WO2000011184A1 - Staphylococcus aureus polypeptides - Google Patents

Abstract

The invention provides methods for the detection, prevention and treatment of pathological conditions resulting from bacterial infection. Provided are isolated polypeptide and nucleic acid sequences derived from Staphylococcus aureus which comprise targets for therapeutic drugs, in particular broad spectrum antibacterial agents. In addition to providing valuable targets for antibacterial agents, the invention provides compositions that are useful in diagnosis and therapy of pathological conditions; antibodies against the polypeptides; and methods for the production of the polypeptides.

Description

STAPHYLOCOCCUS AUREUS POLYPEPTIDES

Field Of The Invention

The invention relates to isolated bacterial-derived nucleic acids and polypeptides. The invention also relates to materials and methods for the diagnosis and treatment of pathological conditions resulting from bacterial infection.

Background Of The Invention

Patient care, as well as the prevention of transmission of bacterial infection, requires sensitive, specific and reliable prevention and treatment regimes.

Unfortunately, bacteria have the ability to acquire resistance factors that suppress their susceptibility to chemotherapeutically useful antibiotics.

Staphylococcus aureus infections, for example, have traditionally been treated with b-lactam antibiotics . Most isolates (85-90 % ) , however , have developed resistance to penicillin and ampicillin. MethiciUin resistance is also a serious problem, with methicillin resistant strains of S. aureus (MRSA) being the first ranking nosocomial pathogens worldwide. While the most "advanced" forms of MRSA carry resistance mechanisms to all but one (vancomycin) of the usable antibacterial agents, vancomycin resistance in MRSA has recently been reported. Moreover, as a result of increased vancomycin usage, a vancomycin resistance mechanism in another nosocomial pathogen (Enterococcus faecium) is now being encountered. Penicillin resistant and multi-resistant pneumococci have also emerged.

Since the development of resistance to current antibiotics is universally considered almost inevitable, there continues to be a need for alternative preventive and therapeutic agents, including effective anti-bacterial agents and immunotherapeutics, for the prevention and/or treatment of disease caused by such microorganisms. Summary Of The Invention

The present invention fulfills this need by providing compositions and methods for diagnosing, treating and preventing bacterial infections, in particular, bacterial infections caused by pathogenic strains that have become resistant to commerically available antibiotics.

In one series of embodiments , the invention provides isolated bacterial-specific polypeptide and nucleic acid sequences that are bacterial specific and essential to the viability of a bacterial species. The sequences of the invention find use in diagnostic applications and as targets to screen for therapeutic agents. The invention provides polypeptide sequences comprising at least five, more preferably at least about 8 or more consecutive amino acid residues derived from any open reading frame (ORF) including complete protein-coding sequences contained within SEQ ID NO: 1 or contained within any of SEQ ID NO: 2-11. Function- conservative variants and homologs are included in the scope of the invention. Also provided are methods for producing such polypeptides.

The invention further provides nucleic acid sequences comprising at least about 12, more preferably at least about 18, and most preferably at least about 20-35 or more consecutive nucleotides shown in SEQ ID NO: 1, including complete protein-coding sequences, and complements thereof. The invention encompasses sequence- conservative variants and function-conservative variants of these sequences. The nucleic acids may be DNA, RNA, DNA/RNA duplexes, protein-nucleic acid (PNA), or derivatives thereof. The invention also encompasses recombinant DNA vectors (including DNA expression vectors) comprising these sequences; cells comprising such vectors, including bacterial, fungal, plant, insect, and mammalian cells; and methods for producing expression products comprising RNA and polypeptides encoded by the sequences.

A particularly preferred embodiment of the invention is directed to a method of screening test compounds for antibacterial activity, in particular broad spectrum antibacterial activity, which method comprises selecting a target sequence; contacting a test compound with the target sequence; and selecting those test compounds which bind to the target sequence as potential antibacterial candidates. The target sequences of the invention may be used alone or in combination.

The invention further provides antibodies, preferably monoclonal antibodies, which specifically bind to a polypeptide having an amino acid shown in SEQ ID NO: 2-11 or fragment thereof. Methods are also provided for producing such antibodies in a host animal.

Yet other aspects of the invention provides methods for diagnosing, preventing or treating disease caused by certain gram-positive and gram-negative bacteria, including, but not limited to, Streptococcus pneumoniae, Staphylococcus aureus

Enterococcus faecium, Bacillus anthracis, Escherichia coli, Haemophilus influenza, and Neisseria meningitidis .

Detailed Description Of The Invention All patent applications, patents, and literature references cited in this specification are hereby incorporated by reference in their entirety.

Definitions

1. "Nucleic acid" or "polynucleotide" refers to purine- and pyrimidine- containing polymers of any length, either polyribonucleotides or polydeoxyribonucleotides or mixed polyribo-polydeoxyribo nucleotides. This includes single- and double-stranded molecules, i.e., DNA-DNA, DNA -RNA and RNA-RNA hybrids, as well as "protein nucleic acids" (PNA) formed by conjugating bases to an amino acid backbone. This also includes nucleic acids containing modified bases. 2. An "open reading frame" (ORF) is a region of a polynucleotide sequence that encodes a polypeptide. This region may represent a portion of a coding sequence or comprise a total coding sequence for the polypeptide.

3. A "coding sequence" or a "protein-coding sequence" is a polynucleotide sequence capable of being transcribed into mRNA and/or capable of being translated into a polypeptide. The boundaries of the coding sequence are typically determined by a translation start codon at the 5 '-terminus and a translation stop codon at the 3'- terminus. 4. A "complement" of a nucleic acid sequence refers to the "antisense" sequence that participates in Watson-Crick base-pairing with the original sequence.

5. An "isolated" nucleic acid or polypeptide as used herein refers to a component that is removed from its original environment (for example, its natural environment if it is naturally occurring). An isolated nucleic acid or polypeptide preferably contains less than about 50% , more preferably less than about 75% , and most preferably less than about 90%, of the cellular components with which it was originally associated. 6. A nucleic acid or polypeptide sequence that is "derived from" a designated sequence refers to a sequence that corresponds to a region of the designated sequence. For nucleic acid sequences, this encompasses sequences that are homologous or complementary to the sequence, as well as "sequence-conservative variants" and "function-conservative variants. " For polypeptide sequences, this encompasses "function-conservative variants. " Sequence-conservative variants are those in which a change of one or more nucleotides in a given codon position results in no alteration in the amino acid encoded at that position. Function-conservative variants are those in which a given amino acid residue in a polypeptide has been changed without substantially altering the overall conformation and function of the native polypeptide, including, but not limited to, replacement of an amino acid with one having similar physico-chemical properties (such as, for example, acidic, basic, hydrophobic, and the like). "Function-conservative" variants also include any polypeptides that have the ability to elicit antibodies specific to a designated polypeptide. 7. An "S. aureu -derived" nucleic acid or polypeptide sequence refers to the source from which the sequence was originally isolated. Thus, an S. aureus derived polypeptide, as used herein, may be used, e.g., as a target to screen for a broad spectrum antibacterial agent, or to search for homologous proteins in other species of bacteria. 8. A "probe" refers to a nucleic acid or oligonucleotide that forms a hybrid structure with a sequence in a target region due to complementarity of at least one sequence in the probe with a sequence in the target.

9. Nucleic acids are "hybridizable" to each other when at least one strand of nucleic acid can anneal to another nucleic acid strand under defined stringency conditions. Stringency of hybridization is determined, e.g., by a) the temperature at which hybridization and/or washing is performed, and b) the ionic strength and polarity (e.g. , formamide) of the hybridization and washing solutions, as well as other parameters. Hybridization requires that the two nucleic acids contain substantially complementary sequences; depending on the stringency of hybridization, however, mismatches may be tolerated . The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementarity, variables well known in the art.

10. An "immunogenic component" is a moiety that is capable of eliciting a humoral and/or cellular immune response in a host animal. 11. An "antigenic component" is a moiety that binds to its specific antibody with sufficiently high affinity to form a detectable antigen-antibody complex.

12. A "sample" refers to a biological sample, such as, for example, tissue or fluid isolated from an individual (including without limitation plasma, serum, cerebrospinal fluid, lymph, tears, saliva, milk, pus, and tissue exudates and sections) or from in vitro cell culture constituents, as well as samples obtained from the environment or laboratory procedures.

Nucleic Acids. Vectors, and Host Cells The invention provides S. αwrews-derived nucleic acid sequences, in particular the nucleic acid sequence of the SlO-spc ribosomal operon of S. aureus ATCC 55748 shown in SEQ ID NO: 1. The location of the ORFs of the ribosomal proteins (L2 to L5) within SEQ ID NO: 1 and reading frame is shown in Table 1.

Table 1

The nucleic acid sequences of the invention comprise at least about 12, more preferably at least about 18, and most preferably at least about 20-35 or more consecutive nucleotides which encode an amino acid sequences shown in any of SEQ ID NO: 2-11, or which are selected from the nucleotides shown in SEQ ID NO: 1, including complete protein-coding sequences, or complements thereof. The invention encompasses sequence-conservative variants and function-conservative variants of these sequences. These nucleic acids sequences find use as probes, primers, and markers, and encode valuable targets for use in screening for therapeutic drugs.

Nucleotide analysis shows high homology (70-90 % ) with the S 10-spc ribosomal protein operons of other bacteria (from RPL2-RPL5), both gram-positive and gram- negative. The bacterial SlO-spc ribosomal operon provides valuable targets for use in screening for new and highly effective antibiotics with a broad spectrum of activity, in particular with respect the gram-positive bacteria, including enterococci, staphylococci and streptococci.

A clinical isolate of Streptococcus pneumoniae with decreased susceptibility to an everninomicin-type antibiotic has been discovered. When the nucleotide sequence of resistant and susceptible isolates were compared, a single point mutation, which resulted in an Ile52Ser substitution in RPL16, was identified. The presence of this mutation in a resistant strain suggests that an alteration in RPL16 contributes to everninomicin resistance and may be a site of interaction of everninomicin. The S10- spc ribosomal protein operon (from RPL2 to RPL5) of S. pneumoniae is disclosed in concurrently filed, commonly assigned application Serial No. (Attorney Docket No. ID0880), the disclosure of which is incorporated herein by reference in its entirety.

Comparison of RPL16 of S. pneumoniae and S. aureus is shown below.

S. aureus MLLPKRVKYRRQHRPKTTGRSKGGNYVTFGEFGLQATTTSWITSRQIESARIAMT S. pneumoniae MLVPKRVKHRREFRGKMRGEAKGGKEVAFGEYGLQATTSHWITNRQIEAARIAMT

S. aureus RYMKRGGKVWIKIFPHTPYTKKPLEVRMGAGKGAVEGWIAWKPGRILFEVAGVS

S. pneumoniae RYMKRGGKVWIKIFPHKSYTAKAIGVRMGSGKGAPEGVJVAPVKRGKVMFEIAGVS

S. aureus EEVAREALRASHKLPVKTKFVKREE GGETNΞS (SEQ ID NO : 6)

S. pneumoniae EEIAREALRASHKLPVKCKFVKREAE (SEQ ID NO: 12)

In practicing the present invention, many conventional techniques in molecular biology, microbiology, and recombinant DNA, are used. Such techniques are well known and are explained fully in, for example, Sambrook et αl. , 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; DNA Cloning: A Practical Approach, Volumes I and II, 1985 (D.N. Glover ed.); Oligonucleotide Synthesis, 1984, (M.L. Gait ed.); Nucleic Acid Hybridization, 1985, (Hames and Higgins); Transcription and Translation, 1984 (Hames and Higgins eds.); Animal Cell Culture, 1986 (R.I. Freshney ed.); Immobilized Cells and Enzymes, 1986 (IRL Press); Perbal, 1984, A Practical Guide to Molecular Cloning; the series, Methods in Enzymology (Academic Press, Inc.); Gene Transfer Vectors for Mammalian Cells, 1987 (J. H. Miller and M. P. Calos eds. , Cold Spring Harbor Laboratory); and Methods in Enzymology Vol. 154 and Vol. 155 (Wu and Grossman, and Wu, eds., respectively).

Nucleic acids comprising any of the sequences disclosed herein or subsequences thereof can be prepared by standard methods using the nucleic acid sequence information provided in SEQ ID NO: 1. For example, DNA can be chemically synthesized using, e.g., the phosphoramidite solid support method of Matteucci et al. , 1981, J. Am. Chem. Soc. 103:3185, the method of Yoo et al. , 1989, J. Biol. Chem. 764: 17078, or other well known methods. This can be done by sequentially linking a series of oligonucleotide cassettes comprising pairs of synthetic oligonucleotides, as described below.

Of course, due to the degeneracy of the genetic code, many different nucleotide sequences can encode polypeptides having the amino acid sequences defined by SEQ ID NO: 2-11 or subsequences thereof. The codons can be selected for optimal expression in prokaryotic or eukaryotic systems. Such degenerate variants are also encompassed by this invention.

In certain embodiments, the invention encompasses isolated nucleic acid fragments comprising all or part of the individual nucleic acid sequences disclosed herein. The fragments are at least about 12 nucleotides in length, preferably at least about 18 nucleotides in length, more preferably at least about 20-25 nucleotides or more in length, and most preferably 35-55 or more nucleotides.

The nucleic acids may be isolated directly from cells. Alternatively, the polymerase chain reaction (PCR) method can be used to produce the nucleic acids of the invention, using either chemically synthesized strands or genomic material as templates. Primers used for PCR can be synthesized using the sequence information provided herein and can further be designed to introduce appropriate new restriction sites, if desirable, to facilitate incorporation into a given vector for recombinant expression.

The encoded S. αwrews-polypeptides may be expressed by using many known vectors, such as pUC plasmids, pET plasmids (Novagen, Inc., Madison, WI), or pRSET or pREP (Invitrogen, San Diego, CA), and many appropriate host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art. The particular choice of vector/host is not critical.

The nucleic acids of the present invention find use as templates for the recombinant production of S. aureus-deήved peptides or polypeptides and as probes and primers for the detection of S. aureus and related homologs.

The nucleic acids described herein may be used as a probe to identify homologous genes in other bacterial species. Homology may be determined experimentally. Alternatively, homology analysis may be performed computationally. In practicing the present invention, a gene that shares at least about 70% DNA sequence homology at the nucleotide level with the genome of another bacterial species is considered to be present in that bacterial species.

The determination that a gene is present in another bacterial species may be achieved using any technique known in the art. Appropriate techniques include without limitation hybridization to genomic DNA, colony hybridization to a genomic or cDNA library, polymerase chain reaction (PCR) using degenerate primers or gene- specific primers and genomic DNA as a template, genetic complementation, antibody cross-reactivity, or biochemical complementation in vitro.

In applying these techniques, conditions are established that discriminate different levels of homology between probe and template. For example, for hybridization of a probe to immobilized DNA (whether in a Southern blot, dot blot, or colony hybridization format), varying the SSC concentration in the buffer allows the detection of hybrids having different levels of homology (IX SSC is 0.15 M NaCl - 0.015 M Na citrate). In a wash buffer containing 6M urea and 0.4% sodium dodecyl sulfate, the presence of 2X SSC, 0.5X SSC, 0. IX SSC, and 0.05X SSC allows the formation of hybrids having threshold homologies of at least 55% + 5% , 65% + 5%, 75% + 5%, and > 85%, respectively.

Preferably, once a gene has been identified in another bacteria by hybridization or PCR, the DNA sequence of the gene is determined directly. It will be understood that some methods that detect homologous sequences may result in the identification or isolation of only a portion of the entire protein-coding sequence of a particular gene. The entire protein-coding sequence can be isolated and identified, for example, by using an isolated nucleic acid encoding the known portion of the sequence, or fragments thereof, to prime a sequencing reaction with genomic DNA as template; this is followed by sequencing the amplified product. The isolated nucleic acid encoding the disclosed sequence, or fragments thereof, can also be hybridized to appropriate genomic libraries to identify clones containing additional complete segments of the protein-coding sequence of which the shorter sequence forms a part. Then, the entire protein-coding sequence, or fragments thereof, or nucleic acids encoding all or part of the sequence, or sequence-conservative or function- conservative variants thereof, may be employed in practicing the present invention. In a similar manner, additional sequences derived from the 5' and 3' flanking regions of sequence encoding the protein, including regulatory sequences, may be isolated, and the nucleotide sequence determined.

Polypeptides The invention provides polypeptide sequences comprising at least five, more preferably at least about 8 or more consecutive amino acid residues derived from any open reading frame (ORF) including complete protein-coding sequences contained within SEQ ID NO: 1 or contained within any of SEQ ID NO: 2-11. Function- conservative variants and homologs are included in the scope of the invention. Also provided are methods for producing such polypeptides.

Both the naturally occurring and recombinant forms of the polypeptides of the invention may be used to prepare antibodies or as targets for drug screening and development.

Many methods of screening for binding activity, such as, for example, the yeast two-hybrid system (Fields and Song, 1989, Nature 340:245-246), are known by those skilled in the art and may be used to practice the invention. Several methods of automated assays have been developed in recent years so as to permit screening of tens of thousands of compounds in a short period of time. Compounds which bind to the polypeptides of the invention are potentially useful as anti-bacterial agents for use in therapeutic compositions.

Polypeptides useful as immunogenic components or as targets are at least five or more residues in length. Preferably, the polypeptides comprise at least about 12, more preferably at least about 20, and most preferably at least about 30 or more residues. Methods for obtaining these polypeptides are well known and are explained in Immunochemical Methods in Cell and Molecular Biology, 1987 (Mayer and Waler, eds; Academic Press, London); Scopes, 1987, Protein Purification: Principles and Practice, Second Edition (Springer-Verlag, N.Y.) and Handbook of Experimental Immunology, 1986, Volumes I-IV (Weir and Blackwell, eds.).

Nucleic acids comprising protein-coding sequences can be used to direct the expression of S. αwrews-derived polypeptides in intact cells or in cell-free translation systems. The known genetic code, tailored if desired for more efficient expression in a given host organism, can be used to synthesize oligonucleotides encoding the desired amino acid sequences. The phosphoramidite solid support method of Matteucci et al. , 1981, J. Am. Chem. Soc. 103:3185, the method of Yoo et al. , 1989, J. Biol. Chem. 764: 17078, or other well known methods can be used for such synthesis. The resulting oligonucleotides can be inserted into an appropriate vector and expressed in a compatible host organism.

The polypeptides of the present invention, including function-conservative variants, may be isolated from wild-type or mutant S. aureus cells, or from heterologous organisms or cells (e.g., bacteria, fungi, insect, plant, and mammalian cells) including S. aureus into which a S. aureus-deήved protein-coding sequence has been introduced and expressed. Furthermore, the polypeptides may be part of recombinant fusion proteins. The polypeptides can also, advantageously, be made by in vitro translation.

Polypeptides may be chemically synthesized by commercially available automated procedures, including, without limitation, exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis.

Methods for polypeptide purification are well-known in the art, including, without limitation, preparative disc-gel electrophoresis, isoelectric focusing, sucrose density gradient centrifugation, HPLC, reversed-phase HPLC, gel filtration, ion exchange and partition chromatography, and countercurrent distribution. For some purposes, it is preferable to produce the polypeptide in a recombinant system in which the S. aureus protein contains an additional equence tag that facilitates purification, such as, but not limited to, a polyhistidine sequence. The polypeptide can then be purified from a crude lysate of the host cell by chromatography on an appropriate solid-phase matrix. Alternatively, antibodies produced against an S. aureus protein or against peptides derived therefrom can be used as purification reagents. Other purification methods are possible.

The present invention also encompasses derivatives and homologues of the S. wrews-encoded polypeptides specifically disclosed herein. For some purposes, nucleic acid sequences encoding the peptides may be altered by substitutions, additions, or deletions that provide for functionally equivalent molecules, i.e. , function-conservative variants. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of similar properties, such as, for example, positively charged amino acids (arginine, lysine, and histidine); negatively charged amino acids (aspartate and glutamate); polar neutral amino acids; and non-polar amino acids. The isolated polypeptides may be modified by, for example, phosphorylation, sulfation, acylation, or other protein modifications. They may also be modified with a label capable of providing a detectable signal, either directly or indirectly, including, but not limited to, radioisotopes and fluorescent compounds.

Antibodies

The present invention encompasses antibodies that specifically recognize bacteria-derived immunogenic components. Such antibodies can be used conventionally, e.g., as reagents for purification of S. aureus cells and components, or in diagnostic applications. Antibodies according to the present invention include polyclonal and monoclonal antibodies. The antibodies may be elicited in an animal host by immunization with immunogenic components of the invention or may be formed by in vitro immunization (sensitization) of immune cells. The immunogenic components used to elicit the production of antibodies may be isolated from bacterial cells (e.g., S. aureus cells) or chemically synthesized. The antibodies may also be produced in recombinant systems programmed with appropriate antibody-encoding DNA. Alternatively, the antibodies may be constructed by biochemical reconstitution of purified heavy and light chains.

The antibodies of this invention can be purified by standard methods, including but not limited to preparative disc-gel electrophoresis, isoelectric focusing, HPLC, reversed-phase HPLC, gel filtration, ion exchange and partition chromatography, and countercurrent distribution. Purification methods for antibodies are disclosed, e.g., in 77ze Art of Antibody Purification, 1989, Amicon Division, W.R. Grace & Co. General protein purification methods are described in Protein Purification: Principles and Practice, R.K. Scopes, Ed. , 1987, Springer-Verlag, New York, NY. The immunogenic components of this invention are useful as antigens for preparing antibodies by standard methods. These antibodies, whether polyclonal or monoclonal, can be used, e.g. , in an immobilized form bound to a solid support by well known methods, to purify the immunogenic components by immunoaffinity chromatography. It is well known in the art that epitopes generally contain at least about five amino acid residues, Ohno et al. , 1985, Proc. Nat I. Acad. Sci. USA 82:2945. Therefore, the immunogenic components of this invention will typically comprise at least five amino acid residues of the sequence of the complete polypeptide chains. Preferably, they will contain at least 7, and most preferably at least about 10 amino acid residues or more to ensure that they will be antigenic. Whether a given component is immunogenic can readily be determined by routine experimentation.

Such immunogenic components can be produced by proteolytic cleavage of larger polypeptides or by chemical synthesis or recombinant technology and are thus not limited by proteolytic cleavage sites. Preferably, smaller immunogenic components will first be rendered more immunogenic by cross-linking or by coupling to an immunogenic carrier molecule (i.e., a macromolecule having the property of independently eliciting an immunological response in a host animal, to which the immunogenic components of the invention can be covalently linked). Cross-linking or conjugation to a carrier molecule may be required because small polypeptide fragments sometimes act as haptens (molecules which are capable of specifically binding to an antibody but incapable of eliciting antibody production, i.e., they are not immunogenic). Conjugation of such fragments to an immunogenic carrier molecule renders them immunogenic through what is commonly known as the "carrier effect".

Suitable adjuvants for the vaccination of animals include but are not limited to Adjuvant 65 (containing peanut oil, mannide monooleate and aluminum monostearate) ; Freund' s complete or incomplete adjuvant; mineral gels such as aluminum hydroxide, aluminum phosphate and alum; surfactants such as hexadecylamine, octadecylamine, lysolecithin, dimethyldioctadecyl-ammonium bromide, N,N-dioctadecyl-N' ,N'-bis(2- hydroxymethyl) propane-diamine, methoxyhexadecylglycerol and pluronic polyols; polyanions such as pyran, dextran sulfate, poly IC, polyacrylic acid and carbopol; peptides such as muramyl dipeptide, dimethylglycine and tuftsin; and oil emulsions. The immunogenic components could also be administered following incorporation into liposomes or other microcarriers. Information concerning adjuvants and various aspects of immunoassays are disclosed, e.g., in the series by P. Tijssen, 1987, Practice and Theory of Enzyme Immunoassays, 3rd Edition, Elsevier, New York. Serum produced from animals thus immunized can be used directly.

Alternatively, the IgG fraction can be separated from the serum using standard methods such as plasmaphoresis or adsorption chromatography using IgG specific adsorbents such as immobilized Protein A.

Hybridomas of the invention used to make monoclonal antibodies against the immunogenic components of the invention are produced by well-known techniques. Usually, the process involves the fusion of an immortalizing cell line with a B- lymphocyte that produces the desired antibody. Alternatively, non-fusion techniques for generating immortal antibody -producing cell lines are possible, and come within the purview of the present invention, e.g. , virally-induced transformation, Casali et al. , 1986, Science 234:476. Immortalizing cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin. Most frequently, rat or mouse myeloma cell lines are employed as a matter of convenience and availability.

Techniques for obtaining the appropriate lymphocytes from mammals injected with the immunogenic components are well known. Generally, peripheral blood lymphocytes (PBLs) are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. A host animal is injected with repeated dosages of a preferably purified immunogenic component, and the animal is permitted to generate the desired antibody-producing cells before these are harvested for fusion with the immortalizing cell line. Techniques for fusion are also well known in the art, and in general involve mixing the cells with a fusing agent, such as polyethylene glycol.

Hybridomas are selected by standard procedures, such as HAT (hypoxanthine- aminopterin-thymidine) selection. From among these hybridomas, those secreting the desired antibody are selected by assaying their culture medium by standard immunoassays, such as Western blotting, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), or the like. Antibodies are recovered from the medium using standard protein purification techniques, Tijssen, 1985, Practice and Theory of Enzyme Immunoassays, Elsevier, Amsterdam.

Many references are available for guidance in applying any of the above techniques: Kohler et al. , 1980, Hybridoma Techniques, Cold Spring Harbor Laboratory, New York; Tijssen, 1985, Practice and Theory of Enzyme Immunoassays , Elsevier, Amsterdam; Campbell, 1984, Monoclonal Antibody Technology, Elsevier, Amsterdam; Hurrell, 1982, Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press, Boca Raton, FL. Monoclonal antibodies can also be produced using well known phage library systems. The use and generation of antibody fragments is also well known, e.g. , Fab fragments: Tijssen, 1985, Practice and Theory of Enzyme Immunoassays, Elsevier, Amsterdam; Fv fragments: Hochman et al , 1973, Biochemistry 12: 1130; Sharon et al , 1976, Biochemistry 15:1591; Ehrlich et al , U.S. Patent No. 4,355,023; and antibody half molecules: Auditore-Hargreaves, U.S. Patent No. 4,470,925. These also may be useful in immunoassays.

Diagnostic Applications

The invention encompasses compositions, methods, and kits useful in clinical settings for the qualitative or quantitative diagnosis of bacterial infection, i.e., the detection of bacterial components in a biological sample. These applications utilize nucleic acids, peptides/polypeptides, or antibodies specific for the bacterial components described herein. The methods may also be used to detect specific strains or to detect new strains in a patient, in particular a human patient. Both antibody- based and nucleic acid-based diagnostic methods, including PCR-based diagnostic methods are contemplated.

In the case of nucleic acid-type diagnostic methods, the sample to be analyzed may be contacted directed with the nucleic acid probes. Probes include oligonucleotides at least 12 nucleotides, preferably at least 18, and most preferably 20- 35 or more nucleotides in length. Alternatively, the sample may be treated to extract the nucleic acids contained therein. It will be understood that the particular method used to extract DNA will depend on the nature of the biological sample. The resulting nucleic acid from the sample may be subjected to gel electrophoresis or other size separation techniques, or, the nucleic acid sample may be immobilized on an appropriate solid matrix without size separation or used for PCR.

Kits suitable for antibody-based diagnostic applications typically include one or more of the following components: (i) Anti-bacterial antibodies : The antibodies may be pre-labeled ; alternatively , the antibody may be unlabelled and the ingredients for labeling may be included in the kit in separate containers, or a secondary, labeled antibody is provided; and

(ii) Reaction components: The kit may also contain other suitably packaged reagents and materials needed for the particular immunoassay protocol, including solid-phase matrices, if applicable, and standards.

Kits suitable for nucleic acid-based diagnostic applications typically include the following components:

(i) Probe DNA: The probe DNA may be pre-labeled; alternatively, the probe DNA may be unlabelled and the ingredients for labeling may be included in the kit in separate containers; and

(ii) Hybridization reagents: The kit may also contain other suitably packaged reagents and materials needed for the particular hybridization protocol, including solid-phase matrices, if applicable, and standards.

PCR based diagnostic kits are also contemplated and are encompassed by the invention. The kits referred to above may include instructions for conducting the test. Furthermore, in preferred embodiments, the diagnostic kits are adaptable to high- throughput and/or automated operation.

Therapeutic Applications

The invention encompasses agents that inhibit the growth of bacteria, including Streptococcus pneumonia, Staphylococcus aureus Enterococcus faecium, Bacillus anthracis, Escherichia coli, Haemophilus influenza, and Neisseria meningitidis. Both bacteriocidal and bacteriostatic agents, and that are identified by the methods described herein. The inhibitory agents may comprise nucleic acids, particularly antisense oligonucleotides; peptides; oligosaccharides; lipids; derivatives of any of the foregoing, or other molecules.

The inhibitory agents may be identified using methods well-known in the art, such as, for example, by screening chemical or natural product libraries for the ability to bind to, and/or inhibit or alter the function of, the nucleic acids or polypeptides of the invention. The inhibitory agents may comprise nucleic acids, particularly antisense oligonucleotides; peptides; oligosaccharides; lipids; derivatives of any of the foregoing, or other molecules. Such compounds may be found in, for example, natural product libraries, fermentation libraries (encompassing plants and microorganisms), combinatorial libraries, compound files, and synthetic compound libraries. For example, synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, NJ), Brandon Associates (Merrimack, NH), and Microsource (New Milford, CT). A rare chemical library is available from Aldrich Chemical Company, Inc. (Milwaukee, WI). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from, for example, Pan Laboratories (Bothell, WA) or MycoSearch (NC), or are readily producible. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means (Blondelle et al., TibTech 14:60, 1996). Pharmaceutical formulations suitable for antibacterial therapy comprise the antibacterial agent in conjunction with one or more biologically acceptable carriers. Suitable biologically acceptable carriers include, but are not limited to, phosphate- buffered saline, saline, deionized water, or the like. Preferred biologically acceptable carriers are physiologically or pharmaceutically acceptable carriers.

The antibacterial compositions include an antibacterial effective amount of active agent. Antibacterial effect amounts are those quantities of the antibacterial agents of the present invention that afford prophylactic protection against bacterial infections, and which result in amelioration or cure of an existing bacterial infection. This antibacterial effective amount will depend upon the agent, the location and nature of the infection, and the particular host. The amount can be determined by experimentation known in the art, such as by establishing a matrix of dosage amounts and frequencies of dosage administration and comparing a group of experimental units or subjects to each point in the matrix.

The antibacterial active agents or compositions can be formed into dosage unit forms, such as for example, creams, ointments, lotions, powders, inhalants, liquids, tablets, capsules, suppositories, sprays, or the like. If the antibacterial composition is formulated into a dosage unit form, the dosage unit form may contain an antibacterial effective amount of active agent. Alternatively , the dosage unit form may include less than such an amount if multiple dosage unit forms or multiple dosages are to be used to administer a total dosage of the active agent. Dosage unit forms can include, in addition, one or more excipient(s), diluent(s), disintegrant(s), lubricant(s), plasticizer(s), colorant(s), dosage vehicle(s), absorption enhancer (s), stabilizer (s), or the like.

For general information concerning formulations, see, e.g., Gilman et al. (eds.), 1990, Goodman and Gilman ' : The Pharmacological Basis of Therapeutics, 8th ed. , Pergamon Press; and Remington's Pharmaceutical Sciences, 17th ed., 1990, Mack Publishing Co., Easton, PA; Avis et al. (eds.), 1993, Pharmaceutical Dosage Forms: Parenteral Medications , Dekker, New York; Lieberman et al. (eds), 1990, Pharmaceutical Dosage Forms: Disperse Systems, Dekker, New York.

The antibacterial agents and compositions of the present invention are useful for preventing or treating bacterial infections. Infection prevention methods incorporate a prophylactically effective amount of an antibacterial agent or composition. A prophylactically effective amount is an amount effective to prevent bacterial infection and will depend upon the specific bacterial strain, the agent, and the host. These amounts can be determined experimentally by methods known in the art and as described above.

Bacterial infection treatment methods incorporate a therapeutically effective amount of an antibacterial agent or composition. A therapeutically effective amount is an amount sufficient to ameliorate or eliminate the infection. The prophylactically and/or therapeutically effective amounts can be administered in one administration or over repeated administrations. Therapeutic administration can be followed by prophylactic administration, once the initial bacterial infection has been resolved.

The antibacterial agents and compositions can be administered topically or systemically. Topical application is typically achieved by administration of creams, ointments, lotions, or sprays as described above. Systemic administration includes both oral and parental routes. Parental routes include, without limitation, subcutaneous, intramuscular, intraperitoneal, intravenous, transdermal, and intranasal administration.

Claims

1. An isolated polypeptide comprising an amino acid sequence shown in SEQ ID NO: 2-11.

2. The polypeptide of claim 1 which is encoded by an open reading frame contained in SEQ ID NO: 1.

3. An isolated nucleic acid encoding an amino acid sequence show in claim 1.

4. The nucleic acid of claim 3 comprising the sequence of SEQ ID: NO 1.

5. A method of screening for candidate antibacterial agents comprising: selecting as a target sequence a microbial specific sequence, which sequence is essential to the viability of a microbial species; contacting a test compound with said target sequence; and selecting as candidate antibacterial agents those test compounds which bind to the target sequence, wherein the target sequence is an isolated polypeptide having the amino acid sequence shown in any one of SEQ ID NO: 2-11 or a bacterial homolog thereof.

6. The method of claim 5 wherein said target sequence is a gram-positive bacterial homolog of any one of SEQ ID NO: 2-11.

7. The method of claim 5 wherein said target sequence is a gram- negative bacterial homolog of any one of SEQ ID NO: 2-11.

8. A method of claim 5 wherein the target sequence is a polypeptide having the amino acid sequence shown in SEQ ID NO: 6 or a derivative, fragment or homolog thereof.

9. A method according to claim 5 wherein the microbe is selected from the group consisting of Streptococcus pneumoniae, Staphylococcus aureus, Enterococcous faecium, Bacillus anthracis, Escherichia coli, Haemophilus influenza, and Neisseria meningitidis

10. A therapeutic composition comprising an agent which binds to a polypeptide sequence having an amino acid sequence shown in any one of SEQ ID NO: 2-11 or homolog thereof.

11. A therapeutic composition comprising an antimicrobial agent selected by the method of claim 5.

12. A method for preventing or treating disease caused by bacteria which comprises administering to an animal in need of such treatment a therapeutic composition defined in claim 12.

13. An antibody which binds to a polypeptide of claim 1.

14. A method for detecting a bacterial-derived nucleic acid sequence in a sample, said method comprising the steps of: (i) contacting a sample suspected to contain a bacterial-derived nucleic acid sequence with a probe comprising a nucleic acid sequence comprising at least 8 consecutive nucleotides selected from SEQ ID NO: 1 under conditions in which a hybrid can form between said probe and the bacterial-derived nucleic acid in said sample; and (ii) detecting any hybrid formed in step (i), wherein detection of said hybrid indicates the presence of a bacterial-derived nucleic acid sequence in said sample

15. The method of claim 16 further comprising amplifying said bacterial-derived sequence in said sample prior to said detecting step.

16. A method for detecting bacterial-derived antigenic components in a sample, said method comprising the steps of: (i) contacting a sample suspected to contain a bacterial- derived antigenic component with an antibody specific for said component, under conditions in which a stable antigen-antibody complex can form between said antibody and a bacterial antigenic component in said sample; and (ii) detecting any antigen-antibody complex formed in step (i), wherein detection of an antigen-antibody complex indicates the presence of at least one bacterial antigenic component in said sample, and wherein said bacterial antigenic component is encoded by a sequence contained in a sequence defined in claim 1.