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WO2000032825A2 - Developpement de nouveaux agents antimicrobiens bases sur des genomes de bacteriophages - Google Patents

Developpement de nouveaux agents antimicrobiens bases sur des genomes de bacteriophages Download PDF

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Publication number
WO2000032825A2
WO2000032825A2 PCT/IB1999/002040 IB9902040W WO0032825A2 WO 2000032825 A2 WO2000032825 A2 WO 2000032825A2 IB 9902040 W IB9902040 W IB 9902040W WO 0032825 A2 WO0032825 A2 WO 0032825A2
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Prior art keywords
bacteriophage
target
orf
phage
sequence
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PCT/IB1999/002040
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WO2000032825A3 (fr
Inventor
Jerry Pelletier
Phillippe Gros
Michael Dubow
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Targanta Therapeutics Inc
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Targanta Therapeutics Inc
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Priority claimed from US09/407,804 external-priority patent/US6982153B1/en
Application filed by Targanta Therapeutics Inc filed Critical Targanta Therapeutics Inc
Priority to JP2000585456A priority Critical patent/JP2002531107A/ja
Priority to CA002353563A priority patent/CA2353563A1/fr
Priority to AU15815/00A priority patent/AU774841B2/en
Priority to EP99958449A priority patent/EP1135535A2/fr
Publication of WO2000032825A2 publication Critical patent/WO2000032825A2/fr
Publication of WO2000032825A3 publication Critical patent/WO2000032825A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10041—Use of virus, viral particle or viral elements as a vector
    • C12N2795/10043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to the field of antibacterial agents and the treatment of infections of animals or other complex organisms by bacteria.
  • the goal is to identify, through sequencing, unique biochemical pathways or intermediates that are unique to the microorganism. Knowledge of this may, in turn, form the rationale for a drug discovery program based on the mechanism of action of the identified enzymes/proteins. Genome Therapeutics Corp., The Institute for Genome Research, Human Genome Sciences Inc., and other companies have such sequencing programs in place. However, one of the most critical steps in this approach is the ascertainment that the identified proteins and biochemical pathways are 1) non- redundant and essential for bacterial survival, and 2) constitute suitable and accessible targets for drug discovery.
  • bacteriophage or phages are viruses that infect and kill bacteria. They are natural enemies of bacteria and, over the course of evolution, have developed proteins (products of DNA sequences) which enable them to infect a host bacteria, replicate their genetic material, usurp host metabolism, and ultimately kill their host.
  • proteins products of DNA sequences
  • the scientific literature well documents the fact that many known bacteria have a large number of such bacteriophages (Ackermann and DuBow, 1987) that can infect and kill them (for example, see the ATCC bacteriophage collection at http://www.atcc.org).
  • This invention utilizes the observation that bacteriophages successfully infect and inhibit or kill host bacteria, targeting a variety of normal host metabolic and physiological traits, some of which are shared by all bacteria, pathogenic and nonpathogenic alike.
  • pathogenic denotes a contribution to or implication in disease or a morbid state of an infected organism.
  • the invention thus involves identifying and elucidating the molecular mechanisms by which phages interfere with host bacterial metabolism, an objective being to provide novel targets for drug design.
  • the basic blueprint for a phage 's bacteria-inhibiting ability is encoded in its genome and can be unlocked using bioinformatics, functional genomics, and proteomics.
  • the invention utilizes sequence information from the genomics of bacteriophage to identify novel antimicrobials that can be further used to actively and/or prophylactically treat bacterial infection.
  • Two important components of the invention thus are: i) the identification of bacteria-inhibiting phage open reading frames ("ORF's) and corresponding products that can be used to develop antibiotics based on amino acid sequence and secondary structural characteristics of the ORF products, and ii) the use of bacteriophages to map out essential bacterial target genes and homologs, which can in turn lead to the development of suitable anti-microbial agents.
  • ORF's bacteria-inhibiting phage open reading frames
  • bacteriophages to map out essential bacterial target genes and homologs
  • the invention thus concerns the identification of bacteriophage ORFs that supply bacteria-inhibiting functions.
  • use of the terms “inhibit”, “inhibition”, “inhibitory”, and “inhibitor” all refer to a function of reducing a biological activity or function.
  • Such reduction in activity or function can, for example, be in connection with a cellular component, e.g., an enzyme, or in connection with a cellular process, e.g., synthesis of a particular protein, or in connection with an overall process of a cell, e.g., cell growth.
  • an inhibitory effect i.e., a bacteria-inhibiting effect
  • bacteriocidal killing of bacterial cells
  • bacteriostatic i.e., stopping or at least slowing bacterial cell growth
  • the latter slows or prevents cell growth such that fewer cells of the strain are produced relative to uninhibited cells over a given period of time. From a molecular standpoint, such inhibition may equate with a reduction in the level of, or elimination of, the transcription and/or translation of a specific bacterial target(s), or reduction or elimination of activity of a particular target biomolecule.
  • a plurality of different phage ORFs for inhibitory activity that may be from one, but is preferably from a plurality of different phage.
  • ORFs from a number of different phage of the same bacterial host provides at least two advantages. One is that the multiple phages will provide identification of a variety of different targets. Second, it is likely that multiple phage will utilize the same cellular target.
  • the terms "bacteriophage” and "phage” are used interchangeably to refer to a virus which can infect a bacterial strain or a number of different bacterial strains.
  • bacteriophage ORF or ""phage ORF” or similar term refers to a nucleotide sequence in or from a bacteriophage.
  • the terms refer an open reading frame which has at least 95% sequence identity, preferably at least 97% sequence identity, more preferably at least 98% sequence identity with an ORF from the particular phage identified herein (e.g., with an ORF as identified herein) or to a nucleic acid sequence which has the specified sequence identify percentage with such an ORF sequence.
  • a first aspect of the invention thus provides a method for identifying a _ bacteriophage nucleic acid coding region encoding a product active on an essential bacterial target by identifying a nucleic acid sequence encoding a gene product which provides a bacteria-inhibiting function when the bacteriophage infects a host bacterium, preferably one that is an animal or plant pathogen, more preferably a bird or mammalian pathogen, and most preferably a human pathogen.
  • the bacteriophage is an uncharacterized bacteriophage.
  • the method excludes, for example, phage ⁇ , ⁇ xl74, ml 3 and other E. cob-specific bacteriophage that have been studied with respect to gene number and/or function. It also excludes, for example, the nucleic acid coding regions described in Tables 12-14, and in preferred embodiments, excludes the phage in which those regions are naturally located.
  • phage for which the description of genomic or protein sequence was first provided herein are uncharacterized.
  • Phage sequences for which host bacteria- inhibiting functions have been identified prior to the filing of the present application (or alternatively prior to the present invention) are specifically excluded from the aspects involving utilization of sequences from uncharacterized bacteriophage, except that aspects may involve a plurality of phage where one or more of those phage are uncharacterized and one or more others have been characterized to some extent.
  • a number of different bacteria-inhibiting phage ORFs are indicated in Tables 11-14. The phage ORFs or sequences identified therein are not within the term
  • Stating that an agent or compound is "active on" a particular cellular target means that the target is an important part of a cellular pathway which includes that target and that the agent acts on that pathway.
  • the agent may act on a component upstream or downstream of the stated target, including on a regulator of that pathway or a component of that pathway.
  • essential in connection with a gene or gene product, is meant that the host cannot survive without, or is significantly growth compromised, in the ⁇ ss ⁇ ce depletion, or alteration of functional product.
  • An “essential gene” is thus one that encodes a product that is beneficial, or preferably necessary, for cellular growth in vitro in a medium appropriate for growth of a strain having a wild-type allele corresponding to the particular gene in question.
  • an essential gene is inactivated or inhibited, that cell will grow significantly more slowly, preferably less than 20%), more preferably less than 10%, most preferably less than 5% of the growth rate of the uninhibited wild-type, or not at all, in the growth medium.
  • the cell will not grow at all or will be non-viable, at least under culture conditions similar to the in vivo conditions normally encountered by the bacterial cell during an infection. For example, absence of the biological activity of certain enzymes involved in bacterial cell wall synthesis can result in the lysis of cells under normal osmotic conditions, even though protoplasts can be maintained under controlled osmotic conditions.
  • essential genes are generally the preferred targets of antimicrobial agents.
  • Essential genes can encode target molecules directly or can encode a product involved in the production, modification, or maintenance of a target molecule.
  • a "target” refers to a biomolecule that can be acted on by an exogenous agent, thereby modulating, preferably inhibiting, growth or viability of a cell. In most cases such a target will be a nucleic acid sequence or molecule, or a polypeptide or protein. However, other types of biomolecules can also be targets, e.g., membrane lipids and cell wall structural components.
  • bacteria refers to a single bacterial strain, and includes a single cell, and a plurality or population of cells of that strain unless clearly indicated to the contrary.
  • strain refers to bacteria or phage having a particular genetic content.
  • the genetic content includes genomic content as well as recombinant vectors.
  • two otherwise identical bacterial cells would represent different strains if each contained a vector, e.g., a plasmid, with different phage ORF inserts.
  • the phage is Staphylococcus aureus phage 77, 3A, 96, or 44 AHJD, Enterococcus sp. phage 182, or Streptococcus pneumoniae phage Dp-1.
  • the phage is selected from. Preferred embodiments involve expressing at least one recombinant phage ORF(s) in a bacterial host followed by inhibition analysis of that host. Inhibition following expression of the phage ORF is indicative that the product of the ORF is active on an essential bacterial target. Such evaluation can be carried out in a variety of different formats, such as on a support matrix such as a solidified medium in a petri dish, or in liquid culture.
  • a plurality of phage ORFs are expressed in at least one bacterium.
  • the plurality of phage ORFs can be from one or a plurality of phage.
  • the plurality of expressed ORFs preferably represents at least 10%>, more preferably at least 20%, 40%, or 60%, still more preferably at least 80% or 90%, and most preferably at least 95% of the ORFs in the phage genome.
  • the plurality of expressed ORFs preferably represents at least 10%, more preferably at least 20%, 40%), or 60%), still more preferably at least 80%> or 90%, and most preferably at least 95%) of the ORFs in the phage genome of each phage.
  • the plurality of phage ORFs can be expressed in a single bacterium, or in a plurality of bacteria where one ORF is expressed in each bacterium, or in a plurality of bacteria where a plurality of ORFs are expressed in at least one or in all of the plurality of bacteria, or combinations of these.
  • a plurality of phage have the same bacterial host species; have different bacterial host species; or both.
  • the plurality of phage includes at least two different phage, preferably at least 3,4,5,6,8,10,15,20, or more different phage. Indeed, more preferably, the plurality of phage will include 50, 75, 100, or more phage.
  • the larger number of phage is useful to provide additional target and target evaluation information useful in developing antibacterial agents, for example, by providing identification of a larger range of bacterial targets, and/or providing further indication of the suitability of a particular target (for example, utilization of a target by a number of different unrelated phage can suggest that the target is particularly stable and accessible and effective) and/or can indicate alternate sites on a target which interact with different inhibitors.
  • Further embodiments involve confirmation of the inhibitor function of the phage ORF, such as by utilizing or incorporating a control(s) designed to confirm the inhibitory nature of the ORF(s) being evaluated.
  • the control can, for example, be provided by expression of an inactive or partially inactive form of the ORF or ORF product, and/or by the absence of expression of the ORF or ORF product in the same or a closely comparable bacterial strain as that used for expression of the test ORF.
  • the reduced level of activity or the absence of active ORF product in the control will thus not provide the inhibition provided by a corresponding inhibitory ORF, or will provide a distinguishably lower level of inhibition.
  • An inactivated or partially inactivated control has a mutation(s), e.g., in the coding region or in flanking regulatory elements, that reduce(s) or eliminate(s) the normal function of the ORF.
  • the inhibition of a bacterium following expression of a phage ORF is determined by comparison with the effects of expression of an inactivated ORF or the response of the bacteria in the absence of expression in the same or similar type bacterium. Such determination of inhibition of the bacterium following expression of the ORF is indicative of a bacteria-inhibiting function.
  • the bacteria can, for example, contain an empty vector or a vector which allows expression of an unrelated sequence which is preferably non-inhibitory. Alternatively, the bacteria may have no vector at all. Combinations of such controls or other controls may also be utilized as recognized by those skilled in the art.
  • expression is inducible.
  • inducible is meant that expression is absent or occurs at a low level until the occurrence of an appropriate environmental stimulus provides otherwise.
  • induction is preferably controlled by an artificial environmental change, such as by contacting a bacterial strain population with an inducing compound (i.e., an inducer).
  • an inducing compound i.e., an inducer
  • induction could also occur, for example, in response to build-up of a compound produced by the bacteria in the bacterial culture, e.g., in the medium.
  • uncontrolled or constitutive expression of inhibitory ORFs can severely compromise bacteria to the point of eradication, such expression is therefore undesirable in many cases because it would prevent effective evaluation of the strain and inhibitor being studied.
  • a controlled or inducible expression is therefore advantageous and is generally provided through the provision of suitable regulatory elements, e.g. , promoter/operator sequences that can be conveniently transcriptionally linked to a coding sequence to be evaluated.
  • the vector will also contain sequences suitable for efficient replication of the vector in the same or different host cells and/or sequences allowing selection of cells containing the vector, i.e., "selectable markers.”
  • preferred vectors include convenient primer sequences flanking the cloning region from which PCR and/or sequencing may be performed.
  • phage ORFs As knowledge of the nucleotide sequence of phage ORFs is useful, e.g., for assisting in the identification of phage proteins active against essential bacterial host targets, preferred embodiments involve the sequencing of at least a portion of the phage genome in combination with the above methods. This can be done either-before or after or independent of expression and inhibition of the ORF in the bacteria, and provides information on the nature and characteristics of the ORF. Such a portion is preferably at least 10%, 20%, 40%, 80%, 90%, or 100% of the phage genome. For embodiments in which a plurality of phage are utilized, preferably each phage is sequenced to an extent as just specified.
  • Such sequencing is preferably accompanied by computer sequence analysis to define and evaluate ORF(s), ORF products, structural motifs or functional properties of ORF products, and/or their genetic control elements.
  • certain embodiments incorporate computer sequence analyses or nucleic acid and/or amino acid sequences.
  • existing data banks can provide phage sequence and product information which can be utilized for analysis and identification of ORFs in the sequence.
  • Computer analysis may further employ known homologous sequences from other species that suggest or indicate conserved underlying biochemical function(s) for the inhibitory or potentially inhibitory ORF sequence(s) being evaluated. This can include the sequences of signature motifs of identified classes of inhibitors.
  • homolog and “homologous” denote nucleotide sequences from different bacteria or phage strains or species or from other types of organisms that have significantly related nucleotide sequences, and consequently significantly related encoded gene products, preferably having related function.
  • homologous gene sequences or coding sequences have at least 70%> sequence identity (as defined by the maximal base match in a computer-generated alignment of two or more nucleic acid sequences) over at least one sequence window of 48 nucleotides, more preferably at least 80 or 85%, still more preferably at least 90%>, and most preferably at least 95%o.
  • the polypeptide products of homologous genes have at least 35% amino acid sequence identity over at least one sequence window of 18 amino acid residues, more preferably at least 40%>, still more preferably at least 50% or 60%>, and most preferably at least 70%>, 80%>, or 90%.
  • the homologous gene product is also a functional homolog, meaning that the homolog will functionally complement one or more biological activities of the product being compared.
  • the percentage is determined using BLAST programs ( with default parameters (Altschul et al., 1997, "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acid Res.
  • Homo logs may also or in addition be characterized by the ability of two complementary nucleic acid strands to hybridize to each other under appropriately stringent conditions.
  • Hybridizations are typically and preferably conducted with probe-length nucleic acid molecules, preferably 20-100 nucleotides in length.
  • probe-length nucleic acid molecules preferably 20-100 nucleotides in length.
  • Homologs and homologous gene sequences may thus be identified using any nucleic acid sequence of interest, including the phage ORFs and bacterial target genes of the present invention.
  • a typical hybridization utilizes, besides the labeled probe of interest, a salt solution such as 6xSSC (NaCl and Sodium Citrate base) to stabilize nucleic acid strand interaction, a mild detergent such as 0.5%> SDS, together with other typical additives such as Denhardt's solution and salmon sperm DNA.
  • a salt solution such as 6xSSC (NaCl and Sodium Citrate base) to stabilize nucleic acid strand interaction
  • a mild detergent such as 0.5%> SDS
  • Other typical additives such as Denhardt's solution and salmon sperm DNA.
  • the solution is added to the immobilized sequence to be probed and incubated at suitable temperatures to preferably permit specific binding while minimizing nonspecific binding.
  • the temperature of the incubations and ensuing washes is critical to the success and clarity of the hybridization.
  • Stringent conditions employ relatively higher temperatures, lower salt concentrations, and/or more detergent than do non-stringent conditions.
  • Hybridization temperatures also depend on the length, complementarity level, and nature (ie, "GC content") of the sequences to be tested. Typical stringent hybridizations and washes are conducted at temperatures of at least 40°C, while lower stringency hybridizations and washes are typically conducted at 37°C down to room temperature ( ⁇ 25°C).
  • GC content ie, "GC content"
  • stringent hybridization conditions hybridization conditions at least as stringent as the following: hybridization in 50%> formamide, 5X SSC, 50 mM NaH 2 PO 4 , pH 6.8, 0.5% SDS, 0.1 mg/mL sonicated salmon sperm DNA, and 5X Denhart's solution at 42°C overnight; washing with 2X SSC, 0.1% SDS at 45°G; and washing with 0.2X SSC, 0.1% SDS at 45°C.
  • an ORF, or motif, or set of motifs in a bacteriophage sequence can be compared to known inhibitor sequences, e.g., homologous sequences encoding homologous inhibitors of bacterial function.
  • the analysis can include comparison with the structure of essential bacterial gene products, as structural similarities can be indicative of similar or replacement biological function.
  • Such analysis can include the identification of a signature, or characteristic motif(s) of an inhibitor or inhibitor class.
  • the identification of structural motifs in an encoded product can be used to infer a biochemical function for the product.
  • a database containing identified structural motifs in a large number of sequences is available for identification of motifs in phage sequences.
  • the database is PROSITE, which is available at www.expasy.ch/cgi ⁇ bin scanprosite.
  • the identification of motifs can, for example, include the identification of signature motifs for a class or classes of inhibitory proteins. Other such databases may also be used.
  • the bacterium or host bacterium is preferably selected from a pathogenic bacterial species, for example, one selected from Table 1.
  • an animal or plant pathogen is used.
  • the bacterium is a bird or mammalian pathogen, still more preferably a human pathogen.
  • one or more bacteriophage are preferably selected from those listed in Table 1. Those exemplary bacteriophge are readily obtained from the indicated sources.
  • phage with non-pathogenic host bacteria it is advantageous to utilize phage with non-pathogenic host bacteria.
  • the genome, structural motif, ORF, homolog, and other analyses described herein can be performed on such phage and bacteria. Such analysis provides useful information and compositions.
  • the results of such analyses can also be utilized in aspects of the present invention to identify homologous ORFs, especially inhibitor ORFs in phage with pathogenic bacterial hosts.
  • identification of a target in a non-pathogenic host can be used to identify homologous sequences and targets in pathogenic bacteria, especially in genetically closely related bacteria.
  • a related aspect of the invention provides methods for identifying a target for antibacterial agents by identifying the bacterial target(s) of at least one uncharacterized or untargeted inhibitor protein or RNA from a bacteriophage. Such identification allows the development of antibacterial agents active on such targets.
  • Preferred embodiments for identifying such targets involve the identification of binding of target and phage ORF products to one another.
  • the phage ORF products may be subportions of a larger ORF product that also binds the host target.
  • the phage protein or RNA is from an uncharacterized bacteriophage in Table 1.
  • This aspect preferably includes the identification of a plurality of such targets in one or a plurality of different bacteria, preferably in one or a plurality of bacteria listed in Table 1.
  • the ORF is Staphylococcus aureus phage 77 ORF 17, 19, 43, 102, 104, or 182 as identified in U.S. application 09/407,804, S. aureus phage 44AHJD ORF 1, 9, or 12, Streptococcus pneumoniae phage Dp-1 ORF 001, 002, 004, 008, 010, 013, 016, 021, 029, 030, 038, or 041, or Enterococcus sp.
  • the method involves the use of a plurality of different phage, and thus a plurality of different phage inhibitors and/or inhibitor ORFs.
  • phage ORF products which are known to be inhibitors of host bacteria, but where the target has not been identified.
  • inhibitors can likewise be utilized as "untargeted" inhibitor phage ORFs and ORF products, e.g., proteins or RNAs.
  • the term "uncharacterized" means that a bacteria-inhibiting function for the protein has not previously been identified.
  • the sequence of the protein or the corresponding coding region or ORF was not described in the art before the filing of the present application for patent (or alternatively prior to the present invention).
  • this term specifically excludes any bacteria-inhibiting phage protein and its associated bacterial target which has been identified as inhibitory before the present invention or alternatively before the filing of the present application, for, example those identified in Tables 12-14 or otherwise identified herein. For example, from E.
  • fragment refers to a portion of a larger molecule or assembly.
  • fragment refers to a molecule which includes at least 5 contiguous amino acids from the reference polypeptide or protein, preferably at least 8, 10, 12, 15, 20, 30, 50 or more contiguous amino acids.
  • fragment refers to a molecule which includes at least 15 contiguous nucleotides from a reference polynucleotide, preferably at least 24, 30, 36, 45, 60, 90, 150, or more contiguous nucleotides.
  • Preferred embodiments involve identification of binding that include methods for distinguishing bound molecules, for example, affinity chromatography, immunoprecipitation, crosslinking, and/or genetic screen methods that permit proteimprotein interactions to be monitored.
  • methods for distinguishing bound molecules for example, affinity chromatography, immunoprecipitation, crosslinking, and/or genetic screen methods that permit proteimprotein interactions to be monitored.
  • Genetic screening for the identification of proteimprotein interactions typically involves the co-introduction of both a chimeric bait nucleic acid sequence (here, the phage ORF to be tested) and a chimeric target nucleic acid sequence that, when co- expressed and having affinity for one another in a host cell, stimulate reporter gene expression to indicate the relationship.
  • a "positive” can thus suggest a potential inhibitory effect in bacteria. This is discussed in further detail in the Detailed Description section below. In this way, new bacterial targets can be identified that are inhibited by specific phage ORF products or derivatives, fragments, mimetics, or other molecules.
  • mutant targets involve the identification and/or utilization of mutant targets by virtue of their host's relatively unresponsive nature in the presence of expression of ORFs previously identified as inhibitory to the non-mutant or wild-type strain.
  • Such mutants have the effect of protecting the host from an inhibition that would otherwise occur and indirectly allow identification of the precise responsible target for follow-up studies and anti-microbial development.
  • rescue from inhibition occurs under conditions in which a bacterial target or mutant target is highly expressed.
  • This is performed, for example, through coupling of the sequence with regulatory element promoters, e.g., as known in the art, which regulate expression at levels higher than wild-type, e.g., at a level sufficiently higher that the inhibitor can be competitively bound to the highly expressed target such that the bacterium is detectably less inhibited.
  • regulatory element promoters e.g., as known in the art, which regulate expression at levels higher than wild-type, e.g., at a level sufficiently higher that the inhibitor can be competitively bound to the highly expressed target such that the bacterium is detectably less inhibited.
  • Identification of the bacterial target can involve identification of a phage- specific site of action. This can involve a newly identified target, or a target where the phage site of action differs from the site of action of a previously known antibacterial agent or inhibitor.
  • phage T7 genes 0.7 and 2.0 target the host RNA polymerase, which is also the cellular target for the antibacterial agent, rifampin.
  • aspects of the present invention can utilize those new, phage- specific sites for identification and use of new agents.
  • the site of action can be identified by techniques well-known to those skilled in the art, for example, by mutational analysis, binding competition analysis, and/or other appropriate techniques.
  • a bacterial host target protein or nucleic acid or mutant target sequence has been identified and/or isolated, it too can be conveniently sequenced, sequence analyzed (e.g., by computer), and the underlying gene(s), and corresponding translated product(s) further characterized.
  • Preferred embodiments include such analysis and identification.
  • a target has not previously been identified as an appropriate target for antibacterial action.
  • Certain embodiments include the identification of at least one inhibitory phage
  • ORF or ORF product e.g., as described for the above aspect, and thus are a combination of the two aspects.
  • the invention provides methods for identifying targets for antibacterial agents by identifying homologs of a bacterial target e.g., S. aureus, Enterococcus faecalis or other Enterococci, and Streptococcus pneumoniae of a bacteriophage inhibitory ORF product.
  • a bacterial target e.g., S. aureus, Enterococcus faecalis or other Enterococci, and Streptococcus pneumoniae of a bacteriophage inhibitory ORF product.
  • homologs may be utilized in the various aspects and embodiments described herein as describded for the host Enterococcus sp. for bacteriophage 182.
  • sequences do not include sequences identified in any of Tables 11-14.
  • Nucleotide sequences of this aspect are at least 15 nucleotides in length, preferably at least 18, 21, 24, or 27 nucleotides in length, more preferably at least 30, 50, or 90 nucleotides in length. In certain embodiments, longer nucleic acids are preferred, for example those of at least 120, 150, 200, 300, 600, 900 or more nucleotides.
  • Such sequences can, for example, be amplification oligonucleotides (e.g., PCR primers), oligonucleotide probes, sequences encoding a portion or all of a phage-encoded protein, or a fragment or all of a phage-encoded protein.
  • the nucleic acid sequence contains a sequence which is within a length range with a lower length as specified above, and an upper length limit which is no more than 50, 60, 70, 80, or 90%> of the length of the corresponding full-length ORF.
  • the upper length limit can also be expressed in terms of the number of base pairs of the ORF (coding region).
  • the nucleic acid sequence is from Staphylococcus aureus phage 77 ORF 17, 19, 43, 102, 104, or 182 as identified in U.S. application 09/407,804, S.
  • the sequences of this aspect includes nucleic acid sequences utilizing such alternate codon usage for one or more codons of a coding sequence. For example, all four nucleic acid sequences GCT, GCC, GCA, and GCG encode the amino acid, alanine.
  • nucleic acid sequence can be modified (e.g., a nucleic acid sequence from a phage as specified above) to form a second nucleic acid sequence encoding the same polypeptide as encoded by the first nucleic acid sequence using routine procedures and without undue experimentation.
  • nucleic acid sequences that encode the specified amino acid sequences are also fully described herein, as if all were written out in full, taking into account the codon usage, especially that preferred in the host bacterium.
  • alternate sequences are described by reference to the natural sequence with replacement of one or more (up to all e.g., up to 3, 5, 10, 15, 20, 30, 40, 50, or more) of the degenerate codons with alternate codons from the alternate codon table (Table 6), or a modified table applicable to a particular organism that has differing codon usage, preferably with selection according to preferred codon usage for the normal host organism or a host organism in which a sequence is intended to be expressed.
  • Those skilled in the art also understand how to alter the alternate codons to be used for expression in organisms where certain codons code differently than shown in the "universal" codon table.
  • sequences contain at least 5 peptide- linked amino acid residues, and preferably at least 6, 7, 10, 15, 20, 30, or 40, amino acids having identical amino acid sequence as the same number of contiguous amino acid residues in a particular phage ORF product. In some cases longer sequences may be preferred, for example, those of at least 50, 60, 70, 80, or 100 amino acids in length.
  • the amino acid sequence contains a sequence which is within a length range with a lower length as specified above, and an upper length limit which is no more than 50, 60, 70, 80, or 90%> of the length of the corresponding full-length ORF product. The upper length limit can also be expressed in terms of the number of amino acid residues of the ORF product.
  • the amino acid sequence or polypeptide has bacteria-inhibiting function when expressed or otherwise present in a bacterial cell which is a host for the bacteriophage from which the sequence was derived.
  • isolated in reference to a nucleic acid is meant that a naturally occurring sequence has been removed from its normal cellular (e.g., chromosomal) environment or is synthesized in a non-natural environment (e.g., artificially synthesized).
  • the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not imply that the sequence is the only nucleotide chain present, but that it is essentially free (about 90-95% pure at least) of non-nucleotide material naturally associated with it, and thus is distinguished from isolated chromosomes.
  • enriched means that the specific DNA or RNA sequence constitutes a significantly higher fraction (2-5 fold) of the total DNA or RNA present in the cells or solution of interest than in normal or diseased cells or in cells from which the sequence was originally taken. This could be caused by a person by preferential reduction in the amount of other DNA or RNA present, or by a preferential increase in the amount of the specific DNA or RNA sequence, or by a combination of the two. However, it should be noted that enriched does not imply that there are no other DNA or RNA sequences present, just that the relative amount of the sequence of interest has been significantly increased.
  • the term "significant" is used to indicate that the level of increase is useful to the person making such an increase and an increase relative to other nucleic acids of about at least 2-fold, more preferably at least 5- to 10-fold or even more.
  • the term also does not imply that there is no DNA or RNA from other sources.
  • the other source DNA may, for example, comprise DNA from a yeast or bacterial genome, or a cloning vector such as pUC19. This term distinguishes from naturally occurring events, such as viral infection, or tumor type growths, in which the level of one mRNA may be naturally increased relative to other species of mRNA. That is, the term is meant to cover only those situations in which a person has intervened to elevate the proportion of the desired nucleic acid.
  • nucleotide sequence be in purified form.
  • purified in reference to nucleic acid does not require absolute purity (such as a homogeneous preparation). Instead, it represents an indication that the sequence is relatively more pure than in the natural environment (compared to the natural level, this level should be at least 2-5 fold greater, e.g., in terms of mg/mL).
  • Individual clones isolated from a cDNA library may be purified to electrophoretic homogeneity. The claimed DNA molecules obtained from these clones could be obtained directly from total DNA or from total RNA.
  • the cDNA clones are not naturally occurring, but rather are preferably obtained via manipulation of a partially purified naturally occurring substance (messenger RNA).
  • a cDNA library from mRNA involves the creation of a synthetic substance (cDNA) and pure individual cDNA clones can be isolated from the synthetic library by clonal selection of the cells carrying the cDNA library.
  • cDNA synthetic substance
  • the process which includes the construction of a cDNA library from mRNA and isolation of distinct cDNA clones yields an approximately 10 6 -fold purification of the native message.
  • purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.
  • nucleic acids may similarly be used to denote the relative purity and abundance of polypeptides (multimers of amino acids joined one to another by -carboxyl: ⁇ -amino group (peptide) bonds). These, too, may be stored in, grown in, screened in, and selected from libraries using biochemical techniques familiar in the art.
  • polypeptides may be natural, synthetic or chimeric and may be extracted using any of a variety of methods, such as antibody immunoprecipitation, other "tagging" - techniques, conventional chromatography and/or electrophoretic methods. Some of the above utilize the corresponding nucleic acid sequence. As indicated above, aspects and embodiments of the invention are not limited to entire genes and proteins.
  • the invention also provides and utilizes fragments and portions thereof, preferably those which are "active" in the inhibitory sense described above.
  • Such peptides or oligopeptides and oligo or polynucleotides have preferred lengths as specified above for nucleic acid and amino acid sequences from phage; corresponding recombinant constructs can be made to express the encoded same.
  • Nucleic acid sequences of the present invention can be isolated using a method similar to those described herein or other methods known to those skilled in the art. In addition, such nucleic acid sequences can be chemically synthesized by well- known methods. Also, by having particular phage ORFs, e.g., the phage ORFs identified herein (e.g., anti-bacterial ORFs of the present invention, portions thereof, or oligonucleotides derived therefrom as described), other antimicrobial sequences from other bacteriophage sources can be identified and isolated using methods described here or other methods, including methods utilizing nucleic acid hybridization and/or computer-based sequence alignment methods.
  • phage ORFs e.g., the phage ORFs identified herein (e.g., anti-bacterial ORFs of the present invention, portions thereof, or oligonucleotides derived therefrom as described)
  • other antimicrobial sequences from other bacteriophage sources can be identified and isolated using methods described here or
  • the invention also provides bacteriophage antimicrobial DNA segments from other phages based on nucleic acids and sequences hybridizing to the presently identified inhibitory ORF under high stringency conditions or sequences that are highly homologous.
  • the bacteriophage segment from a specific phage e.g., an antimicrobial DNA segment
  • homologous coding sequences and products can be used as antimicrobials, to construct active portions or derivatives, to construct peptidomimetics, and to identify bacterial targets.
  • nucleotide and amino acid sequences identified herein are believed to be correct, however, certain sequences may contain a small percentage of errors, e.g., 1- 5%. In the event that any of the sequences have errors, the corrected sequences can be readily provided by one skilled in the art using routine methods.
  • the nucleotide sequences can be confirmed or corrected by obtaining and culturing the relevant phage, and purifying phage genomic nucleic acids.
  • a region or regions of - interest can be amplified, e.g., by PCR from the appropriate genomic template, using primers based on the described sequence. The amplified regions can then be sequenced using any of the available methods (e.g., a dideoxy termination method).
  • a particular sequence or sequences can be identified and isolated as an insert or inserts in a phage genomic library and isolated, amplified, and sequenced by standard methods. Confirmation or correction of a nucleotide sequence for a phage gene provides an amino acid sequence of the encoded product by merely reading off the amino acid sequence according to the normal codon relationships and/or expressed in a standard expression system and the polypeptide product sequenced by standard techniques.
  • the sequences described herein thus provide unique identification of the corresponding genes, coding sequences, and other sequences, allowing those sequences to be used in the various aspects of the present invention.
  • the invention provides recombinant vectors and cells harboring at least one of the phage ORFs or portion thereof, or bacterial target sequences described herein.
  • vectors may be provided in different forms, including, for example, plasmids, cosmids, and virus- based vectors. See, e.g.. Maniatis. T. et al. ( 1989-) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor University Press, Cold Spring, N.Y.; See also, Ausubel, F.M. et al. (eds.) (1994) Current Protocols in Molecular Biology. John Wiley & Sons, Secaucus, NJ.
  • the vectors will be expression vectors, preferably shuttle vectors that permit cloning, replication, and expression within bacteria.
  • An "expression vector" is one having regulatory nucleotide sequences containing transcriptional and translational regulatory information that controls expression of the nucleotide sequence in a host cell.
  • the vector is constructed to allow amplification from vector sequences flanking an insert locus.
  • the expression vectors may additionally or Codley support expression, and/or replication in animal, plant and/or yeast cells due to the presence of suitable regulatory sequences, e.g., promoters, enhancers, 3' stabilizing sequences, primer sequences, etc.
  • the promoters are inducible and specific for the system in which expression is desired, e.g., bacteria, animal, plant, or yeast.
  • the vectors may optionally encode a "tag" sequence or sequences to facilitate protein purification.
  • Convenient restriction enzyme cloning sites and suitable selective marker(s) are also optionally included.
  • Such selective markers can be, for example, antibiotic resistance markers or markers which supply an essential nutritive growth factor to an otherwise deficient mutant host, e.g., tryptophan, histidine, or leucjng " tn the Yeast Two-Hybrid systems described below.
  • the term "recombinant vector” relates to a single- or double-stranded circular nucleic acid molecule that can be transfected into cells and replicated within or independently of a cell genome.
  • a circular double-stranded nucleic acid molecule can be cut and thereby linearized upon treatment with appropriate restriction enzymes.
  • restriction enzymes An assortment of nucleic acid vectors, restriction enzymes, and the knowledge of the nucleotide sequences cut by restriction enzymes are readily available to those skilled in the art.
  • a nucleic acid molecule encoding a desired product can be inserted into a vector by cutting the vector with restriction enzymes and ligating the two pieces together.
  • the vector is an expression vector, e.g., a shuttle expression vector as described above.
  • recombinant cell is meant a cell possessing introduced or engineered nucleic acid sequences, e.g., as described above.
  • the sequence may be in the form of or part of a vector or may be integrated into the host cell genome.
  • the cell is a bacterial cell.
  • the invention also provides methods for identifying and/or screening compounds "active on" at least one bacterial target of a bacteriophage inhibitor protein or RNA.
  • Preferred embodiments involve contacting such a bacterial target or targets (e.g., bacterial target proteins) with a test compound, and determining whether the compound binds to or reduces the level of activity of the bacterial target (e.g., a bacterial target protein). Preferably this is done either in vivo (i.e., in a cell- based assay) or in vitro, e.g., in a cell-free system under approximately physiological conditions.
  • the compounds that can be used may be large or small, synthetic or natural, organic or inorganic, proteinaceous or non-pro teinaceous.
  • the compound is a peptidomimetic, as described herein, a bacteriophage inhibitor protein or fragment or derivative thereof, preferably an "active portion", or a small molecule.
  • the bacterial target is a target of a phage ORF identified herein, e.g., S. aureus phage 44AHJD ORF 1, 9, or 12, Streptococcus pneumoniae p age Dp-1 ORF 001, 002, 004, 008, 010, 013, 016, 021, 029, 030, 038, or 041, or Enterococcus sp. phage 182 ORF 002, 008, or 014.
  • a phage ORF identified herein, e.g., S. aureus phage 44AHJD ORF 1, 9, or 12, Streptococcus pneumoniae p age Dp-1 ORF 001, 002, 004, 008, 010, 013, 016, 021, 029, 030, 038, or 041, or Enterococcus sp.
  • the methods include the identification of bacterial targets or the site of action of an inhibitor on a bacterial target as described above or otherwise described herein.
  • binding is to a fragment or portion of a bacterial target protein, where the fragment includes less than 90%, 80%, 70%, 60%, 50%, 40%, or 30% of an intact bacterial target protein.
  • the at least one bacterial target includes a plurality of different targets of bacteriophage inhibitor proteins, preferably a plurality of different targets.
  • the plurality of targets can be in or from a plurality of different bacteria, but preferably is from a single bacterial species.
  • a “method of screening” refers to a method for evaluating a relevant activity or property of a large plurality of compounds (e.g., a bacteria-inhibiting activity), rather than just one or a few compounds.
  • a method of screening can be used to conveniently test at least 100, more preferably at least 1000, still more preferably at least 10,000, and most preferably at least 100,000 different compounds, or even more.
  • the term "small molecule” refers to compounds having molecular mass of less than 2000 Daltons, preferably less than 1500, still more preferably less than 1000, and most preferably less than 600 Daltons. Preferably but not necessarily, a small molecule is not an oligopeptide.
  • the invention provides a method of screening for potential antibacterial agents by determining whether any of a plurality of compounds, preferably a plurality of small molecules, is active on at least one target of a bacteriophage inhibitor protein or RNA. Preferred embodiments include those described for the above aspect, including embodiments which involve determining whether one or more test compounds bind to or reduce the level of activity of a bacterial target, and embodiments which utilize a plurality of different targets as described above.
  • the identification of bacteria-inhibiting phage ORFs and their encoded products also provides a method for identifying an active portion of such an encoded product. This also provides a method for identifying a potential antibacterial agent by identifying such an active portion of a phage ORF or ORF product.
  • the identification of an active portion involves one or more of mutational analysis, deletion analysis, or analysis of fragments of such products.
  • the method can also include determination of a 3-dimensional structure of an active portion, such as by analysis of crystal diffraction patterns.
  • the method involves constructing or synthesizing a peptidomimetic compound, where the structure of the peptidomimetic compound corresponds to the structure of the active portion.
  • peptidomimetic compound structure has sufficient similarities to the structure of the active portion that the peptidomimetic will interact with the same molecule as the phage protein and preferably will elicit at least one cellular response in common which relates to the inhibition of the cell by the phage protein. ⁇ >l
  • the ORF or ORF product is or is derived or obtained from S. aureus phage 44AHJD ORF 1, 9, or 12, Streptococcus pneumoniae phage Dp-1 ORF 001, 002, 004, 008, 010, 013, 016, 021, 029, 030, 038, or 041, or Enterococcus sp. phage 182 ORF 002, 008, or 014 or product thereof.
  • the methods for identifying or screening for compounds or agents active on a bacterial target of a phage-encoded inhibitor can also involve identification of a phage-specific site of action on the target.
  • the target is uncharacterized; the target is from an uncharacterized bacterium from Table 1 ; the site of action is a phage-specfic site of action.
  • Further embodiments include the identification of inhibitor phage ORFs and bacterial targets as in aspects above.
  • an “active portion” as used herein denotes an epitope, a catalytic or regulatory domain, or a fragment of a bacteriophage inhibitor protein that is responsible for, or a significant factor in, bacterial target inhibition.
  • the active portion preferably may be removed from its contiguous sequences and, in isolation, still effect inhibition.
  • peptidomimetic is meant a compound structurally and functionally related to a reference compound that can be natural, synthetic, or chimeric.
  • a “peptidomimetic,” for example is a compound that mimics the activity- related aspects of the 3-dimensional structure of a peptide or polyeptide in a non- peptide compound, for example mimics the structure of a peptide or active portion of a phage- or bacterial ORF-encoded polypeptide.
  • a related aspect provides a method for inhibiting a bacterial cell by contacting the bacterial cell with a compound active on a bacterial target of a bacteriophage inhibitor protein or RNA, where the target was uncharacterized.
  • the compound is such a protein, or a fragment or derivative thereof; a structural mimetic, e.g., a peptidomimetic, of such a protein or fragment; a small molecule;
  • the contacting is performed in vitro, the contacting is performed in vivo in an infected or at risk organism, e.g., an animal such as a mammal or bird, for example, a human, or other mammal described herein;
  • the bacterium is selected from a genus and or species listed in Table 1 ;
  • the bacteriophage inhibitor protein is uncharacterized;
  • the bacteriophage inhibitor protein is from an uncharacterized phage listed in Table 1 ;
  • the phage inhibitor protein is from one of S.
  • aureus phage 44AHJD ORF 1 9, or 12
  • Streptococcus pneumoniae phage Dp-1 ORF 001, 002, 004, 008, 010, 013, 016 ⁇ 02 ⁇ 029, 030, 038, or 041, ox Enterococcus sp. phage 182 ORF 002, 008, or 014.
  • the term "uncharacterized" means that the target was not recognized as an appropriate target for an antibacterial agent prior to the filing of the present application or alternatively prior to the present invention.
  • Such lack of recognition can include, for example, situations where the target and/or a nucleotide sequence encoding the target were unknown, situations where the target was known, but where it had not been identified as an appropriate target or as an essential cellular component, and situations where the target was known as essential but had not been recognized as an appropriate target due to a belief that the target would be inaccessible or otherwise that contacting the cell with a compound active on the target in vitro would be ineffective in cellular inhibition, or ineffective in treatment of an infection.
  • bacterial targets e.g., for inhibiting bacteria or treating bacterial infections
  • the phage-specific site has different functional characteristics from the previously utilized site.
  • the term "phage-specific" indicates that the target or site is utilized by at least one bacteriophage as an inhibitory target and is different from previously identified targets or target sites.
  • bacteriophage inhibitor protein refers to a protein encoded by a bacteriophage nucleic acid sequence which inhibits bacterial function in a host bacterium. Thus, it is a bacteria-inhibiting phage product.
  • phrase "contacting the bacterial cell with a compound active on a bacterial target of a bacteriophage inhibitor protein” or equivalent phrases refer to contacting with an isolated, purified, or enriched compound or a composition including such a compound, but specifically does not rely on contacting the bacterial cell with an intact phage which encodes the compound. Preferably no intact phage are involved in the contacting.
  • bacteriophage inhibitor protein or RNA a compound active on a target of a bacteriophage inhibitor protein or RNA, or as described for the previous aspect.
  • the bacterium involved in the infection or risk of infection produces the identified target of the bacteriophage inhibitor protein or alternatively produces-a homologous target compound.
  • the host organism is a plant or animal, preferably a mammal or bird, and more preferably, a human or other mammal described herein. Preferred embodiments include, without limitation, those as described for the preceding aspect.
  • Compounds useful for the methods of inhibiting, methods of treating, and pharmaceutical compositions can include novel compounds, but can also include compounds which had previously been identified for a purpose other than inhibition of bacteria. Such compounds can be utilized as described and can be included in pharmaceutical compositions.
  • the target sequence is encoded by a Staphylococcus nucleic acid coding sequence, preferably S. aureus, a Streptococcus nucleic acid coding sequence, preferably Streptococcus pneumoniae, or Enterococcus nucleic acid coding sequence.
  • a Staphylococcus nucleic acid coding sequence preferably S. aureus
  • Streptococcus nucleic acid coding sequence preferably Streptococcus pneumoniae
  • Enterococcus nucleic acid coding sequence Possible target sequences are described herein by reference to sequence source sites.
  • the amino acid sequence of a polypeptide target is readily provided by translating the corresponding coding region.
  • the sequences are not reproduced herein.
  • the sequences are described by reference to the GenBank entries instead of being written out in full herein.
  • the complete sequence can be readily obtained by routine methods, e.g., by isolating a clone in a phage host genomic library, and sequencing the clone insert to provide the relevant coding region.
  • the boundaries of the coding region can be identified by conventional sequence analysis and/or by expression in a bacterium in which the endogenous copy of the coding region has been inactivated and using subcloning to identify the functional start and stop codons for the coding region.
  • the term "corresponding" indicates that the sequence is at least 95% identical, preferably at least 97% identical, and more preferably at least 99%> identical to a sequence from the specified phage genome, a ribonucleotide equivalent, a degenerate equivalent (utilizing one or more degenerate codons), or a homologous sequence, where the homolog provides functionally equivalent biological function.
  • treatment or “treating” is meant administering a compound or pharmaceutical composition for prophylactic and/or therapeutic purposes.
  • prophylactic treatment refers to treating a patient or animal that is not yet infected but is susceptible to or otherwise at risk of a bacterial infection.
  • therapeutic treatment refers to administering treatment to a patient already suffering from, infection.
  • bacterial infection refers to the invasion of the host organism, animal or plant, by pathogenic bacteria. This includes the excessive growth of bacteria which are normally present in or on the body of the organism, but more generally, a bacterial infection can be any situation in which the presence of a bacterial population(s) is damaging to a host organism.
  • an organism suffers from a bacterial population when excessive numbers of a bacterial population are present in or on the organism's body, or when the effects of the presence of a bacterial population(s) is damaging to the cells, tissue, or organs of the organism.
  • administer refers to a method of giving a dosage of a compound or composition, e.g., an antibacterial pharmaceutical composition, to an organism. Where the organism is a mammal, the method is, e.g., topical, oral, intravenous, transdermal, mtraperitoneal, intramuscular, or intrathecal.
  • the preferred method of administration can vary depending on various factors, e.g., the components of the pharmaceutical composition, the site of the potential or actual bacterial infection, the bacterium involved, and the infection severity.
  • mammamal has its usual biological meaning referring to any organism of the Class Mammalia of higher vertebrates that nourish their young with milk secreted by mammary glands, e.g., mouse, rat, and, in particular, human, bovine, sheep, swine, dog, and cat.
  • a "therapeutically effective amount” or “pharmaceutically effective amount” indicates an amount of an antibacterial agent, e.g., as disclosed for this invention, which has a therapeutic effect. This generally refers to the inhibition, to some extent, of the normal cellular functioning of bacterial cells that renders or contributes to bacterial infection.
  • the dose of antibacterial agent that is useful as a treatment is a "therapeutically effective amount.”
  • a therapeutically effective amount means an amount of an antibacterial agent that produces the desired therapeutic effect as judged by clinical trial results and/or animal models. This amount can be routinely determined by one skilled in the art and will vary depending on several factors, such as the particular bacterial strain involved and the particular antibacterial agent used.
  • a compound active on a target of a bacteriophage inhibitor protein or terms of equivalent meaning differ from administration of or contactwTth an intact phage naturally encoding the full-length inhibitor compound. While an intact phage may conceivably be inco ⁇ orated in the present methods, the method at least includes the use of an active compound as specified different from a full length inhibitor protein naturally encoded by a bacteriophage and/or a delivery or contacting method different from administration of or contact with an intact phage encoding the full-length protein.
  • compositions described herein at least include an active compound different from a full-length inhibitor protein naturally encoded by a bacteriophage or such a full-length protein is provided in the composition in a form different from being encoded by an intact phage.
  • the methods and compositions do not include an intact phage.
  • the invention also provides antibacterial agents and compounds active on bacterial targets of bacteriophage inhibitor proteins or RNAs, where the target was uncharacterized as indicated above.
  • active compounds include both novel compounds and compounds which had previously been identified for a purpose other than inhibition of bacteria.
  • the targets, bacteriophage, and active compound are as described herein for methods of inhibiting and methods of treating.
  • the agent or compound is formulated in a pharmaceutical composition which includes a pharmaceutically acceptable carrier, excipient, or diluent.
  • the invention provides agents, compounds, and pharmaceutical compositions where an active compound is active on an uncharacterized phage-specific site.
  • the target is as described for embodiments of aspects above.
  • the invention provides a method of making an antibacterial agent.
  • the method involves identifying a target of a bacteriophage inhibitor polypeptide or protein or RNA, screening a plurality of compounds to identify a compound active on the target, and synthesizing the compound in an amount sufficient to provide a therapeutic effect when administered to an organism infected by a bacterium naturally producing the target.
  • the identification of the target and identification of active compounds include steps or methods and/or components as described above (or otherwise herein) for such identification.
  • the active compound can be as described above, including fragments and derivatives of phage inhibitor proteins, peptidomimetics, and small molecules.
  • peptides can be synthesized by expression systems and purified, or can be synthesized artificially.
  • the inhibitory phage ORF- products is from S. aureus phage 44AH D ORF 1, 9, or 12, Streptococcus pneumoniae phage Dp-1 ORF 001, 002, 004, 008, 010, 013, 016, 021, 029, 030, 038, or 041, or Enterococcus sp.
  • sequence analysis of nucleotide and/or amino acid sequences can beneficially utilize computer analysis.
  • the invention provides computer-related hardware and media and methods utilizing and incorporating sequence data from uncharacterized phage, e.g., uncharacterized phage listed in Table 1, preferably at least one of Staphylococcus aureus phage S. aureus phage 44AHJD ORF 1, 9, or 12, Streptococcus pneumoniae phage Dp-1 ORF 001, 002, 004, 008, 010, 013, 016, 021, 029, 030, 038, or 041, or Enterococcus sp. phage 182 ORF 002, 008, or 014, or 44 AHJD, Enterococcus sp. phage 182, or
  • Streptococcus pneumoniae phage Dp-1 can facilitate the above-described aspects.
  • Various embodiments involve the analysis of genetic sequence and encoded products, as applied to the evaluating bacteriophage inhibitor ORFs and compounds and fragments related thereto.
  • sequence analyses, as well as function analyses can be used separately or in combination, as well as in preceding aspects and embodiments. Use in combination is often advantageous as the additional information allows more efficient prioritizing of phage ORFs for identification of those ORFs that provide bacteria-inhibiting function.
  • the invention provides a computer-readable device which includes at least one recorded amino acid or nucleotide sequence corresponding to one of the specified phage and a sequence analysis program for analyzing a nucleotide and/or amino acid sequence.
  • the device is arranged such that the sequence information can be retrieved and analyzed using the analysis program.
  • the analysis can identify, for example, homologous sequences or the indicated %s of the phage genome and structural motifs.
  • the sequence includes at least 1 phage ORF or encoded product, more preferably at least 10%, 20%, 30%, 40%, 50%, 70%, 90%, or 100%o of the genomic phage ORFs and/or equivalent cDNA, RNA, or amino acid sequences.
  • sequence or sequences in the device are recorded in a medium such as a floppy disk, a computer hard drive, an optical disk, computer random access memory (RAM), or magnetic tape.
  • the program may also be recorded in such medium.
  • the sequences can also include sequences from a plurality of different phage.
  • the term "corresponding" indicates that the sequence is at least 95% identical, preferably at least 97% identical, and more preferably at least 99% identical to a sequence from the specified phage genome, a ribonucleotide equivalent, a degenerate equivalent (utilizing one or more degenerate codons), or a homologous sequence, where the homolog provides functionally equivalent biological function.
  • the invention provides a computer analysis system for identifying biologically important portions of a bacteriophage genome.
  • the system includes a data storage medium, e.g., as identified above, which has recorded thereon a nucleotide sequence corresponding to at least a portion of at least one uncharacterized bacteriophage genome, a set of program instructions to allow searching of the sequence or sequences to analyze the sequence, and an output device where the portion includes at least the sequence length as specified in the preceding aspect.
  • the output device is preferably a printer, a video display, or a recording medium. More one than one output device may be included.
  • the bacteriophage are preferably selected from the uncharacterized phage listed in Table 1, more preferably from bacteriophage 77, 3 A, 96, 44 AHJD (S. aureus), Dp-1 (Streptococcus pneumoniae), or 182 (Enterococcus).
  • the invention also provides a method for identifying or characterizing a bacteriophage ORF by providing a computer-based system for analyzing nucleotide or amino acid sequences, e.g., as describe above.
  • the system includes a data storage medium which has recorded a sequences or sequences as described for the above devices, a set of instructions as in the preceding aspect, and an output device as in the preceding aspect.
  • the method further involves analyzing at least one sequence, and outputting the analysis results to at least one output device.
  • the analysis identifies a sequence similarity or homology with a sequence or sequences selected from bacterial ORFs encoding products with related biological function; ORFs encoding known inhibitors; and essential bacterial ORFs.
  • the analysis identifies a probable biological function based on identification of structural elements or characteristic or signature motifs of an encoded product or on sequence similarity or homology.
  • the uncharacterized bacteriophage is from Table 1 , more preferably at least one of bacteriophage 77, 3 A, 96, 44 AHJD (S. aureus), Dp-1 (Streptococcus pneumoniae), or 182 (Enterococcus).
  • the method also involves determining at least a portion of the nucleotide sequence of at least one uncharacterized bacteriophage as indicated, and recording that sequence on data storage medium of the computer-based system.
  • the analysis identifies a sequence similarity of homology with a S. aureus phage 44AHJD ORF 1, 9, or 12, Streptococcus pneumoniae phage Dp-1 ORF 001, 002, 004, 008, 010, 013, 016, 021, 029, 030, 038, or 041, or Enterococcus sp. phage 182 ORF 002, 008, or 014.
  • “comprising” means including, but not limited to, whatever follows the word “comprising”. Thus, use of the term “comprising” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By “consisting of is meant including, and limited to, whatever follows the phrase “consisting of. Thus, the phrase “consisting of indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.
  • FIGURE 1 A and IB are flow schematics showing the manipulations used to convert pT0021, an arsenite inducible vector containing the luciferase gene, into pTHA or pTM, two ars inducible vectors.
  • Vector pTHA contains BamH I, Sal I, and Hind III cloning sites and a downstream HA epitope tag.
  • Vector pTM contains Bam HI and Hind III cloning sites and no HA epitope tag.
  • FIGURE 2 is a schematic representation of the cloning steps involved to place the DNA segments of any of ORFs 17/ 19/ 43/ 102/104/182 or other sequences into pTHA to assess inhibitory potential.
  • Individual ORFs were amplified by the PCR using oligonucleotides targeting the ATG and stop codons of the ORFs. Using this strategy, Bam HI and Hind III sites were positioned immediately upstream or downstream, respectively of the start and stop codons of each ORF. Following digestion with Bam HI and Hind III, the PCR fragments were _ subcloned into the same sites of pT0021 or pTM.
  • FIGURE 3 shows a schematic representation of the functional assays used to characterize the bactericidal and bacteriostatic potential of all predicted ORFs (>33 amino acids) encoded by bacteriophage 77.
  • Fig. 3A Functional assay on semi-solid support media.
  • Fig. 3B Functional assay in liquid culture.
  • FIGURE 4A, B, and C is a bar graph showing the results of a screen in liquid media to assess bacteriostatic or bactericidal activity of 93 predicted ORFs (>33 amino acids) encoded by bacteriophage 77. Growth inhibition assays were performed as detailed in the Detailed Description. The relative growth of Staphylococcus aureus transformants harboring a given bacteriophage 77 ORF (identified on the bottom of the graph), in the absence or presence of arsenite, is plotted relative to growth of a Staphylococcus aureus transformant containing ORF 5, a non-toxic bacteriophage 77 ORF (which is set at 100%). Each bar represents the average obtained from three Staph A transformants grown in duplicate. Bacteriophage 77 ORFs showing significant growth inhibition consist of ORFs 17, 19, 102, 104, and 182.
  • FIGURE 5 shows a block diagram of major components of a general purpose computer.
  • FIGURE 6 shows an ORF map for Streptococcus pneumoniae bacteriophage Dp-1 showing the ORF identifiers, genomic locations, and orientations of the 85 identified ORFs that were found to have ribosomal binding sites and thus are expected to be expressed.
  • FIGURE 7 shows a schematic representation of the arsenite-inducible expression system present in a shuttle vector designed to express individual Streptococcus bacteriophage Dp-1 ORFs in Streptococcus.
  • Various modifications can be readily made to such a vector, or other vectors can be readily constructed to provide inducible expression of ORFs in a particular host bacterium using well-known techniques.
  • Table 1 is a listing of a large number of available bacteriophage that can be readily obtained and used in the present invention.
  • Table 2 shows the complete nucleotide sequence of the genome of Staphylococcus aureus bacteriophage 77.
  • Table 3 shows a list of all the ORFs from Bacteriophage 77 that were screened in the functional assay to identify those with anti-microbial activity.
  • Table 4 shows the predicted nucleotide sequence, predicted amino acid sequence, and physiochemical parameters of ORF 17/ 19/ 43/ 102/ 104/ 182]. These include the primary amino acid sequence of the predicted protein, the average molecular weight, amino acid composition, theoretical pi, hydrophobicity map, and predicted secondary structure map.
  • Table 5 shows homology search results. BLAST analysis was performed with ORFs 17/ 19/ 43/ 102/ 104/ 182 against NCBI non-redundant nucleotide and Swissprot databases. The results of this search indicate that: I) ORF 17 has no significant homology to any gene in the NCBI non-NCBI non-redundant nucleotide database, II) ORF 19 has significant homology to one gene in the NCBI non- redundant nucleotide database - the gene encoding ORF 59 of bacteriophage phi PVL, III) ORF 43 has significant homology to one gene in the NCBI non-redundant nucleotide database - the gene encoding ORF 39 of phi PVL, IV) ORF 102 has significant homology to one gene in the NCBI non-redundant nucleotide database - the gene encoding ORF 38 of phi PVL, V) ORF 104 has no significant homology to any gene in the NCBI non-redundant nucleotide database
  • Table 7 shows the complete nucleotide sequence of Staphylococcus aureus bacteriophage 3A.
  • Table 8 is a listing of the ORFs identified in Staphylococcus aureus bacteriophage 3A.
  • Table 9 shows the complete nucleotide sequence of Staphylococcus aureus bacteriophage 96.
  • Table 10 is a listing of the ORFs identified in Staphylococcus aureus bacteriophage 96.
  • Table 11 is a listing of sequences deposited in the NCBI public database (GeneBank) for bacteriophage listed in Table 1.
  • Table 12 is a listing of phage which encode a known lysis function , including the identified lysis gene.
  • Table 13 is a listing of bacteriophage which encode holin genes, where holin genes encode proteins which form pores and eventually enable other enzymes to kill the host bacterium.
  • Table 14 is a listing of bacteriophage which encode kil genes.
  • Table 15 is a list of Staphylococcus aureus sequences identified by accession number which may include sequences from genes coding for target sequences for the phage 77-encoded antimicrobial proteins or peptides. The sequences were obtained by searching GenBank for listings.
  • Table 16 shows the nucleotide sequence of the genome of Staphylococcus aureus phage 44 AHJD.
  • Table 17 lists and shows the sequence position of the 73 ORFs predicted to be encoded by Staphylococcus aureus bacteriophage 44 AHJD that are greater than 33 amino acids.
  • Table 18 shows the ORF sequences and putative amino acid sequences for the Staphylococcus aureus bacteriophage 44AHJD ORFs greater than 33 amino acids.
  • Table 19 shows the similarities in sequence identified between predicted
  • Table 20 shows the homology alignments between predicted Staphylococcus aureus bacteriophage 44 AHJD ORFs and the corresponding protein sequences present in public sequence databases.
  • Table 21 shows the complete nucleotide sequence of the genome of
  • Table 22 lists and shows the sequence position of the 80 ORFs identified in bacteriophage 182 and that are greater than 33 amino acids.
  • Table 23 shows the nucleotide and predicted amino acid sequence of all 80 ORFs identified in bacteriophage 182.
  • Table 24 shows the similarities identified to date in sequence between Enterococcus phage 182 ORFs greater than 33 amino acids and sequences present in public sequence databases.
  • Table 25 shows the predicted amino acid sequence as well as the predicted secondary structures map for two Enterococcus bacteriophage 182 ORFs.
  • Table 26 shows the homology alignments between predicted Enterococcus bacteriophage 182 ORFs and the corresponding protein sequences present in public sequence databases.
  • Table 27 list Enterococcus sequences listed in GenBank providing possible Enterococcal target sequences for inhibitory Enterococcus bacteriophage 182 ORFs and other compounds with antibacterial activity.
  • Table 28 shows the complete nucleotide sequence of the genome of Streptococcus bacteriophage Dp- 1.
  • Table 29 lists and shows sequence position of the 273 ORFs identified in Pneumococcal bacteriophage Dp-1 that are greater than 33 amino acids, 85 of which are predicted to be expressed in Dp-1 as having a ribosomal binding site. That set of 85 ORFs is shown in the attached drawings.
  • Table 30 shows the nucleotide and predicted amino acid sequence of all 273
  • Table 31 shows the similarities identified in sequence between Streptococcus phage Dp-1 ORFs greater than 33 amino acids and sequences present in public sequence databases.
  • Table 32 shows the 4731 bp sequence of Dp-1 published by Sheehan et al.,
  • Table 33 lists Streptococcus pneumoniae sequences listed in GenBank providing possible target sequences for inhibitory Streptococcus pneumoniae bacteriophage Dp-1 ORFs and other compounds with antibacterial activity
  • the present invention is concerned, in part, with the use of bacteriophage coding sequences and the encoded polypeptides or RNA transcripts to _ identify bacterial targets for potential new antibacterial agents.
  • the invention concerns the selection of relevant bacteria.
  • Particularly relevant bacteria are those which are pathogens of a complex organism such as an animal, e.g., mammals, reptiles, and birds, and plants. Examples include Stapylococcus aureus, Enterococcus species, and Streptococcus pneumoniae.
  • the invention can be applied to any bacterium (whether pathogenic or not) for which bacteriophage are available or which are found to have cellular components closely homologous to components targeted by phage of another bacterium.
  • the invention also concerns the bacteriophage which can infect a selected bacterium.
  • Identification of ORFs or products from the phage which inhibit the host bacterium both provides an inhibitor compound and allows identification of the bacterial target affected by the phage-encoded inhibitor.
  • targets are thus identified as potential targets for development of other antibacterial agents or inhibitors and the use of those targets to inhibit those bacteria.
  • a target can still be identified if a homologous target is identified in another bacterium.
  • such another bacterium would be a genetically closely related bacterium.
  • a phage-encoded inhibitor can also inhibit such a homologous bacterial cellular component.
  • the demonstration that bacteriophage have adapted to inhibiting a host bacterium by acting on a particular cellular component or target provides a strong indication that that component is an appropriate target for developing and using antibacterial agents, e.g., in therapeutic treatments.
  • the present invention provides additional guidance over mere identification of bacterial essential genes, as the present invention also provides an indication of accessability of the target to an inhibitor, and an indication that the target is sufficiently stable over time (e.g., not subject to high rates of mutation) as phage acting on that target were able to develop and persist.
  • the present invention identifies a subset of essential cellular components which are particularly likely to be appropriate targets for development of antibacterial agents.
  • the invention also, therefore, concerns the development or identification of inhibitors of bacteria, in addition to the phage-encoded inhibitory proteins (or RNA transcripts), which are active on the targets of bacteriophage-encoded inhibitors.
  • inhibitors of bacteria in addition to the phage-encoded inhibitory proteins (or RNA transcripts), which are active on the targets of bacteriophage-encoded inhibitors.
  • phage-encoded inhibitory proteins or RNA transcripts
  • inhibitors can be of a variety of different types, but are preferably small molecules.
  • the first step involves selecting bacterial hosts of interest.
  • such hosts will be pathogens of clinical importance.
  • these features can be targeted for study in one strain, for example a nonpathogenic one, and extrapolated to similarly succeed in pathogenic ones.
  • Nonpathogenic strains may also exhibit initial advantages in being not only less dangerous, but also, for example, in having better growth and culturing characteristics and/or better developed molecular biology techniques and reagents. Consequently, advantageously the invention provides the ability target virtually any bacteria, but preferably pathogenic bacteria, with antimicrobial compounds designed and/or developed using bacteriophage inhibitory proteins and peptides from phage with nonpathogenic and/or pathogenic hosts.
  • Enterococci and Pseudomonas aeruginosa as initial exemplary pathogens. These bacteria are a major cause of morbidity and mortality in hospital-based infections, and the appearance of antibiotics resistance in all three organisms makes it increasingly difficult to treat benign infections involving these organisms.
  • infections can include, for example, otitis media, sinusitis, and skin, and airway infections (Neu, H.C. (1992). Science 257, 1064-1073).
  • the approach described below is clearly applicable to any human bacterial pathogens including but not restricted to Mycobacterium tuberculosis, Nesseria gonorrhoeae, Haemophilus influenza, Acinobacter, Escherichia coli, Shigella dysenteria, Streptococcus pyogenes, Helicobacter pylori, and Mycoplasma species.
  • This invention can also be applied to the discovery of anti-bacterial compounds directed against pathogens of animals other than humans, for example, sheep, cattle, swine, dogs, cats, birds, and reptiles.
  • the invention is not limited to animals, but also applies to plants and plant pathogens.
  • the bacteria are grown according to standard methodologies -, employed in the art, including solid, semi-solid or liquid culturing, which procedures can be found in or extrapolated from standard sources such as Maloy, S.R., Stewart, V.J., and Taylor, R.K. Genetic Analysis of Pathogenic Bacteria (1996) Cold Spring Harbor Laboratory Press, or Maniatis, T. et al. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor University Press, Cold Spring, N.Y.; or Ausubel, F.M. et al. (1994) Current Protocols in Molecular Biology. John Wiley & Sons, Secaucus, N J. Culture conditions are selected which are adapted to the particular bacterium generally using culture conditions known in the art as appropriate, or adaptations of those conditions.
  • nucleic acids within these bacteria can be routinely extracted through common procedures such as described in the above-referenced manuals and as generally known to those skilled in the art. Those nucleic acid stocks can then be used to practice the other inventive aspects described below.
  • the second step involves assembling a group of bacteriophages (phage collection) for one or more of the targeted bacterial hosts. While the invention can be utilized with a single bacteriophage for a pathogen or other bacterium, it is preferable to utilize a plurality of phage for each bacterium, as comparisons between a plurality of such phage provides useful additional information.
  • phage and sources for some of the above-mentioned pathogenic bacteria are found in Table 1. The criteria used to select such phages is that they are infectious for the microbe targeted, and replicate in, lyse, or otherwise inhibit growth of the bacterium in a measurable fashion.
  • phages can be very different from one another (representing different families), as judged by criteria such as morphology (head, tail, plate, etc.), and similarity of genome nucleotide sequence (cross-hybridization). Since such diverse bacteriophages are expected to block bacterial host metabolism and ultimately inhibit by a variety of mechanisms, their combined study will lead to the identification of different mechanisms by which the phages independently inhibit bacterial targets. Examples include degradation of host DNA (Parson K.A., and Snustad, D.P. (1975). J. Virol. 15, 221-444) and inhibition of host RNA transcription (Severinova, E., Severinov, K. and Darst, S.A. (1998;. J.Mol. Biol.
  • Bacteriophage are generally either of two types, lytic or filamentous, meaning they either outright destroy their host and seek out new hosts after replication, or else continuously propogate and extrude progeny phage from the same host without destroying it. Regardless of the phage life cycle and type, preferred embodiments incorporate phage which impede cell growth in measurable fashion and preferably stop cell growth. To this end, lytic phage are preferred, although certain nonlytic species may also suffice, e.g., if sufficiently bacteriostatic. Various procedures that are commonly understood by those of skill in the art can be routinely employed to grow, isolate, and purify phage.
  • the techniques generally involve the culturing of infected bacterial cells that are lysed naturally and/or chemically assisted, for example, by the use of an organic solvent such as chloroform that destroys the host cells thereby liberating the phage within. Following this, the cellular debris is centrifuged away from the supernatant containing the phage particles, and the phage then subsequently and selectively precipitated out of the supernatant using various methods usually employing the use of alcohols and/or other chemical compounds such as polyethylene glycol (PEG). The resulting phage can be further purified using various density gradient/centrifugation methodologies. The resulting phage are then chemically lysed, thereby releasing their nucleic acids that can be conveniently precipitated out of the supernatant to yield a viral nucleic acid supply of the phage of interest.
  • an organic solvent such as chloroform that destroys the host cells thereby liberating the phage within.
  • Exemplary bacteriophage are indicated in Table 1, along with sources where those phage may be obtained.
  • Exemplary bacteria include the reference bacteria for the identified bacteriophage, available from the same sources.
  • the third step involves systematically characterizing the genetic information contained in the phage genome.
  • this genetic information is the sequence of all RNAs and proteins encoded by the phage, including those that are essential or instrumental in inhibiting their host.
  • This characterization is preferably done in a systematic fashion. For example, this can be done by first isolating high molecular weight genomic DNA from the phage using standard bacterial lysis methods, followed by phage purification using density gradient ultracentrifugation, and extraction of nucleic acid from the purified phage preparation. The high molecular weight DNA is then analyzed to determine its size and to evaluate a proper strategy for its sequencing. The DNA is broken down into smaller size fragments by sonication or partial digestion with frequently cutting restriction enzymes such as Sau3A to yield predominantly 1 to 2 kilobase length DNA, which DNA can then be resolved by gel electrophoresis followed by extraction from the gel.
  • the ends of the fragments are enzymatically treated to render them suitable for cloning and the pools of fragments are cloned in a bacterial plasmid to generate a library of the phage genome.
  • Several hundred of these random DNA fragments contained in the plasmid vector are isolated as clones after introduction into an appropriate bacterium, usually Escherichia coli. They are then individually expanded in culture and the DNA from each individual clone is purified.
  • the nucleotide sequences of the inserts of these clones are determined by standard automated or manual methods, using oligonucleotide primers located on either side of the cloning site to direct polymerase mediated sequencing (e.g., the Sanger sequencing method or a modification of that method).
  • sequence of individual clones is then deposited in a computer, and specific software programs (for example, SequencherTM, Gene Codes Corp.) are used to look for overlap between the various sequences, resulting in ordering of contig sequences and ultimately providing the complete sequence of the entire bacteriophage genome (one such example is given in Table 2 for Staphylococcus aureus bacteriophage 77; others are also provided herein).
  • This complete nucleotide sequence is preferably determined with a redundancy of at least 3- to 5-fold (number of independent sequencing events covering the same region) in order to minimize sequencing errors.
  • the bacterial strain used as a phage host should not possess any other innate plasmids, transposons, or other phage or incompatible sequences that would complicate or otherwise make the various manipulations and analyses more difficult.
  • ORFs identified from phage 77 are cataloged into a phage proteome database (Table 3 lists ORFs identified from phage 77; ORF lists are also provided for other exemplary phage). This analysis is preferably performed for each phage under study.
  • the process of ORF identification can be varied depending on the desired results. For example, the minimum length for the putative encoded polypeptide can be varied, and/or putative coding regions that have an associated Shine-Dalgarno sequence can be selected.
  • phage 77 ORFs such parameter adjustment was performed and resulted in the identification of ORFs as listed herein. Different parameters had resulted in the identification of the ORFs listed in the preceding U.S. Provisional Application 60/110,992, filed December 3, 1998, which is hereby incorporated by reference in its entirety.
  • Exemplary phage 77 ORFs identified in that provisional application and as identified herein are shown in the following table:
  • the fourth step entails identifying the phage protein or proteins or RNA transcripts that have the ability to inhibit their bacterial hosts. This can be accomplished, for example, by either or both of two non-mutually exclusive methods.
  • the first method makes use of bioinformatics. Over the past few years, a large amount of nucleotide sequence information and corresponding translated products have become available through large genome sequencing projects for a variety of organisms including mammals, insects, plants, unicellular eukaryotes (yeast and fungi), as well as several bacterial genomes such as E. coli, Mycobacterium tuberculosis, Bacillus subtilis, Staphylococcus aureus and many others.
  • sequences have been deposited in public databases (for example, non-redundant sequence database at GenBank and SwissProt protein sequence database) (http://www.ncbi.nlm.nih.gov)) and can be freely accessed to compare any specific query sequence to those present in such databases.
  • GenBank contains over 1.6 billion nucleotides corresponding to 2.3 million sequence records.
  • TBLASTN computer programs and servers
  • the antimicrobials of the present invention will preferably target features and targets that are highly characteristic or conserved in microbes, and not higher organisms.
  • sequence homology between individual members of evolutionarily distant members of a protein family is usually not randomly distributed along the entire length of the sequence but is often clustered into "motifs" and "domains". These correspond to key three-dimensional folds that form key catalytic and/or regulatory structures that perform key biochemical function(s) for the group of proteins.
  • Commercially available computer software programs can identify such motifs in a new query sequence, again providing functional information for the query sequence.
  • Such structural and functional motifs have also been derived from the combined analysis of primary sequence databases (protein sequences) and protein structure databases (X-ray crystallography, nuclear magnetic resonance) using so-called “threading” methods (Rost B,l and Sander C. (1996) Ann. Rev. Biophy. Biomol. Struct. 25, 113-136).
  • This analysis can point out phage proteins with similarity to proteins from other phages (such as those for E. coli) playing an important role in the basic biochemical pathways of the phage (such as DNA replication, RNA transcription, tRNAs, coat protein and assembly). Selected examples of such proteins include integrase and capsid protein. Therefore, this analysis enables identification and elimination of non-essential ORFs as candidates for an inhibitor function, as well as the identification of (potentially) useful ones.
  • ORFs may encode proteins or enzymes that alter bacterial cell structure, metabolism or physiology, and ultimately viability.
  • proteins present in the genome of Staphylococcus aureus bacteriophage 77 include orfl4 (deoxyuridine triphosphatase from bacteriophage T5), and orfl5 (sialidase).
  • orfl4 deoxyuridine triphosphatase from bacteriophage T5
  • orfl5 sialidase
  • Other examples include ORFs 9 and 12 of S. aureus phage 44 AHJD, which encode the putative lysis functions found in many bacteriophages - a "holin” and an "amidase”.
  • bacterial and eukaryotic viruses can usurp pathways from their host in order to use them to their advantage in blocking host cellular pathways upon infection.
  • the phage can achieve this by 1) directly producing an inhibitor of a key host pathway (e.g. T7 gene 0.5 and 2), 2) directly producing a novel activity (e.g. T4 DNA polymerase), and 3) altering concentrations of cell components by producing similar functions (e.g. T4 transfer RNAs).
  • a key host pathway e.g. T7 gene 0.5 and 2
  • novel activity e.g. T4 DNA polymerase
  • T4 transfer RNAs e.g. T4 transfer RNAs
  • a homology search may reveal that a given phage ORF is related to a protein present in the databases having an activity known to be inhibitory, (e.gA inhibitor of host RNA polymerase by E. coli bacteriophage T7. Such a finding would implicate the phage ORF product in a related activity.
  • a new antimicrobial could be derived by a mimetic approach (e.g., peptidomimetic) imitating this function or by a small molecule inhibitor to the bacterial target of the phage ORF, or any steps in the relevant host metabolic pathway, e.g., high throughput screening of small molecule libraries.
  • ORFs are expressed, preferably overexpressed, in the host and the effect of this expression or overexpression on host metabolism and viability is measured. This approach can be systematically applied to every ORF of the phage, if necessary, and does not rely on the absolute identification of candidate ORFs by bioinformatics.
  • ORFs are resynthesized from the phage genomic DNA, e.g., by the polymerase chain reaction (PCR), preferably using oligonucleotide primers flanking the ORF on either side.
  • PCR polymerase chain reaction
  • These single ORFs are preferably engineered so that they contain appropriate cloning sites at their extremities to allow their introduction into a new bacterial expression plasmid, allowing propagation in a standard bacterial host such as E. coli, but containing the necessary information for plasmid replication in the target microbe such as S. aureus (hereafter referred to as shuttle vector).
  • shuttle vectors and their use are well known in the art.
  • Such shuttle vectors preferably also contain regulatory sequences that allow inducible expression of the introduced ORF.
  • the candidate ORF may encode an inhibitor function that will eliminate the host, it is beneficial that it not be expressed prior to testing for activity. Thus, screening for such sequences when expressed in a constitutive fashion is less likely to be successful when the inhibitor is lethal.
  • regulatory sequences from the ars operon of S. aureus are used to direct individual ORF expression in S. aureus (or other bacteria in which the ars system is functional).
  • the ars operon encodes a series of proteins which normally mediate the extrusion of arsenite and other trivalent oxyanions from the cells when they are exposed to such toxic substances in their environment.
  • individual phage ORFs can be expressed in S. aureus in an inducible fashion by adding to the culture medium non-toxic arsenite concentrations during the growth of individual S. aureus clones expressing such individual phage ORFs.
  • Toxicity of the phage inhibitor ORF for the host is monitored by reduction or arrest of growth under induction conditions, as measured by optical density in liquid culture or after plating the induced cultures on solid medium.
  • interference of the phage ORF with the host biochemical pathways ultimately leading to reduced or arrested host metabolism can be measured by pulse-chase experiments using radiolabeled precursors of either DNA replication, RNA transcription, or protein synthesis. Similar constructs can be made and used for other bacteria using well- known techniques.
  • shuttle vectors and the selection and use of inducible systems are well known and thus other shuttle vectors appropriate for other bacteria can be readily provided by those skilled in the art, e.g., for use in other bacterial species.
  • phage or other viruses inhibit host cells, at least in part, by producing an antisense RNA which binds to and inhibits translation from a bacterial RNA seqeunce.
  • a strong indicator of a possible inhibitory function is provided by the identification of phage sequence which is the identical to or fully complementary (or with only a small percentage of mismatch, e.g., ⁇ 10%, preferably less than 5%, most preferably less than 3%, to a bacterial sequence. This approaches convenient in the case of bacteria that have been essentially completely sequenced, as the comparison can be performed by computer using public database information.
  • the inhibitory effect of the transcript can be confirmed using expression of the phage sequence in a host bacterium. If needed, such inhibitory can also be tested by transfecting the cells with a vector that will transcribe the phage sequence to form RNA in such manner that the RNA produced will not be translated into a polypeptide. Inhibition under such conditions provides a strong indication that the inhibition is due to the transcript rather than to an encoded polypeptide.
  • the expression of an ORF in a host bacterium is found to be inhibitory, but the inhibition is found to be due to an RNA product of the genomic coding region.
  • the sequence of the bacterial target nucleic acid sequence can be identified by inspection of the phage sequence, and the full sequence of the relevant coding region for the bacterial product can be found from a database of the bacterial genomic sequence or can be isolated by standard techniques (e.g., a clone in a genomic library can be isolated which contains the full bacterial ORF, and then sequenced).
  • the identification of a target which is inhibited by an RNA transcript produced by a phage provides both the possible inhibition of bacteria naturally containing the same target nucleic acid sequence, as well as the ability to use the target sequence in screening for other types of compounds which will act directly on the target nucleic acid sequence or on a polypeptide product expressed or regulated, at least in part, by the target of the inhibitory phage RNA.
  • the target of an inhibitory phage RNA or protein has previously been found to be a target of an inhibitory phage RNA or protein has previously been found to be a target for an antibacterial agent.
  • the phage inhibitor can still provide useful information if it is found that the phage-encoded product acts at a different site than the previously identified antibacterial agent or inhibitor, i.e., acts at a phage-specific site.
  • action at a different site provides highly beneficial characteristics and/or information.
  • an alternate site of inhibitor action can at least partially overcome a resistance mechanism in a bacterium.
  • resistance is due, in large part, to altered binding characteristics of the immediate target to the antibacterial agent.
  • the altered binding is due to a structural change which prevents or destabilizes the binding.
  • the structural change is frequently quite local, so that compounds which bind at different local sites will b unaffected or affected to a much lesser degree. Indeed, in some cases the local sites will be on a different molecule and so may be completely unaffected by the local structural change creating resistance to the original agent(s).
  • An example of resistance due to altered binding is provided by methicillin-resistant Staphylococcus aureus, in which the resistance is due to an altered penicillin-binding protein.
  • a new site of action can have improved accessibility as compared to a site acted on by a previously identified agent. This can, for example, assist in allowing effective treatment at lower doses, or in allowing access by a larger range of types of compounds, potentially allowing identification of more potential active agents.
  • Another advantage is that the structural characteristics of a different site of action will lead to identification and/or development of inhibitors with different structures and different pharmacological parameter. This can allow a greater range of possibilities when selecting an antibacterial agent.
  • inhibition targeting an alternate site can produce more efficacious action, e.g., faster killing, slower development of resistance, lower numbers of surviving cells, and different secondary effects (for example, different nutrient utilization).
  • the present invention is concerned, in part, with the use of bacteriophage 77 coding sequences and the encoded polypeptides or RNA transcripts to identify bacterial targets for potential new antibacterial agents.
  • phage 77 ORFs 17, 19, 43, 102, 104, and 182 have been found to have bacteria inhibiting function.
  • Identification of ORFs 17, 19, 43, 102, 104, and 182 and products from the phage which inhibit the host bacterium both provides an inhibitor compound and allows identification of the bacterial target affected by the phage-encoded inhibitor.
  • Such a target is thus identified as a potential target for development of other antibacterial agents or inhibitors and the use of those targets to inhibit those bacteria.
  • a target can still be identified if a homologous target is identified in another bacterium.
  • such another bacterium would be a genetically closely related bacterium.
  • an inhibitor encoded by phage 77 ORF 17, 19, 43, 102, 104, or 182 can also inhibit such a homologous bacterial cellular component.
  • the sequence encoding the target corresponds to a S. aureus nucleic acid sequence available from numerous sources including S. aureus sequences deposited in GenBank, S. aureus sequences found in European Patent Application No. 97100110.7 to Human Genome Sciences, Inc. filed January 7, 1997, S. aureus sequences available from TIGR at http://www.tigr.org/tdb/mdb/mdb.html. and S. aureus sequences available from the Oklahoma University S. aureus sequencing project at the following URL: http://www.genome.ou.edu/staph new.html.
  • Such possible targets are particularly applicable to S aureus phages 77, 3A, 96, and 44 AHJD.
  • a target sequence corresponds to a S. aureus coding sequence corresponding to a sequence listed in Table 15 herein.
  • Table 15 describes S. aureus sequences currently listed with GenBank.
  • the sequences are described by reference to the database accession numbers instead of being written out in full herein.
  • the complete sequence can be readily obtained by routine methods, e.g., by isolating a clone in a phage host S.
  • aureus genomic library and sequencing the clone insert to provide the relevant coding region.
  • the boundaries of the coding region can be identified by conventional sequence analysis and/or by expression in a bacterium in which the endogenous copy of the coding region has been inactivated and using subcloning to identify the functional start and stop codons for the coding region.
  • Staphyloccus aureus phage 44 AHJD The present invention also can utilize the identification of naturally occuring
  • Such identification can utilize bioinformatics identification of specific proteins
  • ORFs utilized by Staphylococcus aureus bacteriophage 44AHJD during the viral life cycle, resulting in a slowing or arrest of growth of the bacterial host, or in death, of the Staphylococcus aureus host including lysis of the infected bacteria.
  • ORFs DNA sequences encoding these proteins (ORFs) are predicted to encode antimicrobial functions.
  • Information derived from these DNA sequences and translated ORFs can, in turn, be utilized to develop inhibitory __ compounds by peptidomimetics that can also function as antimicrobials.
  • the identification of the host bacterial proteins that are targeted and inhibited by the antimicrobial bacteriophage ORFs can themselves provide novel targets for drug discovery.
  • the methodology described above is used to identify and characterize DNA sequences from Staphylococcus sp. bacteriophage 44 AHJD that have antimicrobial activity.
  • the Staphylococcus aureus propagating strain (PS 44A) obtained from the Felix d'Herelle Reference Centre (#HER 1101), was used as a host to propagate its phage 44AHJD, also obtained from the Felix d'Herelle Reference Centre (#HER 101).
  • PS 44A Staphylococcus aureus propagating strain
  • HER 1101 the Staphylococcus aureus propagating strain
  • bacteriophage 44AHJD consists of 16,668 bp (Table 16) predicted to encode 73 ORFs greater than 33 amino acids (Tables 17 & 18).
  • Computational analysis of the predicted protein products of Staphylococcus aureus bacteriophage 44AHJD identified homolgs in public sequence databases as listed inTable 19 and 20, along with the accompanying list of related proteins.
  • ORF 3 3 genes are related to structural proteins found in other bacteriophages. These include genes predicted to encode a tail protein (ORF 3), an upper collar/connector protein of the phage virion (ORF 7), and a lower collar protein (ORF 8). Bioinformatics has also identified one gene whose product is likely involved in phage DNA synthesis.
  • One gene (ORF 1) shows significant homology to DNA polymerases of a number of bacteriophages, bacteria and fungi, and the product of this gene is likely responsible for replicating the genetic material of bacteriophage 44 AHJD.
  • ORF 2 encodes a protein with homology to the dinC gene of Bacillus subtilis that encodes a protein involved in teichoic acid biosynthesis.
  • Teichoic acid is a polyphosphate polymer found in some, but not all, Gram positive organisms (and not in Gram negative organisms), where it is attached to the peptidoglycan layer.
  • the phage protein may thus be involved in the synthesis of this material for incorporation into the cell wall, allowing enhanced lysis by the phage lysis enzymes or, as many enzymes can function in "reverse reactions", may be involved in its degradation allowing for penetration of the peptidoglycan and phage genome entry into the cell following adsorption.
  • Staphylococcus aureus bacteriophage 44AHJD and E. coli phage T7 indicate that they may share similar mechanisms of replication and growth. Both phages belong! 0 the Pododviridae Family of bacteriophages and are members of the "T7-like" Genus of this Family (Ackermann and DuBow; Vlth ICTV Report). Two genes, ORF 9 and 12, were identified with the potential to encode antimicrobial protein products. The homology alignments are shown in Tables 19 and 20.
  • ORF 9 The predicted product of ORF 9 is related to a class of genes which encodes lysozyme-like functions, enzymes which cleave linkages in the mucopolysaccharide cell wall structure of a variety of micro-organisms, including that from the
  • Staphylococcus aureus bacteriophage Twort Staphylococcus aureus bacteriophage Twort.
  • ORF 12 of Staphylococcus aureus bacteriophage 44AHJD shows homology to a set of lysis proteins from several bacteriophages. These lysis proteins are also referred to as holins, and represent phage-encoded lysis functions required for transit of the phage murein hydrolases (lysozyme) to the periplasm, where it can digest the cell wall and thus lyse the bacterium.
  • the present invention provides a nucleic acid sequence isolated from Staphylococcus aureus bacteriophage 44AHJD comprising at least a portion of one of the genes described above with antimicrobial activity.
  • ORF 1 encodes a DNA polymerase function. This polymerase may utilize host-derived accessory proteins for its activity when replicating the phage template, sequestering such proteins from use by the bacterial polymerase, resulting in inhibition of DNA replication, cell division, and cell growth.
  • ORF 9 directly encodes a polypeptide with antimicrobial activity. ORF 9 is predicted to encode an amidase, a protein known to act as a cell wall degrading enzyme.
  • ORF 12 likely encodes a holin function required for transit of the phage amidase (gene 9 product) to the periplasm.
  • this type of gene product from Bacillus phage phi 29 (gene 14) was cloned in Escherichia coli, cell death ensued (Steiner et al., 1993).
  • the present invention also provides the use of the Staphylococcus bacteriophage 44 AHJD antimicrobial ORFs or ORF products as pharmacological agents, either wholly or in part and derivatives, as well as the use of conesp ⁇ ndThg peptidomimetics, developed from amino acid or nucleotide sequence knowledge derived from Staphylococcus bacteriophage 44 AHJD killer ORFs.
  • Bacteriophage 182 was obtained from the Felix D'Herelle phage collection
  • Enterococcus bacteriophage 182 consists of 17,833 bp (Table 21) and is predicted to encode 80 ORFs greater than 33 amino acids (Tables 22 and 23). Computational analysis of the predicted protein products of Enterococcus bacteriophage 182 was performed in order to identify protein products related to those deposited in public databases. Bacteriophage 182 protein products which detected sequences with significant sequence similarity in public databases are listed in Table 24 and 26, along with the accompanying list of related proteins.
  • ORF 001, 004, 007, 009, and 011 are related to structural proteins of several Bacillus phages - Bacillus bacteriophage PZA, phi-29, and B103. These include genes predicted to encode a tail protein (ORF 001), a head protein (ORF 004), and upper collar protein (ORF 007), a lower collar protein (ORF 009), and a pre-neck appendage protein (ORF 011). Two gene products are predicted to encode genes which direct phage morphogenesis - these are ORF 005 and 019.
  • ORF 002 shows significant homology to DNA polymerases of a number of bacteriophages, and the product of this gene is likely responsible for replicating the genetic material of bacteriophage 182.
  • ORF 006 encodes a protein with homology to the encapsidation proteins of several other bacteriophages, including Bacillus phage phi-29 (PI 1014), PZA (P07541), and B 103 (X99260) and Streptococcus phage CP-1 (Z47794).
  • RNA bacteriophage MS2 interacts with viral RNA to translationally repress replicase synthesis (Pickett and Peabody, 1993). This protein-RNA interaction also plays a role in genome encapsidation, enveloping a single copy of the viral " genome in a protein shell composed of many molecules of coat protein.
  • the bacteriophage ⁇ terminase enzyme can be lethal to E.
  • bacteriophage 182 is also present within bacteriophage 182 that encodes a protein that is related to the terminal proteins of Bacillus phage Nf (P06812), Bacillus phage GA-1 (X96987) and Bacillus phage B103 (X99260). DNA terminal proteins are linked to the 5' ends of both strands of the genome and are essential for DNA replication playing a role in initial priming of DNA replication.
  • the similarity between Enterococcus bacteriophage 182 and Bacillus phages phi-29, PZA, and B103 indicates that they may share similar mechanisms of replication and growth.
  • Protein-primed DNA replication is a well described phenomenon, and in the phi-29-like phages, the ends of the DNA serve as origins and termini of replication (Gutierrez et al., 1986; Yoshikawa et al., 1985).
  • ORF 015 there is also a gene (ORF 015) that encodes a protein showing homology to an early protein product of Bacillus bacteriophage PZA and the single-strand nucleic acid binding protein of bacteriophage B103.
  • Two genes, ORF 008 and 014 were identified with the potential to encode anti-microbial protein products. The homology alignments are shown in Tables 24 & 26 and biochemical features of the predicted polypeptides shown in Table 25.
  • the predicted product of ORF 008 is related to a class of genes which encodes lysozyme- like functions, enzymes which cleave linkages in the mucopolysaccharide cell wall structure of a variety of micro-organisms.
  • ORF 014 of Enterococcus 182 shows homology to a set of lysis proteins from Bacillus bacteriophage phi-29, PZA, and B103. These lysis proteins are also referred to as holins and represent phage encoded lysis functions required for transit of the phage murein hydrolases (lysozyme) to the periplasm, where it can digest the outer cell wall and thus lyse the bacterium.
  • the present invention provides a nucleic acid sequence obtained from
  • Enterococcus bacteriophage 182 comprising at least a portion of a phage 182 ORF, preferably an inhibitory ORF, and more preferably at least a portion of one of the genes described above with anti-microbial activity.
  • ORF 002 encodes a DNA polymerase function. This polymerase may utilize host-derived accessory proteins for its activity when replicating the phage template, sequestering such proteins from use by the bacterial polymerase, resulting in inhibition of DNA replication, cell division, and cell growth.
  • ORFs 008 or 014 directly encode polypeptides with anti-microbial activity.
  • ORF 008 is predicted to encode an autolytic lysozyme, a protein known to have anti-microbial activity (Martin et al, 1998).
  • ORF 014 likely encodes a holin function required for transit of the phage murein hydrolases to the periplasm.
  • the present invention also provides the use of the Enterococcus bacteriophage
  • peptidomimetic compound structure has sufficient similarities to the structure of the active portion of a product of one of the Enterococcus ORFs listed, that the peptidomimetic will interact with the same molecule as the product of the ORF, and preferably will elicit at least one cellular response in common which relates to the inhibition of the cell by the phage protein.
  • ORF As a killer ORF, it is preferably expressed in the host or other test bacterial organism and the effect of this expression on bacterial growth and replication is assessed. Therefore, all individual ORFs identified herein, e.g., those identified above, can be expressed, preferably overexpressed, in a suitable host bacterium e.g., a host Enterococcus and the effect of this expression or overexpression on host metabolism and viability can be measured. _
  • ORFs can be resynthesized from the phage genomic DNA by the polymerase chain reaction (PCR) using oligonucleotide primers flanking the ORF on either side.
  • PCR polymerase chain reaction
  • oligonucleotide primers flanking the ORF on either side Those skilled in the art are familiar with the design and synthesis of appropriate primer sequences.
  • These single ORFs are preferably engineered so that they contain appropriate cloning sites at their extremities to allow their introduction into a new bacterial expression plasmid, allowing propagation in a standard bacterial host such as E. coli, but containing the necessary information for plasmid replication in the target microbe, Enterococcus sp. (hereafter referred to as a shuttle vector).
  • This shuttle vector also preferably contains regulatory sequences that allow inducible expression of the introduced ORF.
  • the candidate ORF may encode a killer function that will eliminate the host, it is highly advantageous that it not be expressed (or at least not expressed at a substantial level) prior to testing for activity; thus screening for such sequences in a constitutive fashion is less likely to be successful (lethality).
  • regulatory sequences from the ars operon are used to direct individual ORF expression in Enterococcus.
  • the ars operon encodes a series of proteins which normally mediate the extrusion of arsenite and several other trivalent oxyanions from the cells when they are exposed to such toxic substances in their environment.
  • the operon encoding this detoxifying mechanism is normally silent and only induced when arsenite-related compounds are present.
  • individual phage ORFs can be expressed in Enterococcus or other suitable host in an inducible fashion by adding to the culture medium non-toxic arsenite concentrations during the growth of individual Enterococcus (or other host cells) clones expressing such individual phage ORFs.
  • Toxicity of the phage killer ORF for the host is monitored by reduction or arrest of growth under induction conditions, as measured by optical density in liquid culture or after plating the induced cultures on solid medium. Subsequently, interference of the phage ORF with the host biochemical pathways ultimately leading to reducing or arresting host metabolism can be measured by pulse chase experiments using radiolabeled precursors of either DNA replication, RNA transcription, or protein synthesis.
  • inducible regulatory sequences e.g., promoters, operators, etc.
  • systems using positive induction of expression or systems using release of repression e.g., systems using positive induction of expression or systems using release of repression.
  • Nucleic acid sequences of the present invention can be isolated using a method similar to those described herein or other methods known to those skilled in the art.
  • such nucleic acid sequences can be chemically synthesized by well- known methods.
  • phage 182 ORFs e.g., anti-bacterial ORFs of the present invention, portions thereof, or oligonucleotides derived therefrom as described
  • other anti-microbial sequences from other bacteriophage sources can be identified and isolated using methods described here or other methods, including methods utilizing nucleic acid hybridization and/or computer-based sequence alignment methods.
  • the invention also provides bacteriophage anti-microbial DNA segments from other phages based on nucleic acids and sequences hybridizing to the presently identified inhibitory ORF under high stringency conditions or sequences which are highly homologous.
  • the bacteriophage anti-microbial DNA segment from bacteriophage 182 can be used to identify a related segment from another unrelated phage based on stringent conditions of hybridization or on being a homolog based on nucleic acid and/or amino acid sequence comparisons.
  • homologous coding sequences and products can be used as antimicrobials, to construct active portions or derivatives, to construct peptidomimetics, and to identify bacterial targets.
  • Enterococcus sequences are listed in Table 27 by accession number, providing identification of possible targets of Enterococcus phage inhibitory ORF products, e.g., from phage 182.
  • the present invention is concerned with the use of Streptococcus sp. bacteriophage Dp-1 coding sequences and the encoded polypeptides or RNA transcripts to identify bacterial targets for potential new antibacterial agents.
  • Streptococcus pneumoniae is an important cause of community-acquired pneumonia and a major cause of otitis media, sinusitis, and meningitis in children and adults.
  • S. pneumoniae In Spain and other Mediterranean countries, the majority of S. pneumoniae are relatively resistant to penicillin (Klugman, 1990; Fenoll et al., 1991; Jorgenserret al., 1990). These strains also have decreased susceptibility to broad-spectrum cephaloporins, which are frequently used in the empiric treatment of meningitis and other serious invasive bacterial infections. High-level resistance of pneumococci has been encountered in Hungary where 10% of children who were colonized with S.
  • pneumoniae carried penicillin resistant strains that were also resistant to tetracycline, erythromycin, trimethoprim/sulfamethoxazole, and 30% resistant to chloramphenicol (Neu, 1992).
  • the resistance of pneumococci to macrolides such as erythromycin averages 20-25% in France, -20% in Japan, and ⁇ 10% in Spain (Neu, 1992).
  • Pneumococcal phages belong to four families and they present a great variety in morphology, including lytic and temperate phages (for a review, see Garcia et al., 1997). Examples of lytic phages are C ⁇ -1 and Dp-1, whereas examples of temperate phages are HB-3, EJ-1, and HB-746. The complete nucleotide sequence and functional organization of Cp-1 has been reported (Martin et al., 1996). Cp-1 has a 19,345 bp double-stranded DNA genome, with a terminal protein covalently linked to its 5' ends, that replicates by a protein primed mechanism. The phage contains 29 ORFs, 23 on one strand and 6 on the opposite.
  • coli results in cell death after 2- hours of induction, but did not lead to lysis (Garcia et al., 1997).
  • Cells harboring a plasmid construction with holin and lysozyme genes together did lyse after induction and the viability loss was similar to that of the culture expressing holin alone.
  • Cloning of these lytic genes in S. pneumoniae showed that both genes had the same effect as in E. coli. That is, holin itself did not lyse the culture but the viability loss was noticeable, whereas both holin and lysozyme together were capable of lysing M31, an amidase deleted mutant (Garcia et al., 1997).
  • Dp-1 Bacteriophage Dp-1 was obtained from Dr. P. Garcia (Departamento de Microbiologia Molecular, Centro de Departamento de Investigaations Biologicas, Consejo Superior de Investigaations Cientificas, Velazquez, Madrid, Spain). We found that Dp-1 has a double-stranded DNA genome of 56,506 bp, predicted to encode 85 ORFs greater than 33 amino acids and with upstream Shine-Dalgarno motifs for translation initiation (Tables 28 & 30, and Fig. 6). Computational analysis of the predicted protein products of Streptococcus bacteriophage Dp-1 protein products, which detected homologs in public databases, are listed inTable 31, along with the accompanying list of related proteins.
  • ORFs 001, 002, 004, and 030 are predicted to encode tail proteins, minor structural proteins, and minor capsid proteins (Table 31).
  • ORF 3 which encodes DNA polymerase
  • ORF 8 which encodes a S WI/SNF helicase-related protein
  • ORF 10 encodes a protein showing homology to recA
  • ORF 13 encodes a dnaZX-like ORF.
  • RapA encodes an RNA polymerase (RNAP)-associated protein with -
  • RapA forms a stable complex with RNAP, as if it were a subunit of RNAP and it is possible that the ORF 8 product behaves similarly or in a dominant-negative fashion to inhibit the activity of RapA. Mutation of the essential E. coli dnaZX results in a block in DNA chain elongation during replication (Maki et al., 1988).
  • the dnaZX gene has only one open reading frame for a 71 -kDa polypeptide from which the two distinct DNA polymerase III holoenzyme subunits, tau (71 kDa) and gamma (47 kDa), are produced.
  • the tau subunit is the precursor of the gamma subunit, and the gamma subunit is produced by a -1 frameshift causing early termination of translation (Tsuchihashi et al., 1990).
  • These proteins show single-strand DNA binding properties that is ATPase (and dATPase) dependent and are thought to increasing the processivity of the core DNA polymerase enzyme (Lee et al., 1987).
  • ORFs 20, 29, 38 There are several Dp-1 ORFs which encode proteins predicted to play a role in cellular metabolic pathways. These include polypeptides involved in coenzyme PQQ synthesis (ORFs 20, 29, 38). Pyrrolo-quinoline quinone (PQQ) is the non-covalently bound prosthetic group of many quinoproteins catalysing reactions in the periplasm of Gram-negative bacteria. Most of these involve the oxidation of alcohols or aldose sugars. Interestingly, ORFs 20, 29, and 30 also show homology to the exoenzyme S regulon (Frank, 1997). Proteins encoded by the P.
  • PQQ Pyrrolo-quinoline quinone
  • aeruginosa exoenzyme S regulon may be involved in a contact-mediated translocation mechanism to transfer anti-host factors directly into eukaryotic cells disrupting eukaryotic signal transduction through ADP-ribosylation (Frank, 1997).
  • GTP cyclohydrolase I is an enzyme that catalyzes the first reaction in the pathway for the biosynthesis of the pteridine, a cofactor of the monooxygenases of the aromatic amino acids. Disruption of the homologous gene in Saccharomyces cerevisiae leads to a recessive conditional lethality due to folinic acid auxotrophy, that can be complemented with the mammalian or bacterial GTP cyclohydrolase I enzymes (Nardese et al., 1996; Mancini et al., 1999).
  • ORF 16 shows high homology to autolysin. This region of the phage sequence was previously reported (Sheehan et al., 1997) and encompasses ⁇ 4 kbp of our sequence. The sequence published by (Sheehan et al., 1997) is shown in Table 32.
  • the present invention provides a nucleic acid sequence obtained from
  • Streptococcus bacteriophage Dp-1 comprising at least a portion of a phage Dp-1 QRF; - preferably an inhibitory ORF, and more preferably at least a portion of one of the genes described above with anti-microbial activity.
  • ORF 013 encodes a protein with homology to the gamma subunit of DNA polymerase (dnaX gene). This protein may act in a dominant-negative fashion to sequester the host DNA polymerase for its own replication, thus inhibiting host DNA replication.
  • the dnaX gene product is essential for E. coli replication (Kodaira et al., 1983).
  • the bacterial target of a bacteriophage inhibitor ORF product e.g., an inhibitory protein or polypeptide
  • a Streptococcus nucleic acid coding sequence from a host bacterium for bacteriophage Dp-1.
  • possible target sequences are described herein by reference to sequence source sites.
  • the sequence encoding the target preferably corresponds to a Streptococcus nucleic acid sequence available from The Institute for Genomic Research (TIGR), or available from GenBank or other public database.
  • TIGR Streptococcus sequences are publicly available at The Institute for Genomics Research at URL: http://www.tigr.org
  • a target sequence corresponds to a Streptococcus pneumoniae coding sequences corresponding to a sequence listed in Table 33 herein. Sequences for other Streptococcal species are also available from TIGR and./or from GenBank. The listing in Table 33 describes Streptococcus sequences currently deposited in GenBank. Again, for the sake of brevity, the sequences are described by reference to the GenBank entries instead of being written out in full herein.
  • the complete sequence can be readily obtained by routine methods, e.g., by isolating a clone in a phage Dp-1 host Streptococcus sp. genomic library, and sequencing the clone insert to provide the relevant coding region.
  • the boundaries of the coding region can be identified by conventional sequence analysis and/or by expression in a bacterium in which the endogenous copy of the coding region has been inactivated and using subcloning to identify the functional start and stop codons for the coding region.
  • the sequence is preferably not contained in the sequence described in Sheehan et al., 1997 (Table 32).
  • a fifth step involves validating the identified phage inhibitor ORF by independent methods, and delineating further possible smaller segments of the ORFs that have inhibitory activity. Several methods exist to validate the role of the identified ORF as an inhibitor ORF.
  • One example utilizes the creation of a mutant variant of the phage ORF in which the candidate ORF carries a partial or complete loss-of-function mutation that is measurable as compared with the non-mutant ORF.
  • Comparison of the effects of expression of the loss of function mutant with the normal ORF provides confirmation of the identification of an inhibitor ORF where the loss-of-function mutant provides a measurably lower level of inhibition, preferably no inhibition.
  • the loss of function may be conditional, e.g., temperature sensitive.
  • This may be carried out by a variety of means, e.g., by exonuclease or PCR methodologies, and is used to determine if a relatively small segment of the ORF (i.e., the product of the ORF) still possesses inhibitory activity when isolated away from its native sequence. If so, a portion of the ORF encoding this "active portion" can be used as a template for the synthesis of novel anti-microbial agents and further allowing derivation of the peptide sequence, e.g., using modified peptides and or peptidomimetics.
  • the peptide backbone is transformed into a carbon-based hydrophobic structure that can retain inhibitor activity against the bacterium. This is done by standard medicinal chemistry methods, typically monitored by measuring growth inhibition of the various molecules in liquid cultures or on solid medium. These mimetics can also represent lead compounds for the development of novel antibiotics. Recently, a major effort has been undertaken by the pharmaceutical industry and their biotechnology partners for the sequencing of bacterial pathogen genomes. The rationale is that the systematic sequencing of the genome will identify all of the bacterial proteins and therefore this proteome will be the target for designing novel inhibitor antibiotics. Although systematic, this approach has several major problems.
  • the first is that analysis of primary amino acid sequences of bacterial proteins does not immediately reveal which protein will be essential for viability of the bacterium, and target validation is thus a major issue.
  • the second problem is one of redundancy, as several biochemical pathways are either structurally duplicated in bacteria (different iso forms of the same enzyme), or functionally duplicated by the presence of salvage pathways in the event of a metabolic block in one pathway (different nutritional conditions).
  • the third is that even a valid target may not be structurally or functionally amenable to inhibition by small molecules because of inaccessibility (sequestration of target).
  • the phages herein described have, over millions of years, evolved specific mechanisms to target such key biochemical pathways and proteins.
  • inhibition by phages has been elucidated (e.g., see ref. 3)
  • such bacterial targets are invariably rate-limiting in their respective biochemical pathways, are not redundant, and/or are readily accessible for inhibition by the phage (or by another inhibitory compound). Therefore, the sixth step of this invention involves identifying the host biochemical pathways and proteins that are targeted by the phage inhibitory mechanisms.
  • a rationale for this step is that the inhibitor ORF product from the phage physically interacts with and/or modifies certain microbial host components to block their function.
  • Exemplary approaches which can be used to identify the host bacterial pathways and proteins that interact with, and preferably also are inhibited by, phage ORF product(s) are described below.
  • One approach is a genetic screen to determine physiological protein:protein interaction, for example, using a yeast two hybrid system.
  • the phage ORF is fused to the carboxyl terminus of the yeast Gal4 activation domain II (amino acids 768-881) to create a bait vector.
  • a cDNA library of cloned S. aureus sequences which have been engineered into a plasmid where the S. aureus sequences are fused to the DNA binding domain of Gal4 is also generated. These plasmids are introduced alone, or in combination, into yeast strain Y190 - previously engineered with chromosomally integrated copies of the E.
  • coli lacZ and the selectable HIS3 genes both under Gal4 regulation (Durfee, T., Becherer, K., Chen, P.-L., Yeh, S.-H., Yang, Y., Kilburn, A.E., Lee, W.-H., and Elledge, S J. (1993). Genes & Dev. 1, 555-569). If the two proteins expressed in yeast interact, the resulting complex will activate transcription from promoters containing Gal4 binding sites.
  • a lacZ and His3 gene, each driven by a promoter containing Gal4 binding sites, have been integrated into the. . genome of the host yeast system used for measuring protein-protein interactions. Such a system provides a physiological environment in which to detect potential protein interactions.
  • the non-structural protein NS1 of parvovirus is essential for viral DNA amplification and gene expression and is also the major cytopathic effector of these viruses.
  • a yeast two-hybrid screen with NS 1 identified a novel cellular protein of unknown function that interacts with NS- 1 , called SGT, for small glutamine-rich tetratricopeptide repeat (TPR)-containing protein (Cziepluch C. Kordes E. Poirey R. Grewenig A. Rommelaere, J, and Jauniaux JC. (1998) J Virol. 72, 4149-4156).
  • TPR small glutamine-rich tetratricopeptide repeat
  • the adenovirus E3 protein was recently shown to interact with a novel tumor necrosis factor alpha-inducible protein and to modulate some of the activities of E3 (Li Y. Kang J. and Horwitz M.S. (1998). Mol & Cell Biol. 18, 1601-1610).
  • the herpes simplex virus 1 alpha regulatory protein ICP0 was found to interact with (and stabilize) the cell cycle regulator cyclin D3 (Kawaguchi Y. Van Sant C. and Roizman B. (1997). J Virol. 71,7328-7336).
  • STRATEGENETM CYTO-TRAPTM system
  • the system is a yeast-based method for detecting proteimprotein interactions in vivo, using activation of the Ras signal transduction cascade by localizing a signal pathway component, human Sos (hSos), to its activation site in the yeast plasma membrane.
  • the system uses a temperature-sensitive Saccharomyces cerevisiae mutant, strain cdc25H, which contains a point mutation at amino acid residue 1328 of the cdc25 gene.
  • This gene encodes a guanyl nucleotide exchange factor which binds and activates Ras, leading to cell growth.
  • the mutation in the cdc25 gene prevents host growth at 37°C, but at a permissive temperature of 25°C, growth is normal.
  • the system utilizes the ability of (hSos) to complement the cdc25 defect and activate Fhe yeast Ras signaling pathway.
  • (hSos) is expressed and localized to the plasma membrane, the cdc25H yeast strain grows at 37°C. Localizing hSos to the plasma membrane occurs through a protei protein interaction.
  • a protein of interest, or bait is expressed as a fusion protein with hSos.
  • the library, or target proteins are expressed with the myristylation membrane-localization signal.
  • the yeast cells are then incubated under restrictive conditions (37°C). If the bait and the target protein interact, the hSos protein is recruited to the membrane, activating the Ras signaling pathway and allowing the cdc25
  • the protein targets of phage inhibitory ORFs can also be identified using bacterial genetic screens.
  • One approach involves the overexpression of a phage inhibitory protein in mutagenized bacterial host species, followed by plating the cells and searching for colonies that can survive the antimicrobial activity of the inhibitory ORF. These colonies are then grown, their DNA extracted, and cloned into an expression vector that contains a replicon of a different incompatibility group from the plasmid expressing the original ORF.
  • This library is then introduced into a wild- type host bacterium in conjunction with an expression vector driving synthesis of the phage ORF, followed by selection for surviving bacteria.
  • bacterial DNA fragments from the survivors presumably contain a DNA fragment from the original mutagenized host bacterial genome that can protect the cell from the antimicrobial activity of the inhibitory phage ORF.
  • This fragment can be sequenced and compared with that of the bacterial host to determine in which gene the mutation lies. This approach enables one to determine the targets and pathways that are affected by the killing function.
  • a second approach is based on identifying proteimprotein interactions between the phage ORF product and bacterial S. aureus, e.g., proteins using a biochemical approach based, for example, on affinity chromatography.
  • This approach has been used, for example, to identify interactions between lambda phage proteins and proteins from their E. coli host (Sopta, M., Carthew, R.W., and Greenblatt, J. (1985) J. Biol. Chem. 260, 10353-10369).
  • the phage ORF is fused to a peptide tag (e.g.
  • GST glutathione-S-transferase
  • HIS 6xHIS
  • CPB calmodulin binding protein
  • Target proteins thus recovered should be enriched for the phage protein/peptide of interest and are subsequently electrophoretically or otherwise separated, purified, sequenced, or biochemically analyzed.
  • sequencing entails individual digestion of the proteins to completion with a protease (e.g.-trypsin), followed by molecular mass and amino acid composition and sequence determination using, for example, mass spectrometry, e.g., by MALDI-TOF technology (Qin, J., Fenyo, D., Zhao, Y., Hall, W.W., Chao, D.M., Wilson, C.J., Young, R.A. and Chait, B.T. (1997). Anal. Chem. 69, 3995-4001).
  • a protease e.g.-trypsin
  • the sequence of the individual peptides from a single protein are then analyzed by the bioinformatics approach described above to identify the S. aureus protein interacting with the phage ORF. This analysis is performed by a computer search of the S. aureus genome for an identified sequence. Alternatively, all tryptic peptide fragments of the S. aureus genome can be predicted by computer software, and the molecular mass of such fragments compared to the molecular mass of the peptides obtained from each interacting protein eluted from the affinity matrix.
  • the responsible gene sequence can be obtained, for example by using synthetic degenerate nucleic acid sequences to pull out the corresponding homologous bacterial sequence.
  • antibodies can be generated against the peptide and used to isolate nascent peptide/mRNA transcript complexes, from which the mRNA can be reverse transcribed, cloned, and further characterized using the procedures discussed herein.
  • a variety of other binding assay methods are known in the art and can be used to identify interactions between phage proteins and bacterial proteins or other bacterial cell components. Such methods that allow or provide identification of the bacterial component can be used in this invention for identifying putative targets.
  • Validation of the interaction between the phage ORF product and the bacterial proteins or other components can be obtained by a second independent assay (e.g., co-immunoprecipitation or protein-protein crosslinking experiments (Qiu, H., Garcia- Barrio, M.T., and Hinnebusch, A.G. (1998). Mol & Cell Biology 18, 2697-2711 ; Brown, S. and Blumenthal, T. (1976). Proc. Natl. Acad. Sci. USA 73, 1131-1135)).
  • the essential nature of the identified bacterial proteins is preferably determined genetically by creating a constitutive or inducible partial or complete loss- of-function mutation in the gene encoding the identified interacting bacterial protein. This mutant is then tested for bacterial survival and replication.
  • the protein target of the phage inhibitor function can also be identified using a. _ genetic approach.
  • Two exemplary approaches will be delineated here.
  • the first ⁇ approach involves the overexpression of a predetermined phage inhibitor protein in mutagenized host bacteria, e.g., S. aureus, followed by plating the cells and searching for colonies that can survive the inhibitor. These colonies will then be grown, their DNA extracted and cloned into an expression vector that contains a replicon of a different incompatibility group, and preferably having a different selectible marker than the plasmid expressing the phage inhibitor.
  • host DNA fragments from the mutant that can protect the cell from phage ORF inhibition can be sequenced and compared with that of the bacterial host to determine in which gene the mutation lies. This approach allows rapid determination of the targets and pathways that are affected by the inhibitor.
  • the bacterial targets can be determined in the absence of selecting for mutations using an approach known as "multicopy suppression".
  • multicopy suppression the DNA from the wild type host is cloned into an expression vector that can coexist, as previously described, with one containing a predetermined phage inhibitor.
  • Those plasmids that contain host DNA fragments and genes that protect the host from the phage inhibitor can then be isolated and sequenced to identify putative targets and pathways in the host bacteria.
  • screening assays may additionally utilize gene fusions to specific "reporter genes" to identify a bacterial gene(s) whose expression is affected when the host target pathway is affected by the phage inhibitor.
  • gene fusions can be used to search a number of small molecule compounds for inhibitors that may affect this pathway and thus cause cell inhibition.
  • This approach will allow the screening of a large number of molecules on petri dishes or 96-well format by monitoring for a simple color change in the bacterial colonies. In this manner, we can validate host targets and classes of compounds for further study and clinical development. These inhibitors also represent lead compounds for the development of other antibiotics.
  • Bioinformatics and comparative genomics are preferably then applied to the identified bacterial gene products to predict biochemical function.
  • the biochemical activity of the protein can be verified in vitro in cell free assays or in vivo in intact cells.
  • In vitro biochemical assays utilizing cell-free extracts or purified protein are established as a basis for the screening and development of inhibitors.
  • inhibitors may comprise peptides, antibodies, products from natural sources such as fungal or plant extracts or small molecule organic compounds.
  • small molecule organic compounds are preferred.
  • These compounds may, for example, be identified within large compound libraries, including combinatorial libraries. For example, a plurality of compounds, preferably a large number of compounds can be screened to determine whether any of the compounds binds or otherwise disrupts or inhibits the identified bacterial target. Compounds identified as having any of these activities can then be evaluated further in cell culture and/or animal model systems to determine the pharmacological properties of the compound, including the specific anti-microbial ability of the compound.
  • the active substance can be isolated and identified using techniques well known in the art, if the compound is not already available in a purified form.
  • Identified compounds possessing anti-microbial activity and similar compounds having structural similarity can be further evaluated and, if necessary, derivatized according to synthesis and/or modification methods available in the art selected as appropriate for the particular starting molecule.
  • the in vivo effectiveness of such compounds may be advantageously enhanced by chemical modification using the natural polypeptide as a starting point and incorporating changes that provide advantages for use, for example, increased stability to proteolytic degradation, reduced antigenicity, improved tissue penetration, and/or improved delivery characteristics.
  • inactive modifications or derivatives for use as negative controls or introduction of immunologic tolerance.
  • a biologically inactive derivative which has essentially the same epitopes as the corresponding natural antimicrobial can be used to induce immunological tolerance in a patient being treated. The induction of tolerance can then allow uninterrupted treatment with the active anti-microbial to continue for a significantly longer period of time.
  • Modified anti-microbial polypeptides and derivatives can be produced using a number of different types of modifications to the amino acid chain. Many such methods are known to those skilled in the art. The changes can include, for example, reduction of the size of the molecule, and/or the modification of the amino acid sequence of the molecule. In addition, a variety of different chemical modifications of the naturally occurring polypeptide can be used, either with or without modifications to the amino acid sequence or size of the molecule. Such chemical modifications can, for example, include the inco ⁇ oration of modified or non-natural amino acids or ⁇ i ⁇ n- amino acid moieties during synthesis of the peptide chain, or the post-synthesis modification of inco ⁇ orated chain moieties.
  • the oligopeptides of this invention can be synthesized chemically or through an appropriate gene expression system. Synthetic peptides can include both naturally occurring amino acids and laboratory synthesized, modified amino acids.
  • a functional derivative retains at least a portion of the function of the protein, for example reactivity with a specific antibody, enzymatic activity or binding activity.
  • a “chemical derivative” of the complex contains additional chemical moieties not normally a part of the protein or peptide. Such moieties may improve the molecule's solubility, abso ⁇ tion, biological half-life, and the like. The moieties may alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, and the like. Moieties capable of mediating such effects are disclosed in Alfonso and Gennaro (1995).
  • Cysteinyl residues most commonly are reacted with alpha-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, chloroacetyl phosphate, N- alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloro- mercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-l,3- diazole.
  • Histidyl residues are derivatized by reaction with diethylprocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain.
  • Para- bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.
  • Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues.
  • Other suitable reagents for derivatizing primary amine- containing residues include imidoesters such as methyl _ picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase- catalyzed reaction with glyoxylate.
  • Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high p , of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine alpha-amino group.
  • Tyrosyl residues are well-known targets of modification for introduction of spectral labels by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizol and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.
  • Carboxyl side groups are selectively modified by reaction carbodiimide (R'-N-C-N-R') such as l-cyclohexyl-3-(2-mo ⁇ holinyl(4-ethyl) carbodiimide or l-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.
  • carbodiimide R'-N-C-N-R'
  • aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
  • Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention.
  • Derivatization with bifunctional agents is useful, for example, for cross- linking component peptides to each other or the complex to a water-insoluble support matrix or to other macromolecular carriers.
  • Commonly used cross-linking agents include, for example, 1,1 -bis (diazoacetyl)-2-phenylethane, glutaraldehyde, N- hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobi- functional imidoesters, including disuccinimidyl esters such as 3,3'- dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N- maleimido-l,8-octane.
  • Derivatizing agents such as methyl-3-[p-azidophenyl) dithiolpropioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light.
  • reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Patent Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization.
  • Such derivatized moieties may improve the stability, solubility, abso ⁇ tion, biological half life, and the like.
  • the moieties may alternatively eliminate or attenuate any undesirable side effect of the protein complex.
  • Moieties capable of mediating such effects are disclosed, for example, in Alfonso and Gennaro (1995).
  • fragment is used to indicate a polypeptide derived from the amino acid sequence of the protein or polypeptide having a length less than the full-length polypeptide from which it has been derived.
  • a fragment may, for example, be produced by proteolytic cleavage of the full-length protein.
  • the fragment is obtained recombinantly by appropriately modifying the DNA sequence encoding the proteins to delete one or more amino acids at one or more sites of the C-terminus, N-terminus, and or within the native sequence.
  • Another functional derivative intended to be within the scope of the present invention is a "variant" polypeptide that either lacks one or more amino acids or contains additional or substituted amino acids relative to the native polypeptide.
  • the variant may be derived from a naturally occurring polypeptide by appropriately modifying the protein DNA coding sequence to add, remove, and/or to modify codons for one or more amino acids at one or more sites of the C-terminus, N-terminus, and/or within the native sequence.
  • a functional derivative of a protein or polypeptide with deleted, inserted and/or substituted amino acid residues may be prepared using standard techniques well-known to those of ordinary skill in the art.
  • the modified components of the functional derivatives may be produced using site-directed mutagenesis techniques (as exemplified by Adelman et al., 1983, DNA 2:183; Sambrook et al., 1989) wherein nucleotides in the DNA coding sequence are modified such that a modified coding sequence is produced, and thereafter expressing this recombinant DNA in a prokaryotic or eukaryotic host cell, using techniques such as those described above.
  • components of functional derivatives of complexes with amino acid deletions, insertions and/or substitutions may be conveniently prepared by direct chemical synthesis, using methods well-known in the art.
  • the preferred method of preparation or administration of anti-microbial compounds will generally vary depending on the precise identity and nature of the anti-microbial being delivered. Thus, those skilled in the art will understand that administration methods known in the art will also be appropriate for the compounds of this invention.
  • the particularly desired anti-microbial can be administered to a patient either by itself, or in pharmaceutical compositions where it is mixed with suitable carriers or excipient(s).
  • a therapeutically effective amount of an agent or agents is administered.
  • a therapeutically effective dose refers to that amount of the compound that results in amelioration of one or more symptoms of bacterial infection and or a prolongation of patient survival or patient comfort.
  • Toxicity, therapeutic and prophylactic efficacy of anti-microbials can be determined by standard pharmaceutical procedures in cell cultures and/or experimental organisms such as animals, e.g., for determining the LD 50 (the dose lethal to 50%> of the population) and the ED 50 (the dose therapeutically effective in 50%) of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD J0 /ED 50 .
  • Compounds that exhibit large therapeutic indices are preferred.
  • the data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays. Such information can be used to more accurately determine useful doses in organisms such as plants and animals, preferably mammals, and most preferably humans. Levels in plasma may be measured, for example, by HPLC or other means appropriate for detection of the particular compound.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (see e.g. Fingl et. al., in The Pharmacological Basis of Therapeutics, 1975, Ch. 1 p.l). It should be noted that the attending physician would know how ' and when to terminate, interrupt, or adjust administration due to toxicity, organ dysfunction, or other systemic malady. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity).
  • the magnitude of an administered dose in the management of the disorder of interest will vary with the severity of the condition to be treated and the route of administration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above also may be used in veterinary or phyto medicine.
  • agents may be formulated and administered systemically or locally, i.e., topically.
  • Techniques for formulation and administration may be found in Alfonso and Gennaro (1995). Suitable routes may include , for example, oral, rectal, transdermal, vaginal, transmucosal, intestinal, parenteral, intramuscular, subcutaneous, or intramedullary injections, as well as intrathecal, intravenous, or intraperitoneal injections.
  • the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art.
  • Use of pharmaceutically acceptable carriers to formulate identified antimicrobials of the present invention into dosages suitable for systemic administration is within the scope of the invention.
  • the compositions of the present invention in particular those formulated as solutions, may be administered parenterally, such as by intravenous injection.
  • Appropriate compounds can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration.
  • Such carriers enable the compounds of the invention to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • Agents intended to be administered intracellularly may be administered using techniques well known to those of ordinary skill in the art. For example, such agents may be encapsulated into liposomes, then administered as described above. Liposomes are spherical lipid bilayers with aqueous interiors. All molecules present in an aqueous solution at the time of liposome formation are inco ⁇ orated into the aqueous interior. The liposomal contents are both protected from the external microenvironment and, because liposomes fuse with cell membranes, are efficiently delivered into the cell cytoplasm. Additionally, due to their hydrophobicity, small organic molecules may be directly administered intracellularly.
  • compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended pu ⁇ ose. Determination of the effective amounts is well within the capability of those skilled in the art.
  • these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • the preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions, including those formulated for delayed release or only to be released when the pharmaceutical reaches the small or large intestine.
  • compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levitating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for parenteral administration include aqueous solutions of the active anti-microbial compounds in water-soluble form.
  • suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • compositions for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
  • fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol
  • cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropyl
  • -, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this pu ⁇ ose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. The above methodologies may be employed either actively or prophylactically against an infection of interest.
  • nucleotide sequences, or fragments thereof at least 95%, preferably at least 97%, more preferably at least 99%, and most preferably at least 99.9%> identical to phage inhibitor sequences can also be provided in a variety of additional media to facilitate various uses.
  • nucleotide sequence of the present invention e.g., a nucleotide sequence of an exemplary bacteriophage or a sequence encoding a bacterial target or a fragment thereof, preferably a nucleotide sequence at least 95%, more preferably at least 99%> and most preferably at least 99.9%o identical to such a bacteriophage or bacterial sequence, for example, to a polynucleotide of an unsequenced phage listed in Table 1, preferably of bacteriophage 77 (S.
  • bacteriophage host or bacteriophage 3A (S.aureus host) or bacteriophage 96 (S. aureus host).
  • ORF open reading frame
  • Such an article provides a large portion of the particular bacteriophage genome or bacterial gene and parts thereof (e. ., a bacteriophage open reading frame (ORF)) in a form which allows a skilled artisan to examine and/or analyze the sequence using means not directly applicable to examining the actual genome or gene. _ or subset thereof as it exists in nature or in purified form as a chemical entity.
  • a nucleotide sequence of the present invention can be recorded on computer readable media.
  • computer readable media refers to any medium that can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories, such as magnetic/optical storage media.
  • magnetic storage media such as floppy discs, hard disc storage medium, magnetic tape
  • optical storage media such as CD-ROM
  • electrical storage media such as RAM and ROM
  • hybrids of these categories such as magnetic/optical storage media.
  • recorded refers to a process for storing information on computer readable medium.
  • a skilled artisan can readily adopt any of the presently known methods for recording information on computer readable medium to generate manufactures comprising the nucleotide sequence information of the present invention.
  • a variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon a nucleotide sequence of the present invention.
  • the choice of the data storage structure will generally be based on the means chosen to access the stored information.
  • a variety of data processor programs and formats can be used to store the nucleotide sequence information of the present invention on computer readable medium.
  • the sequence information can, for example, be presented in a word processing test file, formatted in commercially available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like.
  • a skilled artisan can readily adapt any number of data processor structuring formats (e.g., text file or database) in order to obtain computer readable medium having recorded thereon the nucleotide sequence information of the present invention.
  • Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium.
  • nucleotide sequence of an unsequenced bacteriophage such as an exemplary bacteriophage listed in Table 1 or of a sequence encoding a bacterial target or a fragment thereof, preferably a nucleotide sequence at - - least 95%>, more preferably at least 99%> and most preferably at least 99.9%> identical to such a bacteriophage or bacterial sequence, for example, to a polynucleotide of bacteriophage 77 (S. aureus host) or bacteriophage 3A (S.aureus host) bacteriophage 96 (S. aureus host), bacteriophage 44 AHJD (S.
  • bacteriophage Dp-1 Streptococcus pneumoniae host
  • bacteriophage 182 Enterococcus host
  • software can implement a variety of different search or analysis software which implement sequence search and analysis algorithms, e.g., the BLAST (Altschul et al., J. Mol. Biol. 215:403410 (1990) and BLAZE (Brutlag et al., Comp. Chem 17:203-207 (1993)) search algorithms.
  • such search algorithms can be implemented on a Sybase system and used to identify open reading frames (ORFs) within the bacteriophage genome which contain homology to ORFs or proteins from other viruses, e.g, other bacteriophage, and other organisms, e.g., the host bacterium.
  • ORFs open reading frames
  • the ORFs discussed herein are protein encoding fragments of the bacteriophage genomes which encode bacteria-inhibiting proteins or fragments.
  • the present invention further provides systems, particularly computer-based systems, which contain the sequence information described. Such systems are designed to identify, among other things, useful fragments of the bacteriophage genomes.
  • a computer-based system refers to the hardware, software, and data storage media used to analyze the nucleotide sequence information of the present invention.
  • the minimum hardware of the computer-based systems of the present invention comprises a central processing unit (CPU), input device, output device, and data storage medium or media.
  • CPU central processing unit
  • input device input device
  • output device output device
  • data storage medium or media data storage medium
  • the computer-based systems of the present invention comprise data storage media having stored therein a nucleotide sequence of the present invention and the necessary hardware and software for supporting and implementing a search and/or analysis program.
  • data storage media refers to memory which can store nucleotide sequence information of the present invention, or a memory access means which can access manufactures having recorded thereon the nucleotide sequence information of the present invention.
  • search program refers to one or more programs which are implemented on the computer-based system to compare a target sequence or target structural motif with the sequence information stored within the data storage means. Search means are used to identify fragments or regions of the present gnomic sequences which match a particular target sequence or target motif.
  • search program refers to one or more programs which are implemented on the computer-based system to compare a target sequence or target structural motif with the sequence information stored within the data storage means. Search means are used to identify fragments or regions of the present gnomic sequences which match a particular target sequence or target motif.
  • a variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems of the present invention.
  • a target sequence can be any DNA or amino acid sequence of six or more nucleotides or two or more amino acids. A skilled artisan can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occurrence in the database.
  • the target sequence length is preferably selected to include sequence corresponding to a biologically relevant portion of an encoded product, for example a region which is expected to be conserved across a range of source organisms.
  • sequence length of a target polypeptide sequence is from 5- 100 amino acids, more preferably 7-50 or 7-100 amino acids, and still more preferably 10-80 or 10-100 amino acids.
  • sequence length of a target polynucleotide sequence is from 15-300 nucleotide residues, more preferably from 21- 240 or 21-300, and still more preferably 30-150 or 30-300 nucleotide residues.
  • searches for commercially important fragments such as sequence fragments involved in gene expression and protein processing, may be of shorter length. Likewise, it may be desirable to search and/or analyze longer sequences.
  • a target structural motif refers to any rationally selected sequence or combination of sequences in which the sequence(s) are chosen based on a three-dimensional configuration which is formed upon the folding of the target motif.
  • target motifs include, but are not limited to, enzymatic active sites and signal sequences.
  • Nucleic acid target motifs include, but are not limited to promoter sequences, hai ⁇ in structures and inducible expression elements (protein binding sequences).
  • a variety of structural formats for the input and output devices can be used to_ input and output the information in the computer-based systems of the preser_r invention.
  • a preferred format for an output device ranks fragments of the bacteriophage or bacterial sequences possessing varying degrees of homology to the target sequence or target motif. Such presentation provides a skilled artisan with a ranking of sequences which contain various amounts of the target sequence or target motif and identifies the degree of homology contained in the identified fragment.
  • FIG. 6 provides a block diagram of a computer system illustrative of embodiments of this aspect of present invention.
  • the computer system 102 includes a processor 106 connected to a bus 104.
  • main memory 108 preferably implemented as random access memory, RAM
  • secondary storage devices 110 such as a hard drive 112 and a removable medium storage device 114.
  • the removable medium storage device 114 may represent, for example, a floppy disk drive, a CD-ROM drive, a magnetic tape drive, etc.
  • a removable storage medium 116 (such as a floppy disk, a compact disk, a magnetic tape, etc.) containing control logic and/or data recorded therein may be inserted into the removable medium storage device 114.
  • the computer system 102 includes appropriate software for reading the control logic and/or the data from the removable medium storage device 114, once it is inserted into the removable medium storage device 114.
  • a nucleotide sequence of the present invention may be stored in a well-known manner in the main memory 108, any of the secondary storage devices 110, and/or a removable storage medium 116.
  • software for accessing and processing the sequence (such as search tools, comparing tools, etc.) reside in main memory 108, in accordance with the requirements and operating parameters of the operating system, the hardware system and the software program or programs.
  • the data storage medium in which the sequence is embodied and the central processor need not be part of a single stand-alone computer, but may be separated so long as data transfer can occur.
  • the processor or processors being utilized for a search or analysis can be part of one general pu ⁇ ose computer, and the data storage medium can be part of a second general pu ⁇ ose computer connected to_a_ network, or the data storage medium can be part of a network server.
  • the data storage medium can be part of a computer system or network accessible over telephone lines or other remote connection method.
  • Example 1 Growth of Staph A bacteriophage 77 and purification of genomic DNA.
  • the Staphylococcus aureus propagating strain (PS 77; ATCC #27699) was used as a host to propagate its respective phage 77 (ATCC # 27699-B1).
  • Two rounds of plaque purification of phage 77 were performed on soft agar essentially as described in Sambrook et al (1989).
  • phage 77 was subjected to 10-fold serial dilutions using phage buffer (1 mM MgSO 4 , 5 mM MgCl 2 , 80 mM NaCl and 0.1% Gelatin (w/v)) and 10 ⁇ l of each dilution was used to infect 0.5 ml of the cell suspension in the presence of 400 ⁇ g/ml CaCl 2 .
  • 7.5 ml of melted soft agar (NB plus 0.6%> agar) were added to the mixture and poured onto the surface of 150 mm nutrient agar plates and incubated 16 hrs at 30°C.
  • 20 ml of NB were added to each plate and the soft agar layer was collected by scrapping off with a clean microscope slide followed by shaking of the agar suspension for 5 min to break up the agar.
  • the mixture was then centrifuged for 10 min at 4,000 RPM (2,830xg) in a JA-10 rotor- * ' (Beckman) and the supernatant fluid (lysate) was collected and subjected to ⁇ a treatment with 10 ⁇ g /ml of DNase I and RNase A for 30 min at 37°C.
  • the phage suspension was adjusted to 10%> (w/v) PEG 8000 and 0.5 M of NaCl followed by incubation at 4°C for 16 hrs.
  • the phage was recovered by centrifugation at 4,000 ⁇ m (3,500xg) for 20 min at 4°C on a GS-6R table top centrifuge (Beckman).
  • the pellet was resuspended with 2 ml of phage buffer (1 mM MgSO 4 , 5 mM MgCl 2 , 80 mM NaCl and 0.1 %> Gelatin).
  • the phage suspension was extracted with 1 volume of chloroform and further purified by centrifugation on a cesium chloride step gradient as described in Sambrook et al. (1989), using a TLS 55 rotor centrifuged in an Optima TLX ultracentrifuge (Beckman) for 2 h at 28,000 ⁇ m (67,000xg) at 4°C.
  • Phage DNA was prepared from the phage suspension by adding 20 mM EDTA, 50 mg/ml Proteinase K and 0.5% SDS and incubating for 1 h at 65°C, followed by successive extractions with 1 volume of phenol, 1 volume of phenol-chloroform and 1 volume of chloroform. The DNA was then dialyzed overnight at 4°C against 4 L of TE (10 mM Tris pH 8.0, lmM EDTA).
  • phage 77 DNA was diluted in 200 ⁇ l of TE (10 mM Tris, [pH 8.0], 1 mM EDTA) in a 1.5 ml eppendorf tube and sonication was performed (550 Sonic DismembratorTM, Fisher Scientific). Samples were sonicated under an amplitude of 3 ⁇ m with bursts of 5 s spaced by 15 s cooling in ice/water for 3 to 4 cycles. The sonicated DNA was then size fractionated by electrophoresis on 1%> agarose gels utilizing TAE (1 x TAE is: 40 mM Tris-acetate, 1 mM EDTA [pH 8.0]) as the running buffer.
  • TE 10 mM Tris, [pH 8.0], 1 mM EDTA
  • Fractions ranging from 1 to 2 kbp were excised from the agarose gel and purified using a commercial DNA extraction system according to the instructions of the manufacturer (Qiagen), with a final elution of 50 ⁇ l of 1 mM Tris (pH 8.5).
  • the ends of the sonicated DNA fragments were repaired with a combination of T4 DNA polymerase and the Klenow fragment of E. coli DNA polymerase I, as follows. Reactions were performed in a reaction mixture (final volume, 100 ⁇ l) containing sonicated phage DNA, 10 mM Tris-HCl [pH 8.0], 50 mM NaCl, 10 mM MgCl 2 , 1 mM DTT, 50 ⁇ g/ml BSA, 100 ⁇ M of each dNTP and 15 units of T4 DNA polymerase (New England Biolabs) for 20 min at 12°C followed by addition of 12.5 units of Klenow large fragment (New England Biolabs) for 15 min at room- temperature. The reaction was stopped by two phenol chloroform extractions and the DNA was precipitated with ethanol and the final DNA pellet was resuspended in 20 ⁇ l of H 2 O.
  • a typical ligation reaction contained 100 ng of vector DNA, 2 to 5 ⁇ l of repaired sonicated phage DNA (50-100 ng) in a final volume of 20 ⁇ l containing 800 units of T4 DNA ligase (New England Biolabs) and was incubated overnight at 16°C. Transformation and selection of bacterial clones containing recombinant plasmids was performed in E. coli DHlO ⁇ according to standard procedures (Sambrook et al., 1989).
  • Recombinant clones were picked from agar plates into 96-well plates containing 100 ⁇ l LB and 100 ⁇ g/ml ampicillin and incubated at 37°C.
  • the presence of phage DNA insert was confirmed by PCR amplification using T3 and T7 primers flanking the Hinc II cloning site of the pKS 11+ vector.
  • PCR amplification of foreign insert was performed in a 15 ⁇ l reaction volume containing 10 mM Tris (pH 8.3), 50 mM KCl, 1.5 mM MgCl 2 , 0.02% gelatin, 1 ⁇ M primer, 187.5 ⁇ M each dNTP, and 0.75 units Taq polymerase (BRL).
  • thermocycling parameters were as follows: 2 min initial denaturation at 94°C for 2 min, followed by 20 cycles of 30 sec denaturation at 94°C, 30 sec annealing at 57°C, and 2 min extension at 72°C, followed by a single extension step at 72°C for 10 min.
  • Clones with insert sizes of 1 to 2 kbp were selected and plasmid DNA was prepared from the selected clones using QIAprepTM spin miniprep kit (Qiagen).
  • the nucleotide sequence of the extremities of each recombinant clone was determined using an ABI 377-36 automated sequencer with two types of chemistry: ABI prism Big DyeTM primer or ABI prism Big DyeTM terminator cycle sequencing ready reaction kit (Applied Biosystems). To ensure co-linearity of the sequence data and the genome, all regions of phage genome were sequenced at least once from both directions on two separate clones. In areas that this criteria was not initially met, a sequencing primer was selected and phage DNA was used directly as sequencing template employing ABI prism Big DyeTM terminator cycle sequencing ready reaction kit.
  • Phage 77 sequence contigs were assembled using SequencherTM 3.1 software (GeneCodes). To close contig gaps, sequencing primers were selected near the edge of the contigs. Phage DNA was used directly as sequencing template employing ABI prism BIG DYETM terminator cycle sequencing ready reaction kit. The complete sequence of bacteriophage 77 is shown in Table 2.
  • a software program was developed and used on the assembled sequence of bacteriophage 77 to identify all putative ORFs larger than 33 codons.
  • Other ORF identification software can also be utilized, preferably programs which allow alternative start codons.
  • the software scans the primary nucleotide sequence starting at nucleotide #1 for an appropriate start codon. Three possible selections can be made for defining the nature of the start codon; I) selection of ATG, II) selection of ATG or GTG, and III) selection of either ATG, GTG, TTG, CTG, ATT, ATC, and ATA.
  • a counting mechanism is employed to count the number of codons (groups of three nucleotides) between this start codon and the next stop codon downstream of it. If a threshold value of 33 is reached, or exceeded, then the sequence encompassed by these two codons (start and stop codons) is defined as an ORF. This procedure is repeated, each time starting at the next nucleotide following the previous stop codon found, in order to identify all the other putative ORFs. The scan is performed on all three reading frames of both DNA strands of the phage sequence.
  • Sequence homology (BLAST) searches for each ORF are then carried out using an implementation of BLAST programs, although any of a variety of different sequence comparison and matching programs can be utilized as known to those skilled in the art.
  • Downloaded public databases used for sequence analysis include: i) non-redundant GenBank (ftp://ncbi.nlm.nih.gOv/blast/db/nr.Z), ii) Swissprot (ftp://ncbi.nlm.nih.gOv/blast/db/swissprot.Z); iii) vector (ftp://ncbi.nlm.nih.gOv/blast/db/vector.Z); iv) pdbaa databases (ftp://ncbi.nlm.nih.gOv/blast/db/pdbaa.Z); v) S.
  • Example 4 Subcloning of Bacteriophage 77 ORFs into a Staph A inducible expression system.
  • the shuttle vector pT0021 in which the firefly luciferase (lucFF) expression is controlled by the ars (arsenite) promoter/operator (Tauriainen et al., 1997), was modified in the following fashion.
  • Two oligonucleotides corresponding to a short antigenic peptide derived from the heamaglutinin protein of influenza virus (HA epitope tag) were synthesized (Field et al., 1988).
  • the sense strand HA tag sequence (with BamHI, Sail and Hindlll cloning sites) is:
  • 5 '-gatcccggtcgaccaagcttTACCCATACGACGTCCCAGACTACGCCAGCTGA-3 ' (where upper case letters denote the nucletotide sequence of the HA tag);
  • the antisense strand HA tag sequence (with a Hindlll cloning site) is: 5 '-agctTCAGCTGGCGTAGTCTGGGACGTCGTATGGGTAaagcttggtcgaccgg-3 ' (where upper case letters denote the sequence of the HA tag).
  • the two HA tag oligonucleotides were annealed and ligated into pT0021 vector which had been digested with BamHI and Hindlll.
  • This manipulation resulted in replacement of the lucFF gene by the HA tag.
  • This modified shuttle vector containing the arsenite inducible promoter, the arsR gene, and HA tag was named pTHA.
  • a diagram outlining our modification of pT0021 to generate pTHA is shown in Fig. 1A.
  • Each ORF, encoded by Bacteriophage 77, larger than 33 amino acids and having a Shine-Dalgarno sequence upstream of the initiation codon was selected for functional analysis for bacterial inhibition. In total, 98 ORFs were selected and screened as detailed below. A list of these is presented in Table 3.
  • Each individual ORF, from initiation codon to last codon (excluding the stop codon), was amplified from phage genomic DNA using the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • each sense strand primer targets the initiation codon and is preceded by a BamHI restriction site ( 5 cgggatcc 3' .
  • each antisense oligonucleotide targets the pentultimate codon (the one before the stop codon) of the ORF and is preceded by a Sal I restriction site ' gcgtcgaccg 3 ).
  • the PCR product of each ORF was gel purified and digested with BamHI and Sail.
  • the digested PCR product was then gel purified using the Qiagen kit as described, ligated into BamHI and Sail digested pTHA vector, and used to transform E. coli bacterial strain DH10 ⁇ (as described _ - - above).
  • the HA tag is set inframe with the ORF and is positioned at the carboxy terminus of each ORF (pTHA ORF clones).
  • Recombinant pTHA/ORF clones were picked and their insert sizes were confirmed by PCR analysis using primers flanking the cloning site.
  • the names and sequences of the primers that were used for the PCR amplification were: HAF:
  • Staphylococcus aureus strain RN4220 (Kreiswirth et al., 1983) was used as a recipient for the expression of recombinant plasmids. Electoporation was performed essentially as previously described (Schenk and Laddaga, 1992). Selection of recombinant clones was performed on Luria-Broth agar (LB-agar) plates containing 30 ⁇ g/ml of kanamycin.
  • the anti-microbial activity of individual phage 77 ORFs was monitored by two growth inhibitory assays, one on solid agar medium, the other in liquid medium.
  • Staphylococcus bacteria transformed with expression plasmids containing individual ORFs were grown in normal TSA medium and stored in 19% glycerol.
  • arsenite was added to the culture to induce transcription of the phage 77 ORFs cloned immediately downstream from an arsenite-inducible promoter in the pTHA expression plasmid.
  • the effect of ORF induction on bacterial growth characteristics was then monitored and quantitated.
  • the growth inhibition assay on solid medium was performed by streaking pTHA ORF containing S. aureus transformant onto LB-Kn and TSA-Kn plates containing increasing concentrations of sodium arsenite (0; 2.5; 5; and 7.5 ⁇ M).
  • Arsenite is used to induce the expression of cloned DNA in pTHA vector.
  • 3 ⁇ l of 1/10 and 1/100 dilutions of the frozen cultures of the pTHA/ORF transformants were spotted as single drops onto LB-Kn and TSA-Kn plates containing increasing concentration of sodium arsenite (0; 2.5; 5; and 7.5 ⁇ M).
  • the plates were then incubated 16 hrs at 37°C, and the effect of arsenite-induced ORF expression on bacterial growth was monitored and quantitated by comparing the extent to that seen in control plates.
  • the holin/lysin genes of the Sthaphylococcus aureus phage Twort was subcloned into the pTHA ars inducible vector and used.
  • stationary phase cultures were prepared by inoculating 2.5ml TSB-Kn with frozen S. aureus RN4220 transformants containing phage 77 ORFs cloned in pTHA vector followed by incubation for 16 hrs at 37°C. These cultures were then diluted 1/100 in the same medium, and the bacteria were allowed to grow for 2 hrs at 37°C to reach early log phase. 150 ⁇ l of such culture were then mixed with 2.35 ml TSB-Kn medium with or without arsenite (the final concentration of arsenite in the medium was 0 or 5 ⁇ M arsenite).
  • Example 6 Itentification of Cecropin Signature Motif in Staphylococcus aureus Bacteriophage 3A ORF
  • the genome for S. aureus bacteriophage 3A was determined and the sequence was analyzed essentially as described for bacteriophage 77 in the examples above.
  • Upon blast analysis of the identified open reading frames of phage 3A the presence of an amino acid sequence corresponding to a cecropin signature motif was observed. This motif (WDGHKTLEK) is located at position aa 481 -489.
  • Cecropins were originally identified in proteins from the cecropia moth and are recognized as potent antibacterial proteins that constitute an important part of the cell-free immunity of insects.
  • Cecropins are small proteins (31-39 amino acid residues) that are active against both Gram-positive and Gram-negative bacteria by disrupting the bacterial membranes. Although the mechanisms by which the cecropons cause cell death are not fully understood, it is generally thought to involve channel formation and membrane destabilization.
  • Boman & Hultmark 1987, Ann. Rev. Microbiol. 41:103-126. Boman, 1991, Cell 65:205-207.
  • Example 7 Growth of Staphylococcus aureus bacteriophage 44 AHJD: Staphylococcus aureus propagating strain (PS 44A) (Felix d'Herelle Reference
  • phage 44 AHJD (Felix d'Herelle Reference Centre #HER 101).
  • Two rounds of plaque purification of phage 44AHJD were performed on soft agar essentially as described in Sambrook et al. (1989). Briefly, the Staphylococcus aureus PS strain was grown overnight at 37°C in Nutrient Broth [NB: 3 g Bacto Beef Extract, 5 g Bactopeptone per liter, (Difco Laboratories # 0003-17-8), supplemented with 0.5% NaCl]. The culture was then diluted 20 fold in NB and incubated at 37°C until an OD 540 of 0.2.
  • phage 44 AHJD was subjected to 10-fold serial dilutions using the phage buffer (1 mM MgSO 4 , 5 mM MgCl 2 , 80 mM NaCl and 0.1% Gelatin) -ancflO ⁇ l were used to infect 0.5 ml of the cell suspension in the presence of 400 ⁇ g/ml of CaCl 2 .
  • the mixture was then centrifuged for 10 min at 4,000 ⁇ m (2,830 xg) using a JA-10 rotor (Beckman) and the supernatant (lysate) is collected and subjected to a treatment with 10 ⁇ g/ml of DNase I and RNase A for 30 min at 37°C.
  • a treatment with 10 ⁇ g/ml of DNase I and RNase A for 30 min at 37°C.
  • 10% (w/v) of PEG 8000 and 0.5 M of NaCl were added to the lysate and the mixture was incubated on ice for 16 h.
  • the phage was recovered by centrifugation at 4,000 ⁇ m (3,500 xg) for 20 min at 4°C on a GS-6R table top centrifuge (Beckman).
  • the pellet was resuspended with 2 ml of phage buffer (1 mM MgSO 4 , 5 mM MgCl 2 , 80 mM NaCl and 0.1 %> Gelatin).
  • the phage suspension was extracted with 1 volume of chloroform and further purified by centrifugation on a preformed cesium chloride step gradient as described in Sambrook et al. (1989), using a TLS 55,roior and centrifuged in an Optima TLX ultracentrifuge (Beckman) for 2 h at 28,000 ⁇ m (67,000 xg) at 4°C.
  • Phage DNA was prepared from the phage suspension by adding 20 mM EDTA, 50 ⁇ g/ml Proteinase K and 0.5% SDS and incubating for 1 h at 65°C, followed by successive extractions with 1 volume of phenol, 1 volume of phenol-chloroform and 1 volume of chloroform. The DNA was then dialyzed overnight at 4°C against 4 L of TE (10 mM Tris-HCl [pH 8.0], lmM EDTA).
  • Example 8 DNA sequencing of the Bacteriophage 44 AHJD genome.
  • phage DNA was diluted in 200 ⁇ l of TE pH 8.0 in a 1.5 ml eppendorf tube and sonication was performed (550 Sonic Dismembrator, Fisher Scientific). Samples were sonicated under an amplitude of 3 ⁇ m with bursts of 5 s spaced by 15 s cooling in ice/water for 3 to 4 cycles and size fractionated on 1% agarose gels. The sonicated DNA was then size fractionated by gel electrophoresis.
  • Fractions ranging from 1 to 2 kbp were excised from the agarose gel and purified using a coommercial DNA extraction system according to the instructions of the manufacturer (Qiagen) and eluted in 50 ⁇ l of lmMTris-HCl [ pH 8.5]. The ends of the sonicated DNA fragments were repaired with a combination of
  • T4 DNA polymearse and the Klenow fragment of E. coli DNA polymerase 1 as follows. Reactions were performed in a final volume of 100 ⁇ l containing DNA, 10 mM Tris-HCl pH 8.0, 50 mM NaCl, 10 mM MgCl 2 , 1 mM DTT, 5 ⁇ g BSA, 100 ⁇ M of each dNTP and 15 units of T4 DNA polymerase (New England Biolabs) for 20 min at 12°C followed by addition of 12.5 units of Klenow fragment (New England Biolabs) for 15 min at room temperature. The reaction was stopped by two phenol/chloroform extractions and the DNA was ethanol precipitated and resuspended in 20 ⁇ l of H 2 O.
  • Recombinant clones were picked from agar plates into 96-well plates containing 100 ml LB and 100 ⁇ g/ml ampicillin and incubated at 37°C.
  • the presence of phage DNA insert was confirmed by PCR amplification using T3 and T7 primers flanking the Hindi cloning site of the pKS vector.
  • PCR amplification of the potential foreign inserts was performed in a 15 ⁇ l reaction volume containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl 2 , 0.02% gelatin, 1 mM primer, 187.5 ⁇ M each dNTP, and 0.75 units Taq polymerase (BRL).
  • thermocycling parameters were as follows: 2 min initial denaturation at 94°C for 2 min, followed by 20 cycles of 30 sec denaturation at 94°C, 30 sec annealing at 58C, and 2 min extension at 72°C, followed by a single extension step at 72°C for 10 min.
  • Clones with insert sizes of 1 to 2 kbp were selected and plasmid DNA was prepared from the selected clones using the QIAprepTM spin miniprep kit (Qiagen).
  • the nucleotide sequence of the extremities of each recombinant clone was determined using an ABI 377-36 automated sequencer with two types of chemistry: ABI prism BigDyeTM primer cycle sequencing (21M13 primer: #403055)(M13REV primer: #403056) or ABI prism BigDyeTM terminator cycle sequencing ready reaction kit (Applied Biosystems; #4303152).
  • ABI prism BigDyeTM primer cycle sequencing 21M13 primer: #403055)(M13REV primer: #403056)
  • ABI prism BigDyeTM terminator cycle sequencing ready reaction kit Applied Biosystems; #4303152.
  • a software program was used on the assembled sequence of bacteriophage 44AHJD to identify all putative ORFs larger than 33 codons.
  • the software scans the primary nucleotide sequence starting at nucleotide #1 for an appropriate start codon.
  • a counting mechanism is employed to count the number of codons (groups of three nucleotides) between this start codon and the next stop codon downstream of it. If a threshold value of 33 is reached, or exceeded, then the sequence encompassed by these two codons is defined as an ORF. This procedure is repeated, each time starting at the next nucleotide following the previous stop codon found, in order to identify all the other putative ORFs. The scan is performed on all three reading frames of both DNA strands of the phage sequence.
  • GenBank ftp://ncbi.nlm.nih.gOv/blast/db/nr.Z
  • Swissprot ftp://ncbi.nlm.nih.gOv/blast/db/swissprot.Z
  • vector ftp://ncbi.nlm.nih.gOv/blast/db/vector.Z
  • pdbaa databases ftp://ncbi.nlm.nih.gOV/Wast/db/pdbaa.Z
  • Staphylococcus aureus NCTC 8325 ftp://ftp.genome.ou.edu/pub/staph/staph- lk.fa
  • Example 10 Sub-Cloning of Bacteriophage 44 AHJD ORFs.
  • Expression preferably utilizes a shuttle expression vector which is arranged such that expression of the exogenous bacteriophage 44 AHJD ORF sequence is inducible.
  • the shuttle vector pT0021 in which the firefly luciferase (lucFF) expression is controlled by the ars (arsenite) promoter/operator (Tauriainen et al., 1997), can be modified in the following fashion. Two oligonucleotides corresponding to a short antigenic peptide derived from the heamaglutinin protein of influenza virus (HA epitope tag) were synthesized (Field et al., 1988).
  • the sense strand HA tag sequence (with BamHI, Sail and Hindlll cloning sites) is: 5 '-gatcccggtcgaccaagcttTACCCATACGACGTCCCAGACTACGCCAGCTGA-3 ' (where upper case letters denote the nucletotide sequence of the HA tag);
  • the antisense strand HA tag sequence (with a Hindlll cloning site) is:
  • Each ORF encoded by Bacteriophage 44 AHJD, larger than 33 amino acids and having a Shine-Dalgarno sequence upstream of the initiation codon can be selected for functional analysis for bacterial inhibition.
  • Each individual ORF, from initiation codon to last codon (excluding the stop codon), can be amplified from phage genomic DNA using the polymerase chain reaction (PCR).
  • each sense strand primer targets the initiation codon and is preceded by a BamHI restriction site ( 5' cgggatcc 3' ) and each antisense oligonucleotide targets the pentultimate codon (the one before the stop codon) of the ORF and is preceded by a Sal I restriction site ( 5' gcgtcgaccg 3 ).
  • the PCR product of each ORF can be gel purified and digested with BamHI and Sail.
  • the digested PCR product can then be gel purified using the Qiagen kit as described, ligated into BamHI and Sail digested pTHA vector, and used to transform E.
  • HA tag is set inframe with the ORF and is positioned at the carboxy terminus of each ORF (pTHA/ORF clones).
  • Recombinant pTHA ORF clones will be picked and their insert sizes were confirmed by PCR analysis using primers flanking the cloning site.
  • the following primers can be used for PCR amplification: HAF: 5 TATTATCCAAAACTTGAACA 3' ; HAR: 5 CGGTGGTATATCCAGTGATT 3' .
  • the sequence integrity of cloned ORFs can be verified directly by DNA sequencing using primers HAF and HAR. In cases where verification of ORF sequence can not be achieved by one pass with the sequencing primers, additional internal primers will be selected and used for sequencing.
  • Staphylococcus aureus strain RN4220 (Kreiswirth et al., 1983) will be used as a recipient for the expression of recombinant plasmids. ⁇ lectoporation will be performed essentially as previously described (Schenk and Laddaga, 1992). Selection of recombinant clones will be performed on Luria-Broth agar (LB-agar) plates containing 30 ⁇ g/ml of kanamycin.
  • a constitutive promoter can be used to drive expression of the introduced ORF, and compare cell growth to control bacterial cells containing the parental vector lacking any introduced phage ORF.
  • Recombinant plasmids will be introduced into Staphylococcus aureus strain RN4220 (Kreiswirth et al., 1983) using electoporation as previously described (Schenk and Laddaga, 1992). Cloning of ORFs with a Shine-Dalgarno sequence
  • ORFs with a Shine-Dalgarno sequence are selected for functional analysis of bacterial killing.
  • Each ORF, from initiation codon to last codon (excluding the stop codon), can be amplified by PCR from phage genomic DNA.
  • each sense strand primer starts at the initiation codon and is preceded by a restriction site and each antisense strand starts at the last codon (excluding the stop codon) and is preceded by a different restriction site.
  • the PCR product of each ORF will be gel purified and digested with the restriction enzymes with sites contained on the PCR oligonucleotides.
  • the digested PCR product is then gel purified using4he ⁇ Qiagen kit, ligated into the modified shuttle vector, and used to transform bacterial strain DH10. Recombinant clones are then picked and their insert sizes confirmed by PCR analysis using primers flanking the cloning site as well as restriction digestion.
  • the sequence fidelity of cloned ORFs can be verified by DNA sequencing using the same primers as used for PCR. In the cases that the verification of ORFs can not be achieved by one path of sequencing using primers flanking the cloning site internal primers can be selected and used for sequencing.
  • Recombinant plasmids can be introduced into Staphylococcus aureus strain RN4220 (Kreiswirth et al., 1983) using electoporation as previously described (Schenk and Laddaga, 1992). Induction of gene expression from the ars promoter.
  • induction can be assessed, for example, in either of the two methods.
  • the functional identification of killer ORFs can be performed by spreading an aliquot of S. aureus transformed cells containing phage 44 AHJD ORFs onto agar plates containing different concentrations of sodium arsenite (0; 2.5; 5; and 7.5 ⁇ M). The plates are incubated overnight at 37°C, after which a growth inhibition of the ORF transformants on plates that contain arsenite are compared to plates without arsenite.
  • An aliquot of the induced and uninduced culture can also be plated out on agar plates containing an appropriate antibiotic- selection but lacking inducer. Following incubation overnight at 37°C, the number of colonies is counted. Any ORF showing bacteriostatic activity will show a lower, but detectable, number of colonies on the agar plates when grown in the presence of inducer as compared to when grown in the absence of inducer. Any ORF showing full bacteriocidal activity will show no colonies on the agar plates, when grown in the presence of inducer as compared to when grown in the absence of inducer.
  • Example 11 Growth of Enterococcus bacteriophage 182 and purification of genomic DNA.
  • the Enterococcus propagating strain (PS) (Enterococcus sp. Group D, Felix d'Herelle Reference Centre #HER 1080) was used as host to propagate its respective phage 182 (Felix d'Herelle Reference Centre #HER 80). Two rounds of plaque purification of phage 182 were performed on soft agar essentially as described in Sambrook et al. (1989). Briefly, the Enterococcus sp.
  • TAB Tryptic Soy Broth
  • phage 182 was subjected to 10 fold serial dilutions using the phage buffer (1 mM MgSO 4 , 5 mM MgCl 2 , 80 mM NaCl and 0.1% Gelatin (w/v)) and 10 1 of each dilution was used to infect 0.5 ml of the bacterial cell suspension.
  • TSA Tryptone peptone
  • Soytone peptone 5 g Soytone peptone
  • Sodium chloride 15 g of Agar per liter
  • 7.5 ml of melted soft agar (TSB plus 0.6% agar) were added to the mixture and poured onto the surface of 150 mm TSA plates and incubated 16 hrs at 37°C.
  • TSB melted soft agar
  • the mixture was then centrifuged for 10 min at 4,000 ⁇ m (2,830 xg) using a JA-10 rotor (Beckman) and the supernatant fluid (lysate) is collected and subjected to a treatment with 10 ⁇ g /ml of DNase I and RNase A for 30 min at 37°C.
  • the phage suspension was adjusted to 10% (w/v) of PEG 8000 and 0.5 M of NaCl followed by incubation at 4°C for 16 hrs.
  • the phage was recovered by centrifugation at 4,000 ⁇ m (3,500 xg) for 20 min at 4°C on a GS-6R table top centrifuge (Beckman).
  • the pellet was resuspended with 2 ml of phage buffer (1 mM MgSO 4 , 5 mM MgCl 2 , 80 mM NaCl and 0.1% Gelatin).
  • the phage suspension was extracted with 1 volume of chloroform and further purified by centrifugation on a cesium chloride step gradient as described in Sambrook et al. (1989), using a TLS 55 rotor and centrifuged in an Optima TLX ultracentrifuge (Beckman) for 2 hrs at 28,000 ⁇ m (67,000 xg) at 4°C.
  • Phage DNA was prepared from the phage suspension by adding 20 mM EDTA, 50 g/ml Proteinase K and 0.5% SDS and incubating for 1 hr at 65°C, followed by successive extractions with 1 volume of phenol, 1 volume of phenol-chloroform and 1 volume of chloroform. The DNA was then dialyzed overnight at 4°C against 4 L of TE (10 mM Tris-HCl [pH 8.0], lmM EDTA).
  • phage DNA was diluted in 200 ⁇ l of TE (10 mM Tris, [pH 8.0], 1 mM EDTA) in a 1.5 ml eppendorf tube and sonication was performed (550 Sonic Dismembrator, Fisher Scientific). Samples were sonicated under an, amplitude of 3 ⁇ m with bursts of 5 s spaced by 15 s cooling in ice/water for 3 to 4 cycles. The sonicated DNA was then size fractionated by electrophoresis on 1% agarose gels utilizing TAE (1 x TAE is: 40 mM Tris-acetate, 1 mM EDTA [pH 8.0]) as the running buffer.
  • TE 10 mM Tris, [pH 8.0], 1 mM EDTA
  • Fractions ranging from 1 to 2 kbp were excised from the agarose gel and purified using a commercial DNA extraction system according to the instructions of the manufacturer (Qiagen), with a final elution of 50 ⁇ l of 1 mM Tris [pH 8.5].
  • the ends of the sonicated DNA fragments were repaired with a combination of T4 DNA polymerase and the Klenow fragment ofE. coli DNA polymerase I, as follows. Reactions were performed in a reaction mixture (final volume, 100 ⁇ l) containing sonicated phage DNA, 10 mM Tris-HCl [pH 8.0], 50 mM NaCl, 10 mM MgCl 2 , 1 mM DTT, 50 ⁇ g/ml BSA, 100 ⁇ M of each dNTP and 15 units of T4 DNA polymerase (New England Biolabs) for 20 min at 12°C followed by addition of 12.5 units of the Klenow large fragment of DNA polymerase I(New England Biolabs) for 15 min at room temperature. The reaction was stopped by two phenol/chloroform extractions and the DNA was precipitated with ethanol and the final DNA pellet resuspended in 20 ⁇ l of H 2 O.
  • Recombinant clones were picked from agar plates into 96-well plates containing 100 ⁇ l LB and 100 ⁇ g/ml ampicillin and incubated at 37°C.
  • the presence of phage DNA insert was confirmed by PCR amplification using T3 and T7 primers flanking the Hinc II cloning site of the pKS vector.
  • PCR amplification of the potential foreign inserts was performed in a 15 ⁇ l reaction volume containing 10 mM Tris (pH 8.3), 50 mM KCl, 1.5 mM MgCl 2 , 0.02% gelatin, 1 ⁇ M primer, 187.5 ⁇ M each dNT-P, ⁇ and 0.75 units Taq polymerase (BRL).
  • thermocycling parameters were as follows: 2 min initial denaturation at 94°C for 2 min, followed by 20 cycles of 30 sec denaturation at 94°C, 30 sec annealing at 58°C, and 2 min extension at 72°C, followed by a single extension step at 72°C for 10 min.
  • Clones with insert sizes of 1 to 2 kbp were selected and plasmid DNA was prepared from the selected clones using the QIAprepTM spin miniprep kit (Qiagen).
  • the nucleotide sequence of the extremities of each recombinant clone was determined using an ABI 377-36 automated sequencer with two types of chemistry: ABI prism Big DyeTM primer cycle sequencing (21M13 primer: #403055)(M13REV primer: #403056) or ABI prism Big DyeTM terminator cycle sequencing ready reaction kit (Applied Biosystems; #4303152). To ensure co-linearity of the sequence data and the genome, all regions of the phage genome were sequenced at least once from both directions on two separate clones. In areas that this criteria was not initially met, a sequencing primer was selected and phage DNA was used directly as sequencing template employing ABI prism BigDyeTM terminator cycle sequencing ready reaction kit.
  • Example 13 Bioinformatic management of primary nucleotide sequence. Sequence contigs were assembled using SequencherTM 3.1 software (GeneCodes). To close contig gaps, sequencing primers were selected near the edge of the contigs. Phage DNA was used directly as sequencing template employing ABI prism BigDyeTM terminator cycle sequencing ready reaction kit (Applied Biosystems; #4303152). The complete sequence o ⁇ Enterococcus bacteriophage 182 is shown in Table 21.
  • a software program was used on the assembled sequence of bacteriophage 182 to identify all putative ORFs larger than 33 codons.
  • a counting mechanism is employed to count the number of codons (groups of three nucleotides) between this start codon and the next stop codon downstream of it. If a threshold value of 33 is reached, or exceeded, then the sequence encompassed by these two codons is defined as an ORF. This procedure is repeated, each time starting at the next nucleotide following the previous stop codon found, in order to identify all the other putative ORFs. The scan is performed on all three reading frames of both DNA strands of the phage sequence.
  • the predicted ORFs for bacteriophage 182 are listed in Tables 22 & 23.
  • Sequence homology searches for each ORF were carried out using an implementation of BLAST programs.
  • Downloaded public databases used for sequence analysis include: (i) non-redundant GenBank (ftp://ncbi.nlm.mh.gOv/blast db/nr.Z), ii) Swissprot (ftp://ncbi.nlm.nih.g0v/blast/db/swissprot.Z); iii) vector (ftp://ncbi.nlm.nih.gOv/blast/db/vector.Z); iv) pdbaa databases (ftp://ncbi.nlm.r_ih.g0v/blast/db/pdbaa.Z); v) staphylococcus aureus NCTC 8325 (ftp://ftp.genome.ou.edu/pub/staph/staph- lk.fa); vi
  • Example 14 Sub-Cloning of Bacteriophage 182 ORFs. Preparation of the shuttle expression vector
  • Expression preferably utilizes a shuttle expression vector which is arranged such that expression of the exogenous bacteriophage 182 ORF sequence is inducible.
  • the plasmid pND50 replicates in E. coli, E.faecalis, and S. aureus (Yamagishi, J., Kojima, T., Oyamada, Y., Fujimoto, K., Hattori, H., Nakamura, S., and Inoue, M. 1996. Antimocrob. Agents Chemother. 40, 1157-1163).
  • This plasmid— can be modified by conventional techniques to insert the inducible arsenite promoter, derived from the shuttle vector pT0021, in which the firefly luciferase (lucFF) expression is controlled by the ars promoter/operator from a S. aureus plasmid (Tauriainen, S., Ka ⁇ , M., Chang, W and Virta, M. (1997). Recombinant luminescent bacteria for measuring bioavailable arsenite and antimonite. Appl. Environ. Microbiol. 63:4456-4461).
  • This modified shuttle vector will contain the ars promoter, arsR gene and a cloning site for introduction of individual phage ORFs downstream from a shine-delgarno sequence.
  • nisin-inducible system The nisA promoter activity is dependent on the proteins NisR and NisK, which constitute a two-component signal transduction system that responds to the extracellular inducer nisin.
  • the nisin sensitivity and inducer concentration required for maximal induction varies among the strains, but is functional in Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae, Enterococcus faecalis, and Bacillus subtilis.
  • nisA promoter 10- to 60-fold induction
  • a vector containing this promoter was published as Eichenbaum Z, Federle MJ, Marra D, de Vos WM, Kuipers OP, Kleerebezem M, and Scott JR (1998) Appl Environ Microbiol 64, 2763-2769.
  • Other vectors, e.g., plasmids, can also be utilized which will allow replication and transciption in Enterococcus.
  • a constitutive promoter can be used (e.g contun the ⁇ -lactamase promoter is constitutive in E. faecalis - see ref. 1) to drive expression of the introduced ORF, and compare cell growth to control bacterial cells containing the parental vector lacking any introduced phage ORF.
  • Recombinant plasmids are introduced into E. faecalis strain FA2-2 by electroporation, as previously described (Yamagishi, J., Kojima, T., Oyamada, Y., Fujimoto, K., Hattori, H., Nakamura, S., and Inoue, M. 1996. Antimicrob. Agents Chemother. 40, 1157-1163). Cloning of ORFs with a Shine-Dalgarno sequence
  • ORFs with a Shine-Dalgarno sequence are selected for functional analysis of bacterial killing.
  • Each ORF, from initiation codon to last codon (excluding the stop codon), will be amplified by PCR from phage genomic DNA.
  • each sense strand primer starts at the initiation codon and is preceded by a restriction site and each antisense strand starts at the last codon (excluding the sto r ⁇ codon) and is preceded by a different restriction site.
  • the PCR product of each ORF will be gel purified and digested with the restriction enzymes with sites contained on the PCR oligonucleotides.
  • the digested PCR product is then gel purified using the Qiagen kit, ligated into the modified shuttle vector, and used to transform bacterial strain DHlO ⁇ .
  • Recombinant clones are then picked and their insert sizes confirmed by PCR analysis using primers flanking the cloning site as well as restriction digestion.
  • the sequence fidelity of cloned ORFs will be verified by DNA sequencing using the same primers as used for PCR. In the cases that the verification of ORFs can not be achieved by one path of sequencing using primers flanking the cloning site internal primers will be selected and used for sequencing.
  • Recombinant plasmids will be introduced into E.
  • induction can be assessed, for example, in either of the two methods. 1. Screening on agar plates
  • the functional identification of killer ORFs can be performed by spreading an aliquot of E. faecalis transformed cells containing phage 182 ORF onto agar plates containing different concentrations of sodium arsenite (0; 2.5; 5; and 7.5 ⁇ M). The plates are incubated overnight at 37°C, after which a growth inhibition of the ORF transformants on plates that contain arsenite are compared to plates without arsenite. 2. Quantification of growth inhibition in liquid medium
  • the kilA gene of phage lambda (Reisinger, GR., Rietsch, A., Lubitz, W. and - . Blasi, U. 1993 Virology #193: 1033-1036), and the holin/lysin genes Of the Sthaphylococcus aureus phage Twort (Loessner, MJ., Gaeng, S., Wendlinger, G., Maier, SK. and Scherer, S. 1998. FEMS Microbiology Letters #162:265-274) were subcloned into the ars inducible vector.
  • An aliquot of the induced and uninduced culture can also be plated out on agar plates containing an appropriate antibiotic selection but lacking inducer. Following incubation overnight at 37°C, the number of colonies is counted. Any ORF showing bacteriostatic activity will show a lower, but detectable, number of colonies on the agar plates when grown in the presence of inducer as compared to when grown in the absence of inducer. Any ORF showing bacteriocidal activity will show no colonies on the agar plates, when grown in the presence of inducer as compared to when grown in the absence of inducer.
  • Example 15 Growth of Streptococcus bacteriophage Dp-1 and purification of genomic DNA.
  • Streptococcus pneumoniae R6 propagating strain PS (Tomasz, 1966) was used as host to propagate its respective phage Dp-1 (McDonnell et al., 1975).
  • Streptococcus (Diplococcus) pneumoniae R36A could be used.
  • Strain R36A is available from ATCC as #11733 or 27336.
  • Streptococcus pneumoniae is also available from Felix d'Herelle Reference Center in Quebec, Canada as catalog number HER 1054.
  • Other S. pneumoniae strains are also available from ATCC.
  • Two rounds of plaque purification of phage Dp-1 were performed on soft agar essentially as described in Sambrook et al (1989).
  • Dp-1 phage was subjected to 10-fold serial dilutions using the phage buffer (100 mM Tris-HCl [pH 7.5], 100 mM NaCl and 10 mM MgCl 2 )and 10 ⁇ l of each dilution was used to infect 0.5 ml of the cell suspension. After incubation of 15 min at 37°C, 2 ml of melted soft agar (K-CAT supplemented with 0.8%) of agar) were added to the mixture and poured onto the surface of 100 mm K-CAT agar plates [K-CAT supplemented with 1.2 % of agar].
  • K-CAT supplemented with 1.2 % of agar 100 mm K-CAT agar plates
  • 7.5 ml of melted soft agar were added to each plate.
  • 20 ml of K-CAT media were added to each plate and the soft agar layers were collected by scrapping off with a clean microscope slide followed by vigorous shaking of the agar suspension for 5 min to break up the agar.
  • the mixture was then centrifuged for 10 min at 4,000 ⁇ m (2,830 xg) using a JA-10 rotor (Beckman) and the supernatant (lysate) was collected and subjected to a treatment with 10 ⁇ g /ml of DNase I and RNase A for 30 min at 37°C.
  • the phage suspension was adjusted to 10%. (w/v) of PEG 8000 and 0.5 M of NaCl followed by incubation at 4°C for 16 hrs. The phage was recovered by centrifugation at 4,000 ⁇ m (3,500 xg) for 20 min at 4°C on a GS-6R table top centrifuge (Beckman). The pellet was resuspended with 2 ml of phage buffer (100 mM Tris-HCl [pH 7.5], 100 mM NaCl and 10 mM MgCl 2 ).
  • the phage suspension was extracted with 1 volume of chloroform and further purified by centrifugation on a cesium chloride step gradient as described in Sambrook et al. (1989), using a TLS-55 * rotor and centrifuged in an Optima TLX ultracentrifuge (Beckman) for 2 hrs at 28,000 ⁇ m (67,000 xg) at 4°C. Banded phage was collected and ultracentrifuged again on an isopycnic cesium chloride gradient (1.45 g/ml) at 40,000 ⁇ m (64,000 xg) for 24 hrs at 4°C using a TLV rotor (Beckman).
  • the phage was harvested and dialyzed for 4 hrs at room temperature against 4 L of dialysis buffer consisting of 10 mM NaCl, 50 mM Tris-HCl [pH 8] and 10 mM MgCl 2 .
  • Phage DNA was prepared from the phage suspension by adding 20 mM EDTA, 50 ⁇ g/ml Proteinase K and 0.5% SDS and incubating for 1 hr at 65°C, followed by successive extractions with 1 volume of phenol, 1 volume of phenol-chloroform and 1 volume of chloroform. The DNA was then dialyzed overnight at 4°C against 4 L of TE (10 mM Tris-HCl [pH 8.0], lmM EDTA).
  • phage DNA was diluted in 200 ⁇ l of TE (10 mM Tris, [pH 8.0], 1 mM EDTA) in a 1.5 ml eppendorf tube and sonication was performed (550 Sonic Dismembrator, Fisher Scientific). Samples were sonicated under an amplitude of 3 ⁇ m with bursts of 5 sec spaced by 15 sec cooling in ice/water for 3 to 4 cycles. The sonicated DNA was then size fractionated by electrophoresis on 1%> agarose gels utilizing TAE (1 x TAE is: 40 mM Tris-acetate, 1 mM EDTA [pH 8.0]) as the running buffer.
  • TE 10 mM Tris, [pH 8.0], 1 mM EDTA
  • Fractions ranging from 1 to 2 kbp were excised from the agarose gel and purified using a commercial DNA extraction system according to the instructions of the manufacturer (Qiagen), with a final elution of 50 ⁇ l of 1 mM Tris [pH 8.5].
  • the ends of the sonicated DNA fragments were repaired with a combination of T4 DNA polymerase and the Klenow fragment ofE. coli DNA polymerase I, as follows. Reactions were performed in a reaction mixture (final volume, 100 ⁇ l) containing sonicated phage DNA, 10 mM Tris-HCl [pH 8.0], 50 mM NaCl, 10 mM MgCl 2 , 1 mM DTT, 50 ⁇ g/ml BSA, 100 ⁇ M of each dNTP and 15 units of T4 DNA polymerase (New England Biolabs) for 20 min at 12°C followed by addition of 12.5 units of the Klenow large fragment of DNA polymerase I (New England Biolabs) for 15 min at room temperature. The reaction was stopped by two phenol/chloroform extractions and the DNA was precipitated with ethanol and the final DNA pellet resuspended in 20 ⁇ l of H 2 O.
  • Recombinant clones were picked from agar plates into 96-well plates containing 100 ⁇ l LB and 100 ⁇ g/ml ampicillin and incubated at 37°C.
  • the presence of phage DNA insert was confirmed by PCR amplification using T3 and T7 primers flanking the Hinc II cloning site of the pKS vector.
  • PCR amplification of the potential foreign inserts was performed in a 15 ⁇ l reaction volume containing 10 mM Tris (pH 8.3), 50 mM KCl, 1.5 mM MgCl 2 , 0.02% gelatin, 1 ⁇ M primer, 187.5 ⁇ M each dNTP, and 0.75 units Taq polymerase (BRL).
  • thermocycling parameters were as follows: 2 min initial denaturation at 94°C for 2 min, followed by 20 cycles of 30 sec denaturation at 94°C, 30 sec annealing at 58°C, and 2 min extension at 72°C, followed by a single extension step at 72°C for 10 min.
  • Clones with insert sizes of 1 to 2 kbp were selected and plasmid DNA was prepared from the selected clones using the QIAprepTM spin miniprep kit (Qiagen).
  • the nucleotide sequence of the extremities of each recombinant clone was determined using an ABI 377-36 automated sequencer with two types of chemistry: ABI prism Big DyeTM primer cycle sequencing (21M13 primer: #403055)(M13R ⁇ V primer: #403056) or ABI prism Big DyeTM terminator cycle sequencing ready reaction kit (Applied Biosystems; #4303152).
  • ABI prism Big DyeTM primer cycle sequencing 21M13 primer: #403055)(M13R ⁇ V primer: #403056) or ABI prism Big DyeTM terminator cycle sequencing ready reaction kit (Applied Biosystems; #4303152).
  • ABI prism Big DyeTM primer cycle sequencing 21M13 primer: #403055)(M13R ⁇ V primer: #403056)
  • ABI prism Big DyeTM terminator cycle sequencing ready reaction kit Applied Biosystems; #4303152.
  • Example 17 Bioinformatic management of primary nucleotide sequence.
  • Sequence contigs were assembled using SequencherTM 3.1 software (GeneCodes). To close contig gaps, sequencing primers were selected near the edge of the contigs. Phage DNA was used directly as sequencing template employing ABI prism BigDyeTM terminator cycle sequencing ready reaction kit (Applied Biosystems; #4303152). The complete sequence o ⁇ Streptococcus bacteriophage Dp-1 is shown in Table 28.
  • a software program was used on the assembled sequence of bacteriophage Dp-1 to identify all putative ORFs larger than 33 codons.
  • the software scans the primary nucleotide sequence starting at nucleotide #1 for an appropriate start codorrr Three possible selections can be made for defining the nature of the start codon; I) selection of ATG, II) selection of ATG or GTG, and III) selection of either ATG, GTG, TTG, CTG, ATT, ATC, and ATA.
  • a counting mechanism is employed to count the number of codons (groups of three nucleotides) between this start codon and the next stop codon downstream of it. If a threshold value of 33 is reached, or exceeded, then the sequence encompassed by these two codons is defined as an ORF. This procedure is repeated, each time starting at the next nucleotide following the previous stop codon found, in order to identify all the other putative ORFs. The scan is performed on all three reading frames of both DNA strands of the phage sequence.
  • the predicted ORFs for bacteriophage Dp-1 are listed in Tables 29 and 30, and Fig. 6.
  • Sequence homology searches for each ORF were carried out using an implementation of BLAST programs.
  • Downloaded public databases used for sequence analysis include: (i) non-redundant GenBank (ftp://ncbi.nlm.nih.gOv/blast/db/nr.Z), ii) Swissprot (ftp://ncbi.nlm.nih.gOv/blast/db/swissprot.Z); iii) vector (ftp://ncbi.nlm.nih.gOv/blast/db/vector.Z); iv) pdbaa databases (ftp://ncbi.nlm.nih.gOv/blast/db/pdbaa.Z); v) staphylococcus aureus NCTC 8325 (ftp://ftp.genome.ou.edu/pub/staph staph-lk.fa); vi)
  • Example 18 Sub-Cloning of Bacteriophage Dp-1 ORFs.
  • Expression preferably utilizes a shuttle expression vector which is arranged such that expression of the exogenous bacteriophage Dp-1 ORF sequence is inducible.
  • the plasmid pLSE4 replicates in E. coli, and S. pneumoniae (Diaz- and Garcia, 1990).
  • This plasmid can be modified by conventional techniques to insert the inducible arsenite promoter, derived from the shuttle vector pT0021, in which the firefly luciferase (lucFF) expression is controlled by the ars promoter/operator from a S. aureus plasmid (Tauriainen, S., Ka ⁇ , M., Chang, W and Virta, M. (1997).
  • This modified shuttle vector will contain the ars promoter, arsR gene and a cloning site for introduction of individual phage ORFs downstream from a shine-dalgarno sequence.
  • nisin-inducible system The nisA promoter activity is dependent on the proteins NisR and NisK, which constitute a two-component signal transduction system that responds to the extracellular inducer nisin.
  • the nisin sensitivity and inducer concentration required for maximal induction varies among the strains, but is functional in Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae, Enterococcus faecalis, and Bacillus subtilis.
  • nisA promoter 10- to 60-fold induction
  • a vector containing this promoter was published as Eichenbaum Z, Federle MJ, Marra D, de Vos WM, Kuipers OP, Kleerebezem M, and Scott JR (1998) Appl Environ Microbiol 64, 2763-2769.
  • Other vectors, e.g., plasmids, can also be utilized which will allow replication and transcription in Streptococcus.
  • a constitutive promoter can be used to drive expression of the introduced ORF, and compare cell growth to control bacterial cells containing the parental vector lacking any introduced phage ORF.
  • Recombinant plasmids are introduced into S. pneumoniae R6 as previously described (Diaz and Garcia, 1990)
  • ORFs with a Shine-Dalgarno sequence are selected for functional analysis of bacterial killing.
  • Each ORF, from initiation codon to last codon (excluding the stop codon), will be amplified by PCR from phage genomic DNA.
  • each sense strand primer starts at the initiation codon and is preceded by a restriction site and each antisense strand starts at the last codon (excluding the stop codon) and is preceded by a different restriction site.
  • the PCR product of each ORF will be gel purified and digested with the restriction enzymes with sites contained on the PCR oligonucleotides.
  • the digested PCR product is then gel purified using the Qiagen kit, ligated into the modified shuttle vector, and used to transform bacterial strain DHlO ⁇ .
  • Recombinant clones are then picked and their insert sizes confirmed by PCR analysis using primers flanking the cloning site as well as restriction- — digestion.
  • the sequence fidelity of cloned ORFs will be verified by DNA sequencing using the same primers as used for PCR. In the cases that the verification of ORFs can not be achieved by one path of sequencing using primers flanking the cloning site internal primers will be selected and used for sequencing.
  • Recombinant plasmids will be introduced into S. pneumoniae R6 as previously described (Diaz and Garcia, 1990). Induction of gene expression from the ars promoter.
  • induction can be assessed, for example, in either of the two methods.
  • the functional identification of killer ORFs can be performed by spreading an aliquot of S. pneumoniae transformed cells containing phage Dp-1 ORFs onto agar plates containing different concentrations of sodium arsenite (0; 2.5; 5; and 7.5 ⁇ M). The plates are incubated overnight at 37°C, after which a growth inhibition of the ORF transformants on plates that contain arsenite are compared to plates without arsenite.
  • Any ORF showing bacteriostatic activity will show a lower, but detectable, number of colonies on the agar plates when grown in the presence of inducer as compared to when grown in the absence of inducer.
  • Any ORF showing full bacteriocidal activity will show no colonies on the agar plates, when grown in the presence of inducer as compared to when grown in the absence of inducer.
  • the embodiments expressly include any subset or subgroup of those bacteria and/or phage. While each such subset or subgroup could be listed separately, for the sake of brevity, such a listing is replaced by the present description.
  • HER 317 Felix d'Herelle Refrence HER 330 Centre,Quebec,Quebec HER 333 HER 335 HER 334 HER 331 HER 316
  • Mycobacterium 23052-B1 The American Type Culture Collection fortuitum 27207-B1
  • Mycobacterium 25618-B1 The American Type Culture Collection tuberculosis 25618-B2
  • Pseudomonas 12175-B1 The American Type Culture Collection aeruginosa 2 12175-B2
  • Staphylococcus la 2b, 3a, 4b, Can.J.Microbiol.l988.34:1358-1361 epidermidis 5a, 6b, 7b, 8c, 9a, 10a, l ib, 12a & 13b

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Abstract

L'invention concerne une méthode d'identification de cibles appropriées pour des agents antibactériens. Cette méthode consiste à identifier des cibles de protéines codées par des bactériophages. L'invention concerne également des compositions utiles pour ces méthodes d'identification et pour l'inhibition de la croissance des bactéries. L'invention concerne enfin des méthodes de préparation et d'utilisation de ces compositions.
PCT/IB1999/002040 1998-12-03 1999-12-03 Developpement de nouveaux agents antimicrobiens bases sur des genomes de bacteriophages Ceased WO2000032825A2 (fr)

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CA002353563A CA2353563A1 (fr) 1998-12-03 1999-12-03 Developpement de nouveaux agents antimicrobiens bases sur des genomes de bacteriophages
AU15815/00A AU774841B2 (en) 1998-12-03 1999-12-03 Development of novel anti-microbial agents based on bacteriophage genomics
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US09/407,804 US6982153B1 (en) 1998-12-03 1999-09-28 DNA sequences from staphylococcus aureus bacteriophage 77 that encode anti-microbial polypeptides
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WO2002044718A3 (fr) * 2000-12-01 2002-12-12 Phagetech Inc Compositions et procedes faisant intervenir un gene staphylococcus aureus essentiel et la proteine staau_r2 pour laquelle il code
EP1345960A2 (fr) * 2000-12-20 2003-09-24 Phagetech Inc. Compositions et methodes mettant en oeuvre un gene essentiel de staphylococcus aureus et proteine codee staau r4 associee
US6759229B2 (en) 2001-12-18 2004-07-06 President & Fellows Of Harvard College Toxin-phage bacteriocide antibiotic and uses thereof
EP1242611A4 (fr) * 1999-12-22 2004-08-11 Phagetech Inc Compositions et methodes concernant un gene essentiel de staphylococcus aureus et sa proteine codee
WO2003024410A3 (fr) * 2001-09-21 2004-09-23 New Horizons Diagnostics Corp Utilisation d'enzymes lytiques associees a un bacteriophage chimere et brasse pour le traitement prophylactique et therapeutique de colonisation et d'infections causees par le streptococcus pneumoniae
US7101969B1 (en) 1998-12-03 2006-09-05 Targanta Therapeutics Compositions and methods involving an essential Staphylococcus aureus gene and its encoded protein
US7326541B2 (en) 2000-12-19 2008-02-05 Targanta Therapeutics, Inc. Fragments and variants of Staphylococcus aureus DNAG primase, and uses thereof
US7569223B2 (en) 2004-03-22 2009-08-04 The Rockefeller University Phage-associated lytic enzymes for treatment of Streptococcus pneumoniae and related conditions
AU2005219839B2 (en) * 2004-03-01 2011-11-24 Immune Disease Institute, Inc Natural IgM antibodies and inhibitors thereof
US9243059B2 (en) 2013-03-12 2016-01-26 Decimmune Therapeutics, Inc. Humanized anti-N2 antibodies and methods of treating ischemia-reperfusion injury
CN111296493A (zh) * 2020-03-09 2020-06-19 苏州十一方生物科技有限公司 一种噬菌体消毒剂及其制备方法
CN111316999A (zh) * 2020-03-04 2020-06-23 苏州十一方生物科技有限公司 一种含有噬菌体的喷雾型环境消毒剂及其制备方法和应用
CN118240771A (zh) * 2024-03-12 2024-06-25 武汉格瑞农生物科技有限公司 一株可提升鸭疫里默氏杆菌药物敏感性的噬菌体及其应用

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GB201119167D0 (en) * 2011-11-07 2011-12-21 Novolytics Ltd Novel bachteriophages
WO2024263589A1 (fr) 2023-06-20 2024-12-26 Topaz Biosciences, Inc. Hydrolases de paroi cellulaire recombinantes

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CA1207253A (fr) * 1981-08-17 1986-07-08 Lee D. Simon Fragment d'adn t4 comme stabilisateur de proteines exprimees par adn clone
CA1327311C (fr) * 1987-07-06 1994-03-01 Jesse M. Jaynes Polypeptides antimicrobiens therapeutiques, leurs methodes de preparation et leurs usages
CA2186962A1 (fr) * 1994-04-05 1995-10-12 Richard M. Carlton Therapie anti-bacterienne a l'aide de bacteriophages genotypiquement modifies
ATE247711T1 (de) * 1995-06-16 2003-09-15 Nestle Sa Phagenresistenter streptococcus

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US7101969B1 (en) 1998-12-03 2006-09-05 Targanta Therapeutics Compositions and methods involving an essential Staphylococcus aureus gene and its encoded protein
EP1242611A4 (fr) * 1999-12-22 2004-08-11 Phagetech Inc Compositions et methodes concernant un gene essentiel de staphylococcus aureus et sa proteine codee
WO2002044718A3 (fr) * 2000-12-01 2002-12-12 Phagetech Inc Compositions et procedes faisant intervenir un gene staphylococcus aureus essentiel et la proteine staau_r2 pour laquelle il code
US7326541B2 (en) 2000-12-19 2008-02-05 Targanta Therapeutics, Inc. Fragments and variants of Staphylococcus aureus DNAG primase, and uses thereof
EP1345960A2 (fr) * 2000-12-20 2003-09-24 Phagetech Inc. Compositions et methodes mettant en oeuvre un gene essentiel de staphylococcus aureus et proteine codee staau r4 associee
WO2003024410A3 (fr) * 2001-09-21 2004-09-23 New Horizons Diagnostics Corp Utilisation d'enzymes lytiques associees a un bacteriophage chimere et brasse pour le traitement prophylactique et therapeutique de colonisation et d'infections causees par le streptococcus pneumoniae
US6759229B2 (en) 2001-12-18 2004-07-06 President & Fellows Of Harvard College Toxin-phage bacteriocide antibiotic and uses thereof
AU2005219839B2 (en) * 2004-03-01 2011-11-24 Immune Disease Institute, Inc Natural IgM antibodies and inhibitors thereof
AU2005219839B9 (en) * 2004-03-01 2011-12-22 Immune Disease Institute, Inc Natural IgM antibodies and inhibitors thereof
US9657060B2 (en) 2004-03-01 2017-05-23 Children's Medical Center Corporation Natural IgM antibodies and inhibitors thereof
US9914751B2 (en) 2004-03-01 2018-03-13 Children's Medical Center Corporation Natural IGM antibodies and inhibitors thereof
US7569223B2 (en) 2004-03-22 2009-08-04 The Rockefeller University Phage-associated lytic enzymes for treatment of Streptococcus pneumoniae and related conditions
US9243059B2 (en) 2013-03-12 2016-01-26 Decimmune Therapeutics, Inc. Humanized anti-N2 antibodies and methods of treating ischemia-reperfusion injury
US9409977B2 (en) 2013-03-12 2016-08-09 Decimmune Therapeutics, Inc. Humanized, anti-N2 antibodies
CN111316999A (zh) * 2020-03-04 2020-06-23 苏州十一方生物科技有限公司 一种含有噬菌体的喷雾型环境消毒剂及其制备方法和应用
CN111316999B (zh) * 2020-03-04 2022-02-08 苏州十一方生物科技有限公司 一种含有噬菌体的喷雾型环境消毒剂及其制备方法和应用
CN111296493A (zh) * 2020-03-09 2020-06-19 苏州十一方生物科技有限公司 一种噬菌体消毒剂及其制备方法
CN118240771A (zh) * 2024-03-12 2024-06-25 武汉格瑞农生物科技有限公司 一株可提升鸭疫里默氏杆菌药物敏感性的噬菌体及其应用

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