution. O. Methods to Demonstrate Protection Against Conjoint Infections Infections are established and influence ofan inhibitor or combination of inhibitors on outcomes are as outlined below. 20 1nl'ections Intfections with Toxop)/asma goidlii, l'oemnocs'IVti.s curinii, Mtc'obcic'Iern /I IIIuI)CII/0L. . tUoaICIOivin aviim intracellular and ('rip)ro.)poridiu;;m parvumi arc WO 00/66154 PCTUS00/11478 - 158 established alone and together using an immunosuppressed rodent model. Endpoints in these infections are: Survival: Ability of an inhibitor to protect, measured as prolonged survival. Parasitemia: This is measured using isolation of mRNA and RT-PCR with a 5 competitive inhibitor for quantitation. Tissue Parasite Burden: This is determined by quantitating brain and eye cyst numbers. Inflammatory Response: This is noted in histopathologic preparations. Representative combinations of inhibitors are NPMG and sulfadiazine, SHAM and atovaquone, NPMG and pyrimethamine, NPMG and SHAM. 10 P. Testing of Antimicrobial Compounds Presence of inhibitory activity of new antimicrobial compounds is tested in enzymatic assays, in vitro, and in vivo assays as described above and in the literature. Q. Efficacy. Safety. Pharmakokinetics. and Therapeutic/Toxic Index The testing in murine models includes standard Thompson tests. Testing of 15 antimicrobial agents for efficacy and safety in primate models for malaria is performed Dosages are selected based on safety information available from data bases of information concerning herbicides and the literature. Measurements of serum and tissue levels of antimicrobial compounds are performed using assays which detect inhibitor concentrations and concentrations of their metabolites Representative assays 20 are high performance liquid chromatography, and assaying tissues for percentage of radiolabeled compounds administered usinu liquid scintillati7:: and other assays also arc used.

WO 00/66154 PCT/US00/11478 - 159 R. Carcinogenicitv and Teratogenicitv Standard assays to evaluate carcinogenicity include administration of medicines as described above to rodents and observation of offspring for teratogenic effects and carcinogenicity. Observation includes general physical examination, autopsy and 5 histopathologic studies which detect any teratogenic or carcinogenic effects of medicines. S. Constructs to Measure Parasitemia Portions of genes are deleted and the shorter gene is used as an internal standard in RT PCR assays to measure amount of parasites present (Kirisits, Mui, 10 Mack, McLeod, 1996). T. Vaccine Constructs and Proteins and their Administration These are prepared, and sensitivity and specificity are established as is standard in the literature and as described above. Tests and reagents include DNA constructs (Tine el al., 1996) with the appropriate gene or portions of the gene alone or together. 15 with adjuvants. Representative adjuvants include ISCOMS, nonionicsurfactant vesicles, cytokine genes in the constructs and other commonly used adjuvants. Native and recombinant proteins also are used in studies of vaccines. Protection is measured using immunologic in vitro assays, and by assessing survival and reduction of parasitemia and tissue parasite burden and prevention of congenital infection (McLeod 20 cL a/., 19SS) WO 00/66154 PCT/US00/11478 - 160 U. Preparation of Diagnostic Test Reagents and Dianostic Tests: These assays are as described (McLeod and Boyer, 1996). They include ELISAs in which antibodies to the proteins or peptides and recombinant proteins are used and PCR methodology in which primers to amplify DNA which encodes the 5 enzymes or parts of this DNA are used. A test useful in an outpatient setting is based on conjugation of a monoclonal antibody to human red blood cells with antibody to peptides or proteins. The red cells are cross linked if the antibody to the parasite component interacts with the parasite component and agglutinates the red cells in the blood sample. ELISA and PCR can be utilized with samples collected on filter paper 10 as is standard in Newborn Screening Programs and also facilitates outpatient and field use. V. Antisense Antisense oligonucleotides are short synthetic stretches of DNA and RNA designed to block thile action ofthe specific genes described above, for example, 15 chorismate syvnthase of T. gondii or P. falcip)arun, by binding to their RNA transcript. They turn off the genes by binding to stretches of their messenger RNA so that there is breakdown of the mRNA and no translation into protein. Antisense reagents have been found to be active against neoplasms. intlammatory disease of the bowel (Crohn's Disease) and HIV in early trials. Antisense oligonucleotides directed against the 20 nucleic acids which encode the essential parasite metabolic process described herein are cltectivc medicines to trea: these infections A\ntisense olionucleotides also are directed agi:t transit sequencesI1CC in the genes A.\ntisense will not contain c\'tosine WO 00/66154 PCT/US00/11478 - 161 nucleotides followed by guanines as this generates extreme immune responses (Roush, 1997). Antisense oligonucleotides with sequence for thymidine kinase also is used for regulatable gene therapy. W. Ribozvmes and Other Toxic Compounds 5 Ribozymes are RNA enzymes (Mack, McLeod. 1996) and they and toxic compounds such as ricins (Mahal et al, 1997) are conjugated to antisense oligonucleotides (see V, DNA), or intracellular antibodies (see X, for proteins), and these constructs destroy the enzyme. X. Intracellular Antibodies 10 Intracellular antibodies are the Fab portions of monoclonal antibodies directed against the enzymes or portions of them (e.g., anti-transit sequence antibodies) which can be delivered either as proteins or as DNA constructs, as described under vaccines.

WO 00/66154 PCT/US00/11478 - 162 Y. Development of New Antimicrobial Compounds Based on Lead Compounds The herbicide inhibitors comprise lead compounds and are modified as is standard. For example, side chain modifications or substitutions of groups are made to 5 make more active inhibitors. Their mode of action and structure as well as the enzyme and substrate structures are useful in designing related compounds which better abrogate the function of the enzymes. Examples of such substrate or active site targeting are described above. Native or recombinant protein is used in enzymatic assays and in vilrio assays 10 described above are used to test activity of the designed newly synthesized compounds. Subsequently, they will be tested in animals. Z. Trials to Demonstrate Efficacy for Human Disease Trials to demonstrate efficacy for human disease are performed when in vitro and murine and primate studies indicate highly likely efficacy and safety. They are 15 standard Phase I (Safety), Phase II (small efficacy) and Phase III (larger efficacy with outcomes data) trials. For medicines effective against 7 gondii tachyzoites, resolution of intracerebral 7oxoplasmna brain abscess in HIV-infected individuals with no other therapeutic options available due to major intolerance to available medicines is the initial strateev for Phase 11 trials. For medications effective against 7 gonmii 20 bradyzoites, absence of development of toxoplasmic encephalitis in individuals with 1-11 \" int'ection and individuals who are seroposiive for 7 gotoii infection followed ahler a on-lln-illnthi treatmlent for a 2 car period v. ien their CD4 countMs aCre lo WO 00/66154 PCT/US00/11478 - 163 Effective medicines demonstrate efficacy, as 50% of such individuals otherwise develop toxoplasmic encephalitis. When medications efficacious against bradyzoites and recrudescent toxoplasmic encephalitis in patients with AIDS are discovered and found to be safe, similar trials of efficacy and safety for individuals with recurrent 5 toxoplasmic chorioretinitis are performed.

WO 00/66154 PCT/US00/11478 - 164 DEFINITIONS 3-deoxy-d-arabino-heptuloonate 7 phosphate synthase: An enzyme which functions in chorismate synthesis. 3-enolpyruvyshikimnate phosphate synthase (3-phosphoshikinhate-1 5 carboxyvinyltransferase): An enzyme which functions in chorismate synthesis. 3-NPA: An inhibitor of isocitrate lyase in the glyoxylate pathway and also of succinate dehydrogenase. 3-oxtaprenyl-4-hydroxybenzoate carboxylyase: An enzyme which functions in ubiquinone synthesis. 10 4-hydr-oxyvbenzoate octaprenyltransferase: An enzyme which functions in ubiquinone synthesis. 8-OH-quinoline: An inhibitor of the alternative oxidase. Abscissic Acid Metabolism in Plants: A 15-carbon sequiterpenoid synthesized partly in plastids by the mevalonic acid pathway. Abscissic acid protects plants against 15 stress and is.a marker of the plant's maturation and activation of transcription, and causes dormancy. Inhibits protein synthesis and leads to specific activation and deactivation of genes. Acetohydroxy acid synthase: Enzyme which catalyzes production ofacetohydroxy acids (the branched chain amino acids valine. leucine and isoleucine in plants). 20 Alternative oxidase: An enzyme important in the alternative pathway of respiration. There are examples of alternative oxidases in plants and trypanosomes, (Pollakis c aol.. 1995 Rhoads & Mlclntosh. 192 ClarksonI ectc.. 198S9) WO 00/66154 PCT/US00/11478 - 165 Alternative respiration or energy generation: A different pathway for energy generation utilizing the alternative oxidase and election flow in the electron transport chain which is not dependent on conventional cytochromes or heme. Altered gene includes knockouts. 5 Amide: The R portion of the amino group has an amino group connected to a carbonyl carbon. Glutamine and asparagine are amides. Important for nitrogen transport and storage. Amnylopectin: A branched starch of plants. Also found in Tgondii bradyzoites. Amyloplast: Storage granule for starch in plants. Derived from chloroplasts. 10 Aminylose: An unbranched starch of plants. Anabolism: Formation of large molecules such as starch, cellulose, proteins, fats and nucleic acids from small molecules. Requires input of energy. Anthranilate phosporibolsyltransferase: An enzyme which functions in tryptophan synthesis. 15 .Anthranilate synthase component I: An enzyme which functions in tryptophan synthesis. Anthliranilate synthase component II: An enzyme which functions in tryptophan synthesis. Antimicrobial agent: A chemical, for e':ample a protein or antisense nucleic acid 20 which effectively inhibits or kills a pathogenic microbe There are examples (Schwab e i a/., 1994. Strath c/i ad., 1993; Beckers a /.. 1995. Blais ei a/.. 1993; Fichera et al..

WO 00/66154 PCT/US00/11478 -166 1995; Pfefferkorn & Borotz, 1994; Pfefferkorn el al., 1992; Pukivittaykamee et al., 1994). Apicomplex: The common feature of Apicomplexan parasites including a conoid and rhoptry organelles and micronemes at the apical end of the parasite. 5 Apicomplexan parasite: A microorganism that belongs to the Apicomplexan group of parasites. These parasites share a number of morphologic features, including a conoid and rhoptry which are organelles in the cytoplasm at the apical end of the organism and plastids which are multilamellar structures. Representative examples of Apicomplexan parasites include Toxoplasma gondli, Plasmodium, Cryptosporidia and Eimeria. 10 Aromatic acid aminotransferase (aromatic transaminase): An enzyme which functions in tyrosine synthesis. Aspartate, glutamate and glutamnine synthesis: Involve glutamine synthase and glutamate synthetase and are plastid associated in plants. Glutamine synthase in plants is inhibited by the herbicide glufosinate (2 amino-4-[hydroxymethylphosphinyl) 15 butanoic acid. Glutamine synthase also is present in animals. ATP-phosphofructokinase: (ATP-PFK) May exert control over glycolytic pathway because a step when hexoses phosphate cannot also be used to form sucrose or starch. Nearly all animals lack PPi-PFK with plant-like substrate specificity (i.e. PPi, not ATP). 20 Autxins: Growth regulators in plants, which are tryptophan derivatives Herbicides modeled on auxins are stnlictural mimics of these compounds rather than inhibitors of auxin l'unction WO 00/66154 PCT/US00/11478 - 167 Biochemical pathways: Biochemical pathways include metabolic pathways. Any chemical reaction in life. Herein "biochemical pathways" and "metabolic pathways" are used interchangeably. Bradyzoite: The slowly replicating life cycle stage of the Apicomplexan parasite 5 Toxoplasma gondii. This stage is responsible for latent and recrudescent infection due to this parasite. The morphologic features which characterize this parasite stage are electron dense rhoptries and amylopectin granules. Bradyzoites contain a plastid organelle as do other life cycle stages of this parasite. This parasite stage also has specific antigens which other life cycle stages do not have, including bradyzoite surface 10 antigen 4 and bradyzoite antigen 5 (lactate dehydrogenase), which is an intracellular and cyst matrix antigen. Bradyzoites exist together in a structure called a cyst which has a cyst wall and matrix. Cysts contain a few to thousands of bradyzoites. The cyst containing bradyzoites is a major means of transmission of the organism Toxoplasma gondii when it is ingested in meat which is not cooked to well done. It is also a form of 15 the organism responsible for recrudescent eye and brain disease in infants and children who are congenitally infected with the parasite and also in patients whose immune system is not normal. Branched chain amino acid synthesis (valine, leucine and isoleucine) involving acetohvdroxy acid synthase, is the first of the series of reactions. is another metabolic 20 pathway present in plants but not in animals. Branched chain amino acids: Amino acids (valine. leucine a:d isoleucine), the synthesis o1f ,.hich can be inhibited by sul'onylurca and imidaznn!one herbicides WO 00/66154 PCT/US00/11478 - 168 There are examples in plants (Kuriki ei al., 1996: Morell el al.. 1997; Kortostee et al., 1996; Grula et al, 1995; Khoshnoodi et al., 1996). Branching or Q enzyme: Forms branches in amylopectins between C6 of the main chain and Cl I of the branch chain. 5 Catabolism: Degradation or breakdown of large molecules to small molecules, often releasing energy. Calmodulin: is a calcium binding protein (Robson el al., 1993) Catechol 1,2-deoxygenase (phenol hydroxylase): An enzyme which functions in phenylalanine synthesis. 10 Chloroplast: A DNA-containing multilamellar organelle of plants and algae associated with metabolic pathways important for photosynthesis and other energy production. Chloroplasts utilize proteins encoded in their own DNA and also proteins encoded by nuclear DNA. Chorismate: The product of the action of the enzyme EPSP svnthase on shikimate. I5 Chorismate vlyase: An enzyme responsible for the conversion of chorismate to 3,4-dihydroxybenzoate. Chorismate mutase (7-phoslplo-2-delhydro-3-deoxy-arabinlo-lhel)ptulate-ildolase): An enzyme which functions in chorismate synthesis. Chorismate svnthase: An enzyme responsible for the conversion of 3-phospho 5 20 enolpyruvvl shikimate to chorismate. Chorismate: The product of the action of the enzyme EPSP snthase on shikimate.

WO 00/66154 PCT/US00/11478 - 169 Competitive inhibitors: Structures sufficiently similar to the substrate that they compete for the active site of the enzyme. Addition of more natural substrate overcomes effect of the inhibitor. Components: includes nucleic acids, proteins, peptides, enzymes, peptide targeting 5 sequences, transit peptides, carbohydrates, starch, lipids, hormones, for example those listed in Table I and other constituents of metabolic pathways or products derived from these components. Conventional energy generation: Usual pathways of generation of energy in mitochondria utilizing cytochromes for the transfer of electrons. 10 Conversion of Fats to Sugars in Plants: Occurs by oxidation and the glyoxylate cycle. Cryptosporidiosis: The disease due to the Apicomplexan parasite Ciypiosporidium parount. It causes self-limited diarrhea or no symptoms in immunologically normal individuals. In individuals who have immunocompromising illnesses, such as the 15 acquired immune deficiency syndrome, Cryptosporidiosis causes life-threatening, persistent, copious, watery diarrhea. Cryptosporidiumn parmtin: C)ptosp)oridium parvmiun is an Apicomplexan parasite which causes cryptosporidiosis. Cyanide-insensitive, non-heine "alternative" oxidase is a metabolic activity that is 20 found in most cukaryotic plants and algae and is absent from multicellular animals The alternative oxidase is a single polypeptide enzyme that lacks hicne and can serve as the terminal e,ecunn lcceptor to support respiratory gu owth of h". cll in the absence of WO 00/66154 PCT/US00/11478 - 170 heme. The coupling efficiency of this oxidase is lower than that of the cyanide-sensitive cytochrome oxidase. That is, not as many protons are pumped across the mitochondrial inner membrane in parallel with electron transfer through the alternative oxidase as they are through the cytochrome oxidase. The alternative oxidase appears 5 to be used by plants and algae only under certain conditions. The alternative oxidase also is used during different life-cycle stages or under different environmental conditions. Thus, inhibitors of the alternative oxidase may act cooperatively or synergistically with GSAT inhibitors. Cyclohcxadienyl dehydratase: An enzyme which functions in phenylalanine 10 synthesis. Cyclohliexadienyl dehydrogenase: An enzyme which functions in tyrosine synthesis. Cytochrome oxidase: An enzyme utilized in the conventional pathway of energy generation. Dehydroquinate dehydratase: An enzyme which functions in chorismate synthesis 15 Deoxyribonutcleases: Enzymes which are hydrolases which hydrolyze DNA (phosphate esters) Eimneria boris: Causes bovine eimeriosis. Eimeria nIII(LviInaI anl Eincria tenella: Cause eimeriosis in chickens. Eimcria: A group of Apicomplexan parasites which cause gastrointestinal disease in 20 agriculturally important animals including poultry and cattle These economically important parasites include E"Mri IUi Wlella. I". mU\niat and . hoviis WO 00/66154 PCT/US00/11478 - 171 Endosymbiont: An organism which is taken up by another organism and then lives within it. Enzyme: A protein which catalyzes (makes more rapid) the conversion of a substrate into a product. Enzymes are catalysts which speed reaction rates generally by factors 5 between 108 and 1020. They may require ion or protein cofactors. Control is by products and environmental changes. There are more than 5000 enzymes in living systems. Enzymes are named with common or trivial names, and the suffix-ase which characterizes the substrate acted upon (e.g., cytochrome oxidase removes an electron from a cytochrome). Sequential series of steps in a metabolic.pathway. Enzymes that 10 govern the steps in a metabolic pathway are sometimes arranged so that a kind of assembly-line production process occurs. EPSP synthase: An enzyme important in the conversion of shikimate to chorismate. EST: Expressed sequence tag; a short. single pass cDNA sequence generated from randomly selected library clones. 15 Eukaryote: Microorganism or phylogenetically higher organism, the cells of which have a nucleus with a limiting membrane. Fatty Acid Synthesis in Plants: Occurs in chloroplasts of leaves and proplastids of seeds and roots. Mainly palmitic acid and oleic acid. AcetylCo A carboxylases differ in plants and animals. Linoleic acid synthase and linoleneic acid synthase are lipid 20 synthases present in plants and not animals Glvcolysis -- pyruvate ->acetvl CoA WO 00/66154 PCT/US00/11478 - 172 Example: 8 acetyl CoA +7 ATP"+ 14 NADPH + I+H'- palmityl CoA + 7 CoA + 7ADP 2 + 7H 2 PO4 + 14 NADP* + 7H 2 0. Fragment: Refers to a sequence of nucleic acids or aminoacids, where a fragment is 5 sufficient to function as a component of or product derived from an Apicomplexan as defined herein. Gabaculine: An inhibitor of the enzyme GSAT in the heme synthesis pathway. Gene: Nucleotide sequence which encodes an amino acid sequence or another nucleotide sequence. 10 Giberellin Metabolism in Plants: Plant hormones which promote plant growth, overcome dormancy, stimulate GI to S transition and shorten S phase of cell cycle, increase hydrolysis of starch and sucrose into glucose and fructose. They are derivatives of ent-gibberellane skeleton synthesized from a 2acetyl CoA to mevalonic acid to isopenternyl pyrophosphate to 4 isopentenyl pyrophosphate to geranylgeranyl 15 pyrophosphate to copalylpyrophosphate to kaurene to kaurenol to keaurenal to kaurenoic acid to GA 12 aldehyde to other giberellins. These functions are not clearly established but it is hypothesized that hydrolysis of starch to sugar occurs by inducing formation of amylase enzymes. Isoprenoid compounds, diterpenes synthesized from acetate units of acetyl coenzyme A by mevalonic acid pathway stimulate growth. 20 Inhibitors of giberellin synthesis include phosphon D. Amo 1618 (blocks conversion of geranyl pyrophosl)hate to CO palylpyrophosphate). phosplhon D, which also inhibits conversion oI'i0 oxidation) formation oi Kaurcnc. CCC or cycocel, ancvmidol, and WO 00/66154 PCT/US00/1 1478 - 173 pactobutrazol (blocks oxidation of karene and kaurenoic acid). Young leaves are major sites for giberellin synthesis. These plant hormones which induce hydrolysis of polysaccharide into hexoses are used in glycolysis. When hexoses are abundant, glycolysis is more rapid. 5 Glutamyl-tRNA reductase: An enzyme which functions in heme synthesis. Glutamyl-tRNA synthetase: An enzyme which functions in heme synthesis. Glycolysis in Plants: Several reactions of glycolysis also occur in plastids. Glycolysis = lysis of sugar; degradation of hexosis to pyruvic acid in plants. In animals, degradation of glycogen (animal starch) to pyruvate. Plants form no glycogen. 10 Glyoxylate pathway: The pathway important for lipid degradation which takes acetyl CoA and converts it to CoA-SH through the conversion of isocitrate to C4 acids including succinate. This pathway utilizes isocitrate lyase and also converts glyoxylate to malate, a reaction catalyzed by the enzyme malate synthase. The glyoxysome or Glyoxylate pathway which is cytoplasmic in certain algae involves isocitrate lyase and 15 malate synthlase to metabolize lipids and provide C4 acids. A metabolic distinction between autotrophic eukaryotes and heterotrophs is the presence of a glyoxylate cycle. This cycle employs two enzymes, isocitrate lyase and malate synthase, to bypass the two decarboxvlation steps of the TCA cycle and enables the utilization of carbon stored in fatty acids for growth. In plants, the enzymnies of the glyoxvlate cycle are 20 compartmentalized within a unique single-membrane-bound organelle, the glyoxysome. In certain aluae. the cycle is entirely cytoplasmic In plants, these enzymes are most WO 00/66154 PCTIUS00/11478 - 174 abundant during germination and senescence. In animals, the glyoxylate cycle enzymes have been described as being present only during starvation. Glyoxysome: An organelle which in some instances contains enzymes important in the glyoxylate cycle. 5 GSAT: Glutamate-I semialdehyde aminotransferase is the enzyme important in heme synthesis for the conversion of glutamate semialdehyde to ALA (8-aminolevulinic acid). Heme synthesis pathway: A metabolic pathway important for generation of heme, porphyrins and other iron sulfated proteins used in mitochondria in the conventional pathway of energy generation. This pathway occurs in plant chloroplasts and uses the 10 nuclear encoded enzyme GSAT. A metabolic distinction between plants and animals occurs in the heme biosynthesis pathway. Non-photosynthetic eukaryotes, including animals, yeast, fungi and protists, produce 5-aminolevulinic acid (ALA), the common precursor of heme biosynthesis, by condensation of glycine and succinate. In contrast, photosynthetic organisms, including plants, algae and cyanobacteria, E. co/i and some 15 - other bacteria synthesize ALA from glutamate (a 5-carbon pathway). Euglena utilize both condensation of glycine and succinate and the 5 carbon pathway to produce 8 aminolevulinic acid. T. gondii also has the ALA synthase which results in formation of hemne by condensation of glycine and succinate, as does P. fIalcipanun (Surolia and Padmanaban. 1992) Expression of this enzyme is developmentally regulated For 20 example, in plants. GSAT is most abundant in thile leaves There are examples in plants (Matters & Beale. 1995. Elich c't al.. 19S8) I lerhicdle: \ compounLd which kills plants or algae WO 00/66154 PCT/US00/11478 - 175 Hiydrolases: Enzymes which break chemical bonds (e.g., amides, esters, glycosides) by adding the elements of water. Imntidazolinones: Inhibitor of acetohydroxy acid synthase (an enzyme involved in the synthesis of branched chain amino acids, a pathway not in or rarely present in animals, 5 Indole-3-glycerol phosphate synthase (anthranilateisomerase), (indoleglycerol phosphate synthase): An enzyme which functions in tryptophan synthesis. Inhibitor: A compound which abrogates the effect of another compound. A compound which inhibits the replication or survival of a microorganism or the function of an enzyme or key component of a metabolic pathway or otherwise 10 abrogates the function of another key molecule in a microorganism or other organisms or plant. Isocitrate vlyase: An enzyme which functions in glyoxylate cycle. Isomerases: Enzymes which rearrange atoms of a molecule to form a structural isomer. 15 Isoprenoid Mletabolism in Plants: Terpenes are isoprenoids that lack oxygen and are pure hydrocarbons; 5 carbon units with some of the general properties of lipids. Giberellins and abscidic acid are others of this vast complex ofcompounds not found in animals. Isoprene units (head) are CI 2 - CH3C = CH - Cl-H 2 (tail) and are synthesized entirely 20 from acetate of acetyl CoA and restricted to plants Synthesized by mevalonic acid pathxvay because mevalonate is an inm:ortant intermediate.

WO 00/66154 PCT/US00/11478 - 176 Kinases: A subclass of transferases which transfer phosphate groups, especially from ATP. Latency: The dormant form of the parasitic infection. One example is with Toxoplasma gondii in which the infection is not active and the parasite is primarily 5 within cysts in the bradyzoite phase of the life cycle. Another example is the hypnozoite phase of Plasmodiunm falciparum. Ligases or Synthetases: Enzymes which join two molecules coupled with hydrolysis of ATP or other nucleoside triphosphate. Lipases: Enzymes which are hydrolases which hydrolyze fats (esters) 10 Lipid and terpene synthesis associated with plant plastids. Also see fatty acid synthesis and terpenes. Lysases: Enzymes which form double bonds by elimination of a chemical group. Malaria: Disease due to pathogenic Plasmodia. Examples are Plasmodium falciparum, Plasmodium virax, Plasnoditun ovale, Plasmodium malaria, in humans 15 and Plasmodium kn7owlesii in monkeys. Malate synthase: An enzyme which functions in glyoxylate cycle. Metabolic pathways: Both anabolism and catabolism consist of metabolic pathways in which an initial Compound A is converted to another B, then B is converted to C, C to D and so on until a final product is formed. In respiration, glucose is the initial 20 compound, and CO, and 1-:O are the final products There are approximately 50 distinct reactions in respiration but other metabolic pathways have fewer reactions WO 00/66154 PCT/US00/11478 - 177 Herein the phrases "metabolic pathways" and "biochemical pathways" are used interchangeably. Metabolism: Chemical reactions that make life possible. Thousands of such reactions occur constantly in each cell. 5 Microbes: Organisms which are visible only with use of a microscope. Some cause disease (are pathogenic). Microbicidal: An agent (e.g., an antibiotic or antimicrobial compound) which-kills microbes. Mitochondria: An organelle responsible for the generation of energy. 10 Multilamellar: An adjective which refers to the multiple membranes within an organelle. Noncomnipetitive inhibitors: Combine with enzymes at sites other than active site. "Not involve": Are not a starting point, a component, or a product of the metabolic pathways described in relation to this invention. 15 NPMG: An inhibitor ofEPSP svnthase in the shikimate pathway. Nucleic Acid: Deoxyribonucleic acid and ribonucleic acid molecules are constructed of a sugar phosphate backbone and nitrogen bases; important in the encoding, transcription and synthesis of proteins. Oocyst: A life cycle stage of a parasite, e.g.. Tox.oplasma gondi that contains 20 sporozoites 7 gondii sporozoites and oocvsts form only in the cat intestine. This form of the parasite is able to persist in nature in warm. moist soil for up to a year and is highly infectious Sporulation occurs several days after excretion ol'ooc\'sts by WO 00/66154 PCT/US00/11478 - 178 members of the cat family (e.g., domestic cats or wild cats such as lions or tigers). Sporulation must occur before the oocyst becomes infectious. Organelle: A structure within a cell. Examples are plastids, mitochondria, rhoptries, dense granules and micronemes. 5 Oxidoreductases (oxidases, reductases, dehydrogenases): Remove and add electrons or electrons and hydrogen. Oxidases transfer electrons or hydrogen to 02 only. Paraminobenzoic acid (PABA): A product of the shikimate pathway in plants. Parasite: An organism which lives in or on a host for a period of time during at least 10 one life-cycle stage. Phagemid: Plasmid packaged within a filamentous phage particle. Phosphoribosyl anthranilate isomnerase: An enzyme which functions in tryptophan synthesis. Plant-like: Present in algae and higher plants, but not or only rarely, or in unusual 15 circumstances in animals. Plasmodiumnfalcipuarumn: One species of Plasmodium which causes substantial human disease. Plasmnodiunm knoiilesii: A species of Plasmodium which causes malaria in monkeys. Plastid: A mnultilamellar organelle of plants. algae and Apicomplexan parasites which 20 contains its own DNA separate from nuclear DNA. Plastids have been described in studies of Aicomplexan parasites which used electron micrographs (Siddall, 1992, WO 00/66154 PCT/US00/11478 - 179 Williamson et al., 1994; Wilson et al., 1991; Wilson et al., 1994; Wilson et al., 1996; Hackstein et al., 1995; McFadden et al., 1996). Polymerases: Enzymes which link subunits (monomers) into a polymer such as RNA or DNA. 5 PPi phosphofructokinase Type I: An enzyme present in plants that functions in glycolysis and in a number of organisms regulates glycolysis. In plants and protozoans PPi, not ATP (as in animals) is utilized to synthesize Fru-l-6P 2 from Fru 6P. Activity is not stimulated in protozoa by Fru-2-6-P 2 (Peng & Mansour, 1992; Denton et al., 1996a,b). 10 Prephenate dehydratase (phenol 2-monoxygenase): An enzyme which functions in phenylalanine synthesis. Prephenate dehydrogenase: An enzyme which functions in tyrosine synthesis. Product: The end result of the action of an enzyme on a substrate. SProsthetic group: Smaller organic nonprotein portion of an enzyme essential for 15 catalytic activity. Flavin is an example. Proteinases: Enzymes which are hydrolases which hydrolyze proteins (peptide bonds). PS 11: Important alternative means for producing energy within chloroplasts and apparently also described as being present in Apicomniplexans. Pvrimethlamine: An inhibitor of the conversion offolate to folinic acid and thus an 20 inhibitor of nucleic acids production effective against 7Toxop/asma goudii. Recrudescence: Reactivation of the parasite Toxo//asma goidi from its latent phase WO 00/66154 PCTJUS00/11478 - 180-. Respiration: Major catabolic process that releases energy in all cells. It involves breakdown of sugars to CO 2 and H 2 0. Ribonucleases: Enzymes which are hydrolases which hydrolyze RNA (phosphate esters). 5 Salicylic Acid Metabolismni in Plants: Salicylic acid is a plant hormone which promotes activity of cyanide resistant respiration. SHAM: An inhibitor of the alternative oxidase. Shikimate dehydrogenase: An enzyme which functions in chorismate synthesis. Shikimniate kinase: (shikimate 3-phosphotransferase) An enzyme which functions in 10 chorismate synthesis. Shikimniate pathway A pathway that involves the conversion of shikimate to chorismate and subsequently the production of folate, aromatic amino acids, and ubiquinone. This pathway contains enzymes which lead to production of folic acid, ubiquinone, and aromatic amino acids. Folate, ubiquinone, and aromatic amino acids 15 . are products derived from this pathway in plants. There is sequential use of products of these pathways as reactants in subsequent enzymatically catalyzed reactions. For example, ubiquinone is an essential coenzyme for both conventional and alternative respiration. There are examples in plants, bacteria and fungi. (Bornemann et al., 1995: Marzabadi et al., 1996; Ozenberger et al.. 1989; Shah et al.. 1997; Gilchrist & Kosuge. 2 1980; W\alsh et al., 1990; \Veische & Leisterner, 1985; Green c a/., 1992: Youngi ei !L., 1971) ShikiIaMeC: The substrate bfor EPSP synthase WO 00/66154 PCT/US00/11478 - 181 Sporozoite: Another phase of the life cycle of Toxoplasma gondii which forms within the oocyst which is produced only within the cat's intestine. A highly infectious form of the parasite. Stage specific: A characteristic of the parasite which is expressed or present only in a 5 single life cycle stage or in some but not all life cycle stages. Starch Degradation in Plants: 3 enzymes: cc amylase (attack 1, 4 bonds of amylopectin (to maltose) and amylase (to dextrin). Many activated by Ca++. Located in chloroplasts. 3 amylase hydrolyzes starch to maltose; starch phosphorylase degrades starch beginning at nonreducing end. (Starch + H2PO4 *- glucose + 10 Phosphate) Only partially degrades amylopectin debranching enzymes hydroxy 1.6 branch linkage in amylopectin. Hexoses cannot move out of chloroplasts or amrnyloplasts thus must be converted to triose phosphate (3-PG aldehyde and dehydroxyacetone P). sucrose + UDP ** fructose + UDP-glucose, *= sucrose synthase 15 Starchli Formation in Plants: Animals store starch as glycogen and plants store starch as amylose and amylopectin. Starch synthesis is dependent on starch synthase and branching Q enzymes. Mutations in genes encoding these enzymes lead to diminished production of starch In addition, amylopectin synthesis predominates in plant mutants without UDP-glucose-starch glycosyl transferase whereas wild type plants with this 20 enzyme make predominantly amylose and a smaller amount of amylopectin In the mutant UDP-Ulucose-starch glycosyl transferase appears to be transcriptionally related .Amino acid moilfs that target proteins to plant plastid organelles have been WO 00/66154 PCT/US00/11478 - 182 identified in UDP-glucose starch glycosyl transferase, as have other motifs that determine transit into plastids and mitochondria and these have been used to target the transported proteins in plants. Reactions include: ADPG + small amylose (in glucose) *,-larger amylose (N+1 glucose units)+ADP,*= starch synthase K+. Branching or Q 5 enzymes form branches in amylopectins between C6 of the main chain and Cl of the branch chain. There are examples in plants (Abel et al., 1996; Van der Leif et al., 1991; Van der Steege et al., 1992). Starch synthase: catalyzes reaction: ADPG + small amylose (n-glucose units) -- 4 larger amylose n+1 glucose units + ADP and is activated by K+. Thus, sugars not 10 starch accumulate in plants deficient in K+. Starch: Major storage carbohydrate of plants, used for energy regeneration. Two types composed of D glucose connected by 1. 4 bonds which cause starch chains to .coil into helices. The two types are amylose and amylopectin. Amylopectin is highly branched with the branches occurring between C-6 of a glucose in thile main chain and 15 C-I of the first glucose in the branch chain (-1.6 bonds). Amyloses are smaller and have fewer branches. Amylopectin becomes purple or blue when stained with iodine potassium-iodine solution. Amylopectin exhibits a purple red color. Control of starch formation is by K+ and a light activated sucrose phosphate synthase enzyme, invertase enzymes and the allosteric effect of fructose 2. 6 phiphosphate adenosine 20 diphosphoulucose (ADPG) donates glucoses to form starch. Starch in amyloplasts is a principal respiratoi-, substrate for storage organs WO 00/66154 PCT/US00/11478 - 183 Substrate reactant: Enzyme substrates have virtually identical functional groups that are capable of reacting. Specificity results from enzyme substrate combinations similar to a lock and key arrangement. Substrate: The protein on which an enzyme acts that leads to the generation of a 5 product. Sucrose Formation Reactions in Plants: UTP+glucose 1 phosphate -UDPGCr+PPi PPi+H 2 0 +2 Pi UDPG+fructose 6 phosphatesucrose-6-phosphate+UDP Sucrose-6-PHOSPHATE+H 2 0-sucrose+Pi 10 UDP+ATP-UTP+ADP :.glucose- -phosphate+fructose 6 phosphate+2 H 2 0+ATP-sucrose 3Pi+ADP Sulfadiazine: An antimicrobial agent effective against Toxoplasma gondli which competes with para-amninobenzoic acid important in folate synthesis. Sulfonylureas: Inhibitors of acetohydroxy acid synthase (an enzyme involved in the 15 synthesis of branched chain amino acids, a pathway not or rarely present in animals), Synergy: The effect of a plurality of inhibitors or antimicrobial agents which is greater than the additive effect would be combining effects of either used alone. Synergy occurs particularly when the action of an enzyme (which is inhibited) on a substrate leads to a product which is then the substrate for another enzyme which also is 20 inhibited; that is, when the enzymes are in series or follow one another in a pathway. This eTffect occurs because the production of the first enzymatic reaction provides less substrate for the second reaction and thus amplifies the cfect of the second inhibitor or WO 00/66154 PCT/US00/11478 -184 antimicrobial agent. In contrast, an additive effect is when the effect of the compounds used together is simply the sum of the effects of each inhibitory compound used alone. This most often occurs when the pathways are in parallel, for example, when the effect on the first enzyme does not modify the effect ofthe second enzyme. 5 Tachyzoite: The rapidly replicating form of the parasite Toxoplasma gondii. Theileria: An Apicomplexan parasite infecting cattle. Tox.oplasma gondii: A 3-5 micron, obligate, intracellular, protozoan parasite Which is an Apicomplexan. Toxoplasmosis: Disease due to Toxoplasma gondii. 10 Transit (translocation) peptide sequence: Amino acid sequence which results in transit into or out of an organelle. These have been described in plants (Volkner & Schatz, 1997; Theg & Scott, 1993). Herein we also call it a "metabolic pathway," although it is part of a component of a metabolic pathway or may function independently of a metabolic pathway. 15 Triazine: An inhibitor of PS I complex. T-yptophan synthase alpha subunit: An enzyme which functions in tryptophan synthesis. Tryptophan svnthase beta subunit: An enzyme which functions in tryptophan synthesis. 20 Type I PPi lphosphofructokinase is another enzyme present in plants and there is different substrate utilization by phosphofructokinases ot'f animals WO 00/66154 PCT/US00/11478 - 185 UDP glucose starch glycosyl transferase: An enzyme involved in production of amylose in plants. The absence of this enzyme leads to starch formation as amylopectin rather than amylose. USPA: Gene which encodes a universal stress protein. This has been described in 5 E. coli (Nystrom & Neidhardt, 1992).

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Claims (7)

1. A pharmaceutical composition having a parasite with a chorismate synthase gene that is knocked out.

2. An immunogenic composition comprising an attenuated parasite 5 of claim 1.

3. The parasite of claim I is T. gotndii.

4 An immunogenic composition comprising a cDNA molecule encoding chorismate synthase, said molecule complementary to an mRnRNA from T. gondii. 10

5. An assay for a candidate inhibitor of T. gondii, said assay comprising: (a) adding a chorismate synthase - green fluorescent reporter protein construct to a parasite of the T. gondii species; (b) contacting the parasite with the candidate inhibitor; 15 (c) comparing the amounts of green fluorescent reporter protein in the parasite in the presence and absence of the candidate inhibitor; and (d) inferring that the candidate inhibitor is an inhibitor of the parasite if there is significantly less reporter protein 20 when the candidate inhibitor is present.

6. A method for detecting a life cycle stage in a sample tested for T. gondii said method comprising: (a) determining an amount of chorismate synthase present in the sample; and 25 (b) comparing the amount to amounts of standards determined from known life cycle stages.

7. The method of claim 6, wherein the sample is derived from a cat. PmrTIilIn _n-4lFT ('I II Z 011