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WO2006020898A2 - Methodes de criblage d'inhibiteurs de m. tuberculosis, dosages utilises, et methodes et compositions de traitement ou de prevention d'une infection par m. tuberculosis - Google Patents

Methodes de criblage d'inhibiteurs de m. tuberculosis, dosages utilises, et methodes et compositions de traitement ou de prevention d'une infection par m. tuberculosis Download PDF

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WO2006020898A2
WO2006020898A2 PCT/US2005/028795 US2005028795W WO2006020898A2 WO 2006020898 A2 WO2006020898 A2 WO 2006020898A2 US 2005028795 W US2005028795 W US 2005028795W WO 2006020898 A2 WO2006020898 A2 WO 2006020898A2
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tuberculosis
mycobacterial cell
rdl
mycobacterial
cfp
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WO2006020898A3 (fr
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Keith M. Derbyshire
Joseph C. Kowalski
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Health Research Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/35—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycobacteriaceae (F)

Definitions

  • Patent law e.g., they allow for the inclusion of additional ingredients or steps that do not detract from the novel or basic characteristics of the invention, i.e., they exclude additional unrecited ingredients or steps that detract from novel or basic characteristics of the invention, and they exclude ingredients or steps of the prior art, such as documents in the art that are cited herein or are incorporated by reference herein, especially as it is a goal of this document to define embodiments that are patentable, e.g., novel, nonobvious, inventive, over the prior art, e.g., over documents cited herein or incorporated by reference herein.
  • the terms "consists of and “consisting of have the meaning ascribed to them in U.S. Patent law; namely, that these terms are closed ended.
  • the present invention relates to the use of the RDl -encoded secretion apparatus from Mycobacterium tuberculosis that has been functionally expressed in the related mycobacterial species, M. smegmatis. This forms the basis for a high-throughput in vivo screen for the discovery of new anti-tuberculosis drugs.
  • Mycobacteria are gram-positive bacilli, nonmotile rod-shaped organisms that do not form spores.
  • the composition of the cell wall includes a very high concentration of lipids complexed to a variety of peptides and polysaccharides.
  • the unusual structure of the cell wall distinguishes mycobacteria from most other bacteria and is detectable by its resistance to acid-alcohol staining.
  • Mycobacterium tuberculosis The leading cause of death by an infectious agent is Mycobacterium tuberculosis. It is estimated that 2 million people die of tuberculosis every year, and that one-third of the world's population is infected with M. tuberculosis (1). The global health problems associated with the disease have been exacerbated by its deadly synergistic association with HIV and the appearance of multidrug-resistant strains. An additional 8 million people develop the disease annually (Plorde, 1994). Most infections are caused by inhalation of droplet nuclei carrying the mycobacterium. A single cough can generate 3000 infected droplet nuclei and even 10 bacilli may be sufficient to cause a pulmonary infection.
  • tuberculosis would make an excellent drug target for at least two reasons: none of the commonly used antituberculosis drugs in use targets secretion, so the RDl apparatus represents a completely novel target. Also, since the secretion apparatus is at least partly extracellular, inhibitors will not have to cross the permeability barrier posed by the mycobacterial cell envelope or be subject to any efflux systems or modifying enzymes in the cytoplasm.
  • the RDl -encoded secretory apparatus is encoded by a genomic region present in the chromosome of M. tuberculosis, but absent in the attenuated vaccine strains (M. bovis BCG and M. microti).
  • BCG was derived betweenl908 and 1921 after 230 serial passages of M. bovis in vitro, which resulted in an attenuated strain of the normally pathogenic M. bovis. After attenuation, but before the invention of freeze-dried seed lots, serial passage continued and the resulting substrains were distributed around the world for use in vaccination.
  • RDl is deleted in all BCG substrains, but is present in all of the virulent M. tuberculosis strains tested. The fact that RDl is delelted in all BCG substrains suggests it might have been the primary attenuating mutation (Behr et al, 1999).
  • the RDl deletion in BCG is 9.5 kb and affects 9 ORFs, which have been subjected to extensive sequence analyses (Gey Van Pittius et al., 2001; Pallen, 2002). Genes in the region appear to encode an ATP-dependent secretory apparatus that secretes two small proteins, Esat-6 and Cfp-10, both of which elicit potent immune responses. Recent genetic studies with M. tuberculosis and BCG have confirmed the conclusions drawn from sequence analyses. Complementation of the RDl deletion in M. bovis BCG with the RDl region from M. tuberculosis resulted in secretion of both Cfp-10 and Esat-6 (Pym et al., 2003). Deletion of the RDl region in M. tuberculosis resulted in the loss of Esat-6 secretion and attenuation of virulence (in a mouse model) (Hsu et al., 2003).
  • M. tuberculosis strains with a mutant cfplO are defective in lysis of alveolar epithelial cells in vitro and Esat-6 alone is sufficient to disrupt an artificial lipid bilayer in patch-clamp experiments (Hsu et al., 2003).
  • RDl deletions in M. tuberculosis are able to infect and grow within THP-I cells (a human macrophage-like cell line). However, they seem unable to lyse the cells and therefore are deficient at spreading to new cells (Guinn et al., 2004).
  • the RDl region of M. tuberculosis encodes a secretory apparatus responsible for exporting Esat-6, an important virulence factor.
  • the RDl apparatus makes an attractive drug target because it is essential to virulence and partly extracellular. Therefore, inhibitors will not have to cross the mycolic acid layer of the cell envelope.
  • RDl regulates transfer and involves a secretion apparatus, along with the finding by others that at least Esat ⁇ and Cfp-10 are secreted by genes in this region (20, 22), suggests two possibilities compatible with each species.
  • the secreted proteins might coat the cell surface and thereby interfere with formation of specific cell-cell contacts necessary for DNA transfer in M. smegmatis, whereas they may protect M. tuberculosis cells from the hostile macrophage environment.
  • This hypothesis is supported by the observation that, when RDl is expressed in M. bovis bacillus Calmette-Guerin, Esat-6 and Cfp-10 are localized in cell wall fractions, and transformants exhibit an altered colony-morphology phenotype (31).
  • the secreted proteins act as sex pheromones, which negatively regulate transfer until appropriate conditions are met.
  • the secreted proteins may act as a quorum-sensing mechanism, sensing the minimum number of cells before triggering lysis and reinfection of macrophages.
  • This idea is consistent with the observation that attenuated M. tuberculosis RDl mutants replicate and accumulate within macrophages but are unable to spread to uninfected cells (28).
  • Important future goals will be to establish which proteins are secreted by the RDl apparatus, whether that protein-secretion profile varies among species, and, if so, how the different spectrum of proteins accommodate the biology of the mycobacteria.
  • tuberculosis certainly has sinister overtones.
  • the present invention provides a screen designed to find inhibitors of the RDl apparatus that is based on the heterologous expression of the RDl apparatus from M. tuberculosis in M. smegmatis.
  • M. smegmatis is a fast- growing avirulent species of mycobacteria and its chromosome has a region of DNA syntenous with the RDl region of M. tuberculosis, hi M. smegmatis, the RDl secretion apparatus secretes a factor that inhibits the conjugative transfer of DNA.
  • RDl' 6 the secretion apparatus from M. tuberculosis
  • RDl* meg M. smegmatis
  • M. tuberculosis is poorly suited to high-throuput screening because of its virulence and slow growth.
  • M. smemgatis is avirulent, fast growing and much easier to manipulate with modern molecular genetic techniques.
  • the present invention takes advantage of these characteristics to develop a high throuhgput screen to find inhibitors of the RDl secretion apparatus of M. tuberculosis.
  • Conjugation plays a major role in the horizontal transfer of genetic material, especially because many of the plasmids mediating transfer have a broad host range, allowing them to transfer into, and establish themselves in, a wide range of organisms. Conjugation is also an important molecular tool that, along with transformation and transduction, is essential for the genetic analysis of bacteria. Conjugation systems are generally plasmid-borne and require the synthesis of conjugative pili, which are responsible for mating-pair formation (8). These proteins also encode the pore through which DNA is exported into the recipient. More recent studies have shown that this conjugal DNA transport apparatus belongs to a larger family of transport apparatus called type IV secretion systems (9-11).
  • the transfer system is chromosomally encoded and requires recipient recombination functions for both chromosome and plasmid transfer.
  • Cw-acting sequences have been identified that confer mobility on nontransferable plasmids, but these are larger and have different properties to canonical oriT sites found in bacterial plasmids.
  • a library of transposon insertion mutants was generated in the donor strain, and individual mutants were screened for their effect on transfer. From this screen, a collection of insertion mutants was isolated that increased conjugation frequencies relative to wild type. Remarkably, the mutations map to a 25-kb region of the M.
  • smegmatis chromosome that is syntenous with the RDl region of Mycobacterium tuberculosis, which is considered to be the primary attenuating deletion in the related vaccine strain Mycobacterium bovis bacillus Calmette-Guerin.
  • the genes of the RDl region encode a secretory apparatus responsible for exporting CfplO and Esat ⁇ , both potent antigens and virulence factors.
  • donor cells can suppress the elevated transfer phenotype of mutant donors, which is consistent with the secretion of a factor that suppresses conjugation.
  • the RDl region of M is syntenous with the RDl region of Mycobacterium tuberculosis, which is considered to be the primary attenuating deletion in the related vaccine strain Mycobacterium bovis bacillus Calmette-Guerin.
  • the genes of the RDl region encode a secretory apparatus responsible for exporting CfplO and Esat ⁇ , both potent antigens and virulence
  • tuberculosis complements the conjugation phenotype of the RDl mutants in M. smegmatis.
  • M. tuberculosis and M. smegmatis RDl regions are functionally equivalent and provide a unique perspective on the role of this critical secretion apparatus.
  • a chromosomally encoded transfer system in Mycobacterium smegmatis has recently been described which operates by a previously undescribed mechanism (6, 7).
  • M. smegmatis chromosome transfer requires extended contact between two viable bacteria (a donor and a recipient), is unidirectional, and is resistant to DNase I; thus, it meets the working definition of conjugal transfer (12).
  • the present invention pertains to a screening assay involving the RDl secretion activity of M. tuberculosis and is based on the ability of M. smegmatis or variants thereof to express and secrete ESAT6 and CFPlO via the RDl secretion apparatus that is syntenousl in M. tuberculosis and M. smegmatis (M. tuberculosis antigens natively secreted by the RDl apparatus).
  • the method involves screening for agents that induce inhibition of expression and/or secretion of one or more of ESAT6 and CFPlO.
  • M. smegmatis variants M. smegmatis that have been altered to have the RDl secretion apparatus of M. tuberculosis and not have the syntenous
  • RDl -like region native to M. smegmatis to be used in high-throughput screening of small molecules for inhibitors of RDl -encoded / secreted proteins (such as ESTAT6 and CFPlO). It is a further aspect of the invention that such small molecules that inhibit one or more of the proteins ESAT6 and CFPlO would be useful in treating or inhibiting tuberculosis - either in vivo or in vitro.
  • Figure 1 is a schematic outline of the micro titer-mating procedure used to screen for insertion mutants affecting DNA transfer in M. smegmatis.
  • Figure 2 shows microtiter-replica plates used to identify transfer-up mutants. The plate shown is the result of a single 96-well mating assay replica-plated onto trypticase soy agar plates.
  • A Hyg-containing trypticase soy agar medium selecting for donors.
  • B HygSm- containing trypticase soy agar medium selecting for transconjugants.
  • the spot boxed at 8E is an example of an up-mutant. Note the confluent growth of 8E transconjugants compared with the speckled growth of other spots.
  • Controls are indicated in rectangles and were as follows. Column 11 contains wild-type donor cells only, and consequently growth is only observed on plate A. hi row H, wells 1-10 contain recipient cells only, which cannot grow on either plate A or B. In column 1, rows A-G contain wild-type donor and recipient and represent normal levels of Hygr transfer on plate B.
  • Figure 3 depicts the alignment of the M. tuberculosis and M. smegmatis RDl regions.
  • ORPs (prf) are colored according to their putative functions, which were derived from the PFAM and TUBERCULIST databases (18, 19), as follows: red orfs encode AAA domains (PFAM accession no.
  • PF0004 orange orfs encode putative transmembrane proteins
  • yellow orfs encode FtsK SpoIIIE domains PFOl 580, note that the FtsK_SpoIIIE and AAA domains are specific examples of the AAA_ domain
  • green orfs encode PE PF00934 proteins
  • cyan orfs encode PPE PF00823 proteins
  • purple orfs encode members of COG0455 35.
  • ATPases involved in chromosome partitioning are shown. The functions of the gray orfs are unclear.
  • An or/corresponding to the transposase of ISl 549 is shown in black.
  • M. smegmatis orfs (Ms) were given names reflective of the closest M.
  • tuberculosis H37Rv homologue.
  • Ms-orf is a predicted gene with no known M. tuberculosis homologue.
  • the white bar indicates the RDl deletion originally defined in bacillus Calmette-Guerin, which also attenuates M. bovis and M. tuberculosis.
  • Black vertical arrows below the maps represent mariner Kmr insertions, which increaseDNAtransfer.
  • a mycobacterial cell comprising a nucleic acid sequence encoding a protein derived from M. tuberculosis is provided, wherein the protein promotes extracellular secretion from the mycobacterial cell.
  • the mycobacterial cell is not M. tuberculosis, hi a preferred embodiment of the present invention, the mycobacterial cell is M. smegmatis.
  • Another embodiment of the present invention provides for integration of the nucleic acid sequence into the mycobacterial genome.
  • the protein derived from M. tuberculosis is essential for virulence of M. tuberculosis.
  • the protein can be RDl, wherein RDl promotes extracellular secretion of proteins, such as Esat-6 and Cfp- 10.
  • the nucleic acid sequence further comprises sequences encoding recombinant Esat-6 and Cfp- 10.
  • the Esat-6 and Cfp- 10 sequences can further comprise marker sequences selected from the group consisting of sequences encoding fluorescent, luminescent, and epitope markers.
  • a second aspect of the present invention provides a method of identifying potential inhibitors of mycobacterial infection, comprising the steps of growing the mycobacterial cell of the invention in bacterial media containing one or more potential inhibitors, collecting the bacterial media, detecting an increase or decrease in extracellular secretion in the bacterial media, and identifying the potential inhibitor.
  • the bacterial media is collected by centrifugation.
  • detecting an increase or decrease in extracellular secretion comprises identifying the presence of Esat-6 or Cfp-10 in the bacterial media. This can be achieved by methods comprising spectroscopy, microscopy, luminescence, immunohistochemistry, immunoblotting, immunoprecipitation, and ELISA.
  • a kit comprising mycobacterial cells and a nucleic acid sequence encoding a protein derived from M. tuberculosis is provided, wherein the protein promotes extracellular secretion from the mycobacterial cell.
  • the nucleic acid sequence is contained on a plasmid.
  • the nucleic acid sequence can be RDl, wherein RDl promotes extracellular secretion of proteins comprising Esat-6 and Cfp-10.
  • Esat-6 and Cfp-10 can be recombinant and can be on the plasmid comprising RDl .
  • the recombinant Esat-6 and Cfp- 10 can comprise marker sequences selected from the group consisting of fluorescent, luminescent, and epitope markers.
  • the kit of the present invention further comprises reagents useful in identifying extracellular secretion from the mycobacterial cell.
  • reagents useful in identifying extracellular secretion from the mycobacterial cell can comprise antibodies to Esat-6 and Cfp-10.
  • the present invention further provides a high-throughput assay for identifying potential inhibitors of mycobacterial infection, comprising M. smegmatis comprising a nucleic acid sequence encoding a protein derived from M. tuberculosis, wherein the protein promotes extracellular secretion from the Mycobacterium smegmatis.
  • compositions for the treatment and/or prevention of M. tuberculosis may comprise an inhibitor of M. tuberculosis identified by any of the methods or assays provided for herein.
  • Such compositions for treatment (i.e., pharmaceuticals) and/or prevention of M. tuberculosis may additionally comprise an immunologically and pharmaceutically acceptable carrier, vehicle or adjuvant in order to ensure optimum performance of such a composition.
  • An effective vaccine or pharmaceutical wherein an inhibitor, including a polypeptide, of the invention is recognized by the individual receiving the treatment, will be able to decrease bacterial load in target organs, prolong survival times and/or diminish weight loss after challenge with a virulent Mycobacterium, compared to non-vaccinated individuals.
  • Suitable carriers are selected from the group consisting of a polymer to which the polypeptide(s) is/are bound by hydrophobic non-covalent interaction, such as a plastic, e.g. polystyrene, or a polymer to which the polypeptide(s) is/are covalently bound, such as a polysaccharide, or a polypeptide, e.g. bovine serum albumin, ovalbumin or keyhole limpet haemocyanin.
  • Suitable vehicles are selected from the group consisting of a diluent and a suspending agent.
  • the adjuvant is advantageously selected from the group consisting of dimethyldioctadecylammonium bromide (DDA), Quil A, poly I:C, aluminium hydroxide, Freund's incomplete adjuvant, IFN- ⁇ , IL-2, IL- 12, monophosphoryl lipid A (MPL), Treholose Dimycolate (TDM), Trehalose Dibehenate and muramyl dipeptide (MDP).
  • DDA dimethyldioctadecylammonium bromide
  • Quil A Quil A
  • poly I:C aluminium hydroxide
  • Freund's incomplete adjuvant IFN- ⁇
  • IL-2 interleukin-2
  • IL- 12 interleukin-12
  • MPL monophosphoryl lipid A
  • TDM Treholose Dimycolate
  • Trehalose Dibehenate and muramyl dipeptide
  • agents such as aluminum hydroxide or phosphate (alum), synthetic polymers of sugars (Carbopol), aggregation of the protein in the vaccine or pharmaceutical by heat treatment, aggregation by reactivating with pepsin treated (Fab) antibodies to albumin, mixture with bacterial cells such as C. parvum or endotoxins or lipopolysaccharide components of gram- negative bacteria, emulsion in physiologically acceptable oil vehicles such as mannide mono- oleate (Aracel A) or emulsion with 20 percent solution of a perfluorocarbon (Fluosol-DA) used as a block substitute may also be employed.
  • Other possibilities involve the use of immune modulating substances such as cytokines or synthetic IFN- ⁇ inducers such as poly r.C in combination with the above-mentioned adjuvants.
  • a relevant antigen such as an antigen of the present invention can be conjugated to an antibody (or antigen binding antibody fragment) against the Fc ⁇ receptors on monocytes/macrophages .
  • the vaccines or pharmaceuticals are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic.
  • the quantity to be administered depends on the subject to be treated, including, e.g., the capacity of the individual's immune system to mount an immune response, and the degree of protection desired.
  • Suitable dosage ranges are of the order of several hundred micrograms active ingredient per vaccination with a preferred range from about 0.1 ⁇ g to 1000 ⁇ g, such as in the range from about 1 ⁇ g to 300 ⁇ g, and especially in the range from about 10 ⁇ g to 50 ⁇ g.
  • Suitable regimens for initial administration and booster shots are also variable but are typified by an initial administration followed by subsequent inoculations or other administrations.
  • the manner of application may be varied widely. Any of the conventional methods for administration of a vaccine or pharmaceutical are applicable. These are believed to include oral application on a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, by injection or the like.
  • the dosage of the vaccine or pharmaceutical will depend on the route of administration and will vary according to the age of the person to be vaccinated or treated and, to a lesser degree, the size of the person to be vaccinated or treated.
  • the vaccines and pharmaceuticals are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations.
  • binders and carriers may include, for example, polyalkalene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, advantageously 1-2%.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and advantageously contain 10-95% of active ingredient, advantageously 25-70%.
  • vaccines can be administered to prevent an infection with virulent mycobacteria and/or to treat established mycobacterial infection.
  • the vaccine is given prophylactically, before definitive clinical signs or symptoms of an infection are present.
  • the vaccine and pharmaceuticals according to the invention may comprise several different polypeptides or other active ingredients, including inhibitors identified through the methods herein, in order to increase the immune response.
  • Another aspect of the present invention are methods for treating an individual for infection by M. tuberculosis comprising administering a composition containing one or more inhibitors of M. tuberculosis identified through the methods and assays described herein.
  • yet a further aspect of the present invention are methods for preventing infection by M. tuberculosis comprising administering a vaccine or immunological composition containing one or more inhibitors of M. tuberculosis identified through the methods and assays described herein.
  • Another aspect of the present invention is the use of inhibitors of M. tuberculosis identified through the methods and assays provided herein in vitro.
  • inhibitors may be used in environments where it would be advantageous to prohibit growth of M. tuberculosis, such as in hospital or clinical settings.
  • the inhibitors identified by the methods and assays of the present invention can be included in sterilizing or cleaning compositions, or in hygienic items such as mouth wash.
  • Example 1 Isolation of Insertion Mutants with an Elevated-Transfer Frequency
  • a library of transposon insertions was generated in the donor strain mc2155.
  • a library of insertion mutations was generated in an Hyg r derivative of the donor strain, mc2155 (MKD 158), in which a Hyg r gene is integrated at the L5 attB site (13).
  • Southern blot and DNA sequencing analyses confirmed the randomness of our library. The location of each insertion was identified by DNA sequence analysis after the isolation of plasmid DNA containing the transposon and flanking chromosomal DNA. These plasmid clones were isolated by taking advantage of the R6K plasmid origin of replication, which is contained within the mariner transposon. The sequence was then compared to the unannotated M. smegtnatis genome sequence. Preliminary sequence data were obtained from The Institute for Genomic Research. ORFs were assigned putative functions based on BLAST searches and analyses using the PFAM and TUBERCULIST databases (17-19).
  • the resuspended cells were spotted in arrays onto trypticase soy agar and incubated at 30°C for 18 h to allow transfer to take place.
  • the cells were then replicated onto media selective for transconjugants (containing HygSm) or donors (containing Hyg) and incubated for 3-4 days at 37°C before scoring for relative transfer efficiencies by comparison with appropriate controls (Fig. 2).
  • Conjugation frequencies were determined in filter matings as previously described
  • the donor fraction consisted of a 10-fold excess of MKD 158 (a wild-type donor, Hyg r ) over a Km r up- mutant donor, hi all cases, the mating mix consisted of an equal mixture of donor and recipient cells.
  • MKDlO (12) wild-type donor, Km 1
  • Km 1 wild-type donor
  • the mutation (Km 1 ) was transduced into the wild-type mc2155 background to separate it from the Hyg r gene, which was used in the original mutant screen (see above).
  • the transduction was carried out by using a generalized transducing mycobacteriophage, Bxzl (S. Lee, J. Kriakov, and W. Jacobs, personal communication). Successful transduction of each mutation was confirmed by Southern analysis comparing original and transduced chromosomal DNAs and by a mating-out assay that showed the inheritance of the elevated transfer phenotype.
  • Bxzl generalized transducing mycobacteriophage
  • Example 2 Insertion Mutations Map to the RDl Region of M. smegmatis
  • the location of each mariner insertion was determined by DNA sequence analysis and compared to the M. smegmatis genome sequence. Significantly, 14 of the 20 insertion mutations were mapped to a ⁇ 30-kb locus, indicating that this region plays an important role in regulating DNA transfer in M. smegmatis ( Figure 3). Even more surprisingly, this locus is syntenous with the RDl region of M. tuberculosis, a region shown to be critical for full virulence of this pathogen (Table 1 and refs. 20-22).
  • RDl is one of several regions of difference identified by genetic comparisons of virulent strains of Mycobacterium bovis and the attenuated vaccine strain, M. bovis bacillus Calmette-Guerin (bacillus Calmette-Guerin) (23).
  • M. bovis bacillus Calmette-Guerin bacillus Calmette-Guerin
  • the RDl locus is deleted in all bacillus Calmette-Guerin strains when sequences are compared with those of virulent M. bovis and M. tuberculosis strains and is thought to be the primary attenuating mutation (23, 24).
  • the RDl deletion in bacillus Calmette-Guerin is 9.5 kb and includes M. tuberculosis genes Rv3871-3979.
  • the deletion of the RDl -like region in M. smegmatis was made using a system described originally in Pelicic et al., 1997, Proceedings of the National Academy of Sciences, vol. 94, pp. 10955-10960.
  • the sequences kofragl (SEQ ID NO: 2) and kofrag2 (SEQ ID NO: 3) were cloned into pPR23 to make the knockout, mc 2 155: ⁇ RDl ⁇ meg .
  • RDl' 6 has been introduced into mc 2 155: ⁇ RDl imeg on the integrating vector, pRDl-2F9, which contains a ⁇ 32kb fragment encompassing RDl' 6 (SEQ ID NO: 1).
  • the recombinant vector pRDl-2F9 was first described in Pym et al., 2003, Nature Medicine, vol.9(5),pp.533-539.
  • the approximate sequence of the deleted RDl-like region is shown in SEQ ID NO
  • Recombinant versions of both Esat-6 and Cfp-10 were constructed with His 6 tags, his- tagged proteins secreted by mc 2 155: ⁇ RDl 5meg /pRDl-2F9 were detected using anti-His 6 antibodies, which are commercially available, sensitive and highly specific. Initially, filtered culture supernatants were subjected to SDS-PAGE and blotting before detection. In addition to monitoring secretion by antibody-binding to epitope tags, detection by recombinant fluorescent proteins was investigated. Recombinant versions of both Esat-6 and Cfp-10 were constructed as fusions with green fluorescent protein and expressed from pMV261, allowing for detection by spectroscopy and elimination of the blotting step required for antibody detection.
  • Example 3 Evidence for Transfer Regulation by a Secreted Factor
  • Example 4 The RDl Regions of M tuberculosis and M. smeematis Are Functionally Equivalent
  • the similar genetic organizations (Fig. 3) and the high degree of homology between the RDl regions of M. tuberculosis and M. smegmatis (Table 1) suggest that the locus performs similar functions in the two species, despite their very different lifestyles and habitats. This inference is supported by the results above, which show that M. smegmatis RDl mutants can be suppressed by a secreted factor.
  • pRDl-2F9 plasmid (pRDl-2F9) (31) encompassing the entire region from M.
  • tuberculosis (Rv3860-Rv3885) to complement the M. smegmatis insertion mutants. Transfer of each insertion mutant was reduced by the presence of the plasmid, indicating that the regions are functionally equivalent (Fig. 3). Although the degree of complementation varied and was only reduced to wild-type levels in a few cases, the overall trend is clear and reproducible. We suspect that this incomplete complementation is a consequence of the fact that these experiments were designed to suppress, rather than rescue, the transfer phenotype, and it may also reflect the natural variations between the two secretion systems.
  • the phenotypes of the M. smegmatis donor mutants are consistent with either, or both, of the following two hypotheses.
  • Secreted proteins may decorate the surfaces of the bacteria and interfere with the formation of mating pairs.
  • the secreted factor may act as an extracellular pheromone, repressing transfer until conditions are suitable for it to occur.
  • Ms3870 and Ms3871 encode putative homologues of FtsK SpoIIIE, which are proteins involved in chromosome partitioning and translocation, and this suggests a direct role in DNA transfer (29).
  • a SpoIIIE homologue is required for DNA transfer of the plasmid pSAM2 from Streptomyces (30).
  • the insertion mutants have an effect opposite to that expected for a knockout, and the FtsK_SpoIIIE homologies include ATPase motifs common to macromolecular motors. It has been hypothesized, therefore, that Rv3870 and Rv3871 are more likely to provide energy for secretion rather than the translocation of DNA (26).

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  • General Health & Medical Sciences (AREA)
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  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
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Abstract

La présente invention concerne des méthodes de criblage d'inhibiteurs de M. tuberculosis, des dosages utilisés dans lesdites méthodes, ainsi que des méthodes et des compositions de traitement ou de prévention d'une infection par M. tuberculosis contenant des inhibiteurs de M. tuberculosis identifiés à l'aide des méthodes et des dosages de la présente invention.
PCT/US2005/028795 2004-08-11 2005-08-11 Methodes de criblage d'inhibiteurs de m. tuberculosis, dosages utilises, et methodes et compositions de traitement ou de prevention d'une infection par m. tuberculosis Ceased WO2006020898A2 (fr)

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WO2006020898A8 WO2006020898A8 (fr) 2006-04-27
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007010413A3 (fr) * 2005-06-22 2007-08-30 Pasteur Institut Molecules esat-6 modifiees et souches de vaccin ameliorees de mycobacterium bovis bcg
CN102565393A (zh) * 2012-01-15 2012-07-11 中国人民解放军第四军医大学 一种检测体液细胞中结核杆菌特异性分泌抗原的免疫细胞化学染色试剂盒

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DAUGELAT ET AL.: 'The RD1 proteins of Mycobacterium tuberculosis: expression in Mycobacterium smegmatis and biochemical characterization' MICROBES AND INFECTION vol. 5, no. 12, October 2003, pages 1082 - 1095, XP003001070 *
LE BORGNE ET AL.: 'pBRINT-Ts: a plasmid family with a temperature-sensitive replicon, designed for chromosomal integration into the lacZ gene of Escherichia coli' GENE vol. 223, no. 1-2, November 1998, pages 213 - 219, XP002942944 *
PYM ET AL.: 'Recombinant BCG exporting ESAT-6 confers enhanced protection against tuberculosis' NATURE MEDICINE vol. 9, no. 5, May 2003, pages 533 - 539, XP003001069 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007010413A3 (fr) * 2005-06-22 2007-08-30 Pasteur Institut Molecules esat-6 modifiees et souches de vaccin ameliorees de mycobacterium bovis bcg
CN102565393A (zh) * 2012-01-15 2012-07-11 中国人民解放军第四军医大学 一种检测体液细胞中结核杆菌特异性分泌抗原的免疫细胞化学染色试剂盒

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WO2006020898A8 (fr) 2006-04-27

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