ribA

Abstract: The invention provides ribA polypeptides and polynucleotides encoding ribA polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing ribA polypeptides to screen for antibacterial compounds.

Description: ribA RELATED APPLICATIONS This Application claims benefit to US Patent Application Number 08/978,458 filed November 25, 1997 FIELD OF THE INVENTION This invention relates to newly identified polynucleotides and polypeptides. and their production and uses, as well as their vanants, agonists and antagonists, and their uses In particular the invention relates to novel polynucleotides and polypeptides of the GTP cyclohydrolase II family, hereinafter referred to as "nbA" BACKGROUND OF THE INVENTION The Streptococci make up a medically important genera of microbes known to cause several types of disease in humans, including, for example, otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid Since its isolation more than 100 years ago Streptococcus pneumomae has been one of the more intensively studied microbes For example, much of our early understanding that DNA is, in fact, the genetic matenal was predicated on the work of Griffith and of Avery, Macleod and McCarty using this microbe Despite the vast amount of research with Streptococcus pneumomae, many questions concerning the virulence of this microbe remain It is particularly preferred to employ Streptococcal genes and gene products as targets for the development of antibiotics Riboflavin (vitamin B2) is a member of the B complex of vitamins which function as coenzymes m metabolic reactions Riboflavin has two coenzyme forms, flavin mononucleotide (FMN) and flavm adenme dmucleotide (FAD) which act in oxidation-reduction reactions such as the cytochrome system of electron transport and the oxidative degradation of pyruvate, fatty acids and ammo acids The first committed step in the biosynthesis of nboflavm is the opening of the lnudazole ring of GTP In the presence of 3 H2O and Mg ++ , the C-8 of GTP is released as formate accompanied by the release of pyrophosphate by the action of GTP cyclohyrolase II (GCH2, EC 3 5 4 25) This enzyme function is encoded by ribA in bacteria and rώl in yeast species Through a series of steps, involving 3.4-dιhydroxy-2-butanone 4 phosphate synthase (ribA), 6,7-dιmethyl-8-πbιtyllumazιne synthase (ribH), riboflavin synthase (rώB), pynmidine deammase and pynmidine reductase (ribG), enzymes encoded by genes within the riboflavin biosynthesis operon, riboflavin is formed Because GCH2 catalyzes the first committed step in the biosynthesis of riboflavin (Richter G, et al (1993) Biosynthesis of riboflavin cloning, sequencing, mapping, and expression of the gene coding for GTP cyclohydrolase II in Escherichia coli J Bactenol 175 4045-4051), is present in many pathogenic microorganisms, and since riboflavin biosynthesis has shown to be required for virulence in the swme pathogen Actinobacillus pleuropneumomae (Fuller, TE, et al (1996) A riboflavin auxotroph of Actinobacillus pleuropneumomae is attenuated in swine Infect Immun 64 4659-4664). it represents a broad spectrum antibacterial as well as antifungal target The frequency of Streptococcus pneumomae infections has nsen dramatically in the past few decades This has been attributed to the emergence of multiply antibiotic resistant strains and an increasing population of people with weakened immune systems It is no longer uncommon to isolate Streptococcus pneumomae strains which are resistant to some or all of the standard antibiotics This phenomenon has created a demand for both new anti-microbial agents, and diagnostic tests for this organism Clearly, there exists a need for factors, such as the πbA embodiments of the invention, that have a present benefit of being useful to screen compounds for antibiotic activity Such factors are also useful to determine their role in pathogenesis of infection, dysfunction and disease There is also a need for identification and characterization of such factors and their antagonists and agonists to find ways to prevent, ameliorate or correct such infection, dysfunction and disease Certain of the polypeptides of the invention possess amino acid sequence homology to a known Actinobacillus pleuropneumomae nbA protein (Swiss Prot Accession no P50855, GenBank Accession no U27202, Richter.G et al , J Bactenol 175 4045-4051 (1993), Fuller,T E et al , Infect Immun 64 4659-4664 (1996), Foor, F et al , Methods Enzymol 66 303-307 (1980) ) SUMMARY OF THE INVENTION It is an object of the invention to provide polypeptides that have been identified as novel nbA polypeptides by homology between the amino acid sequence set out in Table 1 [SEQ ID NO 2 or 4] and a known amino acid sequence or sequences of other proteins such as Actinobacillus pleuropneumomae nbA protein It is a further object of the invention to provide polynucleotides that encode πbA polypeptides, particularly polynucleotides that encode the polypeptide herein designated nbA In a particularly preferred embodiment of the invention, the polynucleotide compnses a region encoding nbA polypeptides compnsing a sequence set out in Table 1 [SEQ ID NO 1, 3 or 7] which includes a full length gene, or a vanant thereof In another particularly preferred embodiment of the invention, there is a novel nbA protein from Streptococcus pneumomae compnsing the ammo acid sequence of Table 1 [SEQ ID NO 2 or 4], or a variant thereof As a further aspect of the invention there are provided isolated nucleic acid molecules encoding nbA, particularly Streptococcus pneumomae nbA, including mRNAs, cDNAs, genomic DNAs Further embodiments of the invention include biologically, diagnosttcally, prophylactically, clinically or therapeuttcally useful vanants thereof, and compositions compnsing the same In accordance with another aspect of the invention, there is provided the use of a polynucleotide of the invention for therapeutic or prophylactic purposes, in particular genetic immunization Among the particularly prefened embodiments of the invention are naturally occurring allelic vanants of nbA and polypeptides encoded thereby As another aspect of the invention, there are provided novel polypeptides oi Streptococcus pneumomae refened to herein as nbA as well as biologically, diagnosttcally, prophylactically, clinically or therapeuttcally useful vanants thereof, and compositions compnsing the same Among the particularly prefened embodiments of the invention are vanants of nbA polypeptide encoded by naturally occurring alleles of the nbA gene In a prefened embodiment of the invention, there are provided methods for producing the aforementioned nbA polypepttdes In accordance with yet another aspect of the invention, there are provided inhibitors to such polypeptides, useful as antibacterial agents, including, for example, antibodies In accordance with certain prefened embodiments of the invention, there are provided products, compositions and methods for assessing nbA expression, treating disease, assaying genetic vanatton, and administering a nbA polypeptide or polynucleotide to an organism to raise an lmmunological response against a bactena, especially a Streptococcus pneumomae bactena In accordance with certain prefened embodiments of this and other aspects of the mvention. there are provided polynucleotides that hybndize to nbA polynucleotide sequences, particularly under stringent conditions In certain prefened embodiments of the mvention, there are provided antibodies against nbA polypepttdes In other embodiments of the mvention, there are provided methods for identifying compounds which bind to or otherwise interact with and inhibit or activate an acttvitv of a polypeptide or polynucleotide of the mvention compnsmg contactmg a polypeptide or polynucleotide of the mvention with a compound to be screened under conditions to permit binding to or other mteractton between the compound and the polypeptide or polynucleotide to assess the binding to or other mteractton with the compound, such binding or mteractton bemg associated with a second component capable of providing a detectable signal m response to the binding or mteractton of the polypepttde or polynucleotide with the compound, and determining whether the compound bmds to or otherwise interacts with and activates or inhibits an activity of the polypeptide or polynucleotide by detecting the presence or absence of a signal generated from the binding or interaction of the compound with the polypeptide or polynucleotide In accordance with yet another aspect of the mvention, there are provided nbA agonists and antagonists, preferablv bactenostattc or bactenocidal agonists and antagonists In a further aspect of the mvention, there are provided compositions compnsmg a nbA polynucleotide or a nbA polypepttde for administration to a cell or to a multicellular organism Vanous changes and modi-ficattons within the spmt and scope of the disclosed mvention will become readily apparent to those skilled m the art from reading the following descnpttons and from reading the other parts of the present disclosure DESCRIPTION OF THE INVENTION The mvention relates to novel nbA polypepttdes and polynucleotides as descnbed m greater detail below In particular, the mvention relates to polypeptides and polynucleotides of a novel nbA of Streptococcus pneumomae, which is related by ammo acid sequence homology to Actmobacillus pleuropneumomae ribA polypeptide. The invention relates especially to ribA having the nucleotide and amino acid sequences set out in Table 1 as SEQ ID NO: 1 and SEQ ED NO: 2 respectively. TABLE 1 ribA Polynucleotide and Polypeptide Sequences (A) Sequences from Streptococcus pneumoniae ribA polynucleotide sequence [SEQ ID NOT]. 5 ' - ATGGATTTTAGCAGAAAGGATAATCAGTCAATGGAATATCGAAAAATGACAAGAAGCATTGAGGAAGCA TTGCAGAAGGGACGACTTGTTCTTGTTATAGACGACAAGGATAGAGAAAATGAAGGAGACTTAATTTGT TCTGCACAAGCAGCTACAACAGAAAATGTTAATTTTATGGCTACTTATGCCAAAGGATTAATTTGTATG CCTATGAGCGAAAGTTTAGCTAATCAATTAATGCTTTCACCTATGGTTGAAAACAATACAGATAATCAT AAGACTGCTTTTACAGTTTCAATTGATTATAAAGAAACGACCACAGGTATTTCTGCCGAGGAAAGAGGA CTGACCGCACGTATGTGTGTAGCTGAAGATATAACACCCTCTGATTTTCGCAGGCCAGGACACATGTTT CCTTTAATTGCAAAAAAAGGTGGTGTTCTAGAAAGAAATGGACACACAGAAGCAACTGTTGATTTATTA AAATTAGCTGGACTAAAAGAGTGTGGCCTATGTTGTGAAATAATGAATCATGATGGCAAAATGATGAGA ACAGATGA TTTAATTCAGTTCTCGAAGAAACACAACATTCCACTAATTACCATCAAAGAATTACAAGAATATAGAAA AGTATATGATCAGCTGGTAGAACGAGTTTCAACTGTCAATATGCCTACTAGATACGGTAATTTCAAAGC AATTAGCTATATAGATAAACTAAATGGGGAACATCATCTTGCTCTTATTATGGGAAACATAGAGGATGA AGCCAATGTATTATGTCGGGTCCACTCCGAATGTTTAACAGGAGATGTTTTAGGCTCTTTACGTTGCGA TTGTGGACAGCAATTCGATAAAGCTATGAAAATGATTGTTGAGAATGGTTCGGGTGTCTTACTTTACTT GCGACAGGAGGGACAAGGAATTGGACTTATCAATAAATTAAAAGCCTATCATTTACAAAATCAAGGCAT GGATACGCTTGATGCCAATCTTGCATTAGGCTTTGAAGGTGATTTAAGAGAATATCATATTGGAGCACA AATGCTTAAAGATCTGGGACTTCAGTCACTTCATTTACTGACAAATAATCCTGACAAGGTTGAACAGTT AGAAAAAT ATGGAATTACCATTTCCAGTAGAATATCAATCGAAATAGAAGCCAATCCTTACGATAGTTTTTATTTAG AAACAAAGAA-AATCGAATGGGTCACATTTTAAATATGGAGGAAAAATAA -3 ' (B) Streptococcus pneumoniae ribA polypeptide sequence deduced from the polynucleotide sequence in this table [SEQ ID NO:2], NH 2 - MDFSRKDNQS!-3YRK TRSIEEALQKGRLVLVIDDKDRENEGDLICSAQAATTENVNFMATYAKGLI CM PMSESLANQU:LS PMVENNTDNHKTAFTVSI DYKETTTGI SAEERGLTARMCVAEDITPSDFRRPGHMF PLIAKKGGVLERNGHTEATVDLLKLAGLKECGLCCEIMNHDGKMMRTDDLIQFSKKHNI PLITIKELQE YRKVYDQLVERVSTVNMPTRYGNFKAI SYIDKLNGEHHLALIMGNI EDEANVLCRVHSECLTGDVLGSL RCDCGQQFDKAMKMIVENGSGVLLYLRQEGQGIGLINKLKAYHLQNQGMDTLDANLALGFEGDLREYHI GAQMLKDLGLQSLHLLTNNPDKVEQLEKYGITISSRISIEIEANPYDSFYLETKKNR GHILNMEEK- COOH (C) Polynucleotide sequences compnsmg Streptococcus pneumoniae nbA ORF sequence [SEQ ID NO 3] 5 ' - CTCCCAAGCAGATAATTGCATCATATTTTTTACTTTTTGCCATTTTTGATGCAATCAGTGGTATTTCAA AAGCTCCTGGAACCCAGGCTACCTCGATATCTTTCTCGTTTACATTCTCTCTTTTGAGATTATCTAGTG CTCCAGATAATAATTTTGAAGTTATAAATTCATTAAATCTCGCTACAACAATACCTATTTTAATATTGT TTGCTACTAAATTACCTTCATAAGTGTTCATTTATTTTCCCTCCATATTTAAAATGTGACCCATTCGAT TTTTCTTTGTTTCTAAATAAAAACTATCGTAAGGATTGGCTTCTATTTCGATTGATATTCTACTGGAAA TGGTAATTCCATATTTTTCTAACTGTTCAACCTTGTCAGGATTATTTGTCAGTAAATGAAGTGACTGAA GTCCCAGATCTTTAAGCATTTGTGCTCCAATATGATATTCTCTTAAATCACCTTCAAAGCCTAATGCAA GATTGGCATCAAGCGTATCCATGCCTTGATCTTGTAAATGATAGGCTTTTAATTTATTGATAAGTCCAA TTCCTCGTCCCTCCGGTCGCAAGTAAAGTAAGACACCCGAACCATTCTCAACAATCATTTTCATAGCTT TATCGAATTGCTGTCCACAATCGCAACGTAAAGAGCCTAAAACATCTCCTGTTAAACATTCGGAGTGGA CCCGACATAATACATTGGCTTCATCCTCTATGTTTCCCATAATAAGAGCAAGATGATGTTCCCCATTTA GTTTATCTATATAGCTAATTGCTTTGAAATTACCGTATTTAGTAGGCATATTGACA-3' (D) Streptococcus pneumoniae nbA polypeptide sequence deduced from the polynucleotide ORF sequence in this table [SEQ ID NO 4] NH 2 - MGNIEDEA VLCRVHSECLTGDVLGSLRCDCGQQFDKAMKMIVENGSGVLLYLRPEGRGIGLINKLKAY HLQDQGMDTLDANLALGFEGDLREYHIGAQMLKDLGLQSLHLLTNNPDPVEQLEKYGITISSRISIEIE ANPYDSFYLETKKNRMGHILNMEGK-COOH Deposited materials A deposit contammg a Streptococcus pneumoniae 0100993 strain has been deposited with the National Collections of Industnal and Marme Bactena Ltd (here "NCIMB"), 23 St Machar Dnve, Aberdeen AB2 1RY, Scotland on 11 Apnl 1996 and assigned deposit number 40794 The deposit was descnbed as Streptococcus pneumomae 0100993 on deposit On 17 Apnl 1996 a Streptococcus pneumomae 0100993 DNA library m E coli was similarly depositedwith the NCEVIB and assigned deposit number 40800 The Streptococcus pneumoniae strain deposit is referred to herem as "the deposited strain" or as "the DNA of the deposited strain " The deposited strain contains the full length nbA gene The sequence of the polynucleotides contained m the deposited strain, as well as the ammo acid sequence of the polypeptide encoded thereby, are controlling in the event of any conflict with any descnptton of sequences herem The deposit of the deposited strain has been made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for Purposes of Patent Procedure The strain will be nrevocably and without restnctton or condition released to the public upon the issuance of a patent The deposited strain is provided merely as convemence to those of skill m the art and is not an admission that a deposit is required for enablement such as that required under 35 U S C §112 A license may be required to make, use or sell the deposited strain, and compounds denved therefrom, and no such license is hereby granted One aspect of the mvention there is provided an isolated nucleic acid molecule encoding a mature polypepttde expressible by the Streptococcus pneumoniae 0100993 strain contained m the deposited strain Further provided by the mvention are nbA nucleottde sequences of the DNA m the deposited strain and am o acid sequences encoded thereby Also provided by the mvention are nbA polypepttde sequences isolated from the deposited stram and ammo acid sequences denved therefrom Polypeptides The polypeptides of the mvention mclude a polypepttde of Table 1 [SEQ ID NO 2 or 4] (m particular the mature polypeptide) as well as polypepttdes and fragments, particularly those which have the biological activity of nbA. and also those which have at least 70% identity to a polypeptide of Table 1 [SEQ ID NO 1. 3 or 7] or the relevant portion, preferably at least 80% identity to a polypeptide of Table 1 [SEQ ID NO 2 or 4] and more preferably at least 90% similaπty (more preferably at least 90% identity) to a polypeptide of Table 1 [SEQ ID NO 2 or 4] and still more preferably at least 95% similanty (still more preferably at least 95% identity) to a polypepttde of Table 1 [SEQ ID NO 2 or 4] and also mclude portions of such polypepttdes with such portion of the polypeptide generally containing at least 30 ammo acids and more preferably at least 50 ammo acids The mvention also mcludes polypepttdes of the formula X-(Rι) m -(R 2 )-(R 3 )n-Y wherein, at the ammo termmus, X is hydrogen, and at the carboxyl terrmnus, Y is hydrogen or a metal, R\ and R3 are any ammo acid residue, m is an mteger between 1 and 1000 or zero, n is an mteger between 1 and 1000 or zero, and R 2 is an ammo acid sequence of the mvention, particularly an ammo acid sequence selected from Table 1 In the formula above R 2 is oπented so that its ammo terminal residue is at the left, bound to R\ and its carboxy terminal residue is at the nght bound to R3 Any stretch of ammo acid residues denoted by either R group, where m and/or n is greater than 1 , may be either a heteropolymer or a homopolymer, preferably a heteropolymer A fragment is a vanant polypepttde having an ammo acid sequence that entirely is the same as part but not all of the a mo acid sequence of the aforementioned polypepttdes As w th πbA polypepttdes fragments may be "free-standing." or compnsed within a larger polypeptide of which they form a part or region, most preferably as a smgle contmuous region, a smgle larger polypeptide Prefened fragments mclude, for example, truncation polypepttdes having a portion of an ammo acid sequence of Table 1 [SEQ ID NO 2 or 4], or of vanants thereof, such as a contmuous senes of residues that mcludes the ammo terrmnus. or a contmuous senes of residues that mcludes the carboxyl termmus Degradation forms of the polypepttdes of the mvention m a host cell, particularly a Streptococcus pneumoniae, are also prefened Further prefened are fragments characterized by structural or functional attributes such as fragments that compnse alpha-he x and alpha-helix formmg regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forrmng regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic mdex regions Also prefened are biologically active fragments which are those fragments that mediate activities of nbA. mcludmg those with a similar activity or an improved activity, or with a decreased undesirable activity Also included are those fragments that are antigenic or immunogenic m an animal, especially m a human Particularly prefened are fragments compnsmg receptors or domains of enzymes that confer a function essential for viability of Streptococcus pneumoniae or the ability to initiate, or maintain cause disease m an individual, particularly a human Vanants that are fragments of the polypeptides of the mvention may be employed for producmg the coπesponding full-length polypeptide by peptide synthesis, therefore, these vanants may be employed as intermediates for producmg the full-length polypepttdes of the mvention In addition to the standard single and triple letter representations for ammo acids, the term "X" or "Xaa" may also be used in describing certain polypeptides of the mvention "X" and "Xaa" mean that any of the twenty naturally occuring amino acids may appear at such a designated position in the polypeptide sequence Polynucleotides Another aspect of the mvention relates to isolated polynucleotides, mcludmg the full length gene, that encode the nbA polypepttde having a deduced ammo acid sequence of Table 1 [SEQ ID NO 2 or 4] and polynucleotides closely related thereto and vanants thereof Usmg the information provided herein, such as a polynucleotide sequence set out m Table 1 [SEQ ID NO 1, 3 or 7], a polynucleotide of the mvention encoding nbA polypeptide may be obtained usmg standard cloning and screening methods, such as those for cloning and sequencing chromosomal DNA fragments from bactena usmg Streptococcus pneumoniae 0100993 cells as starting matenal, followed by obtaining a full length clone For example, to obtain a polynucleotide sequence of the invention, such as a sequence given in Table 1 [SEQ ID NO 1. 3 or 7], typically a library of clones of chromosomal DNA of Streptococcus pneumoniae 0100993 in E coh or some other suitable host is probed with a radiolabeled o gonucleotide, preferably a 17-mer or longer, denved from a partial sequence Clones carrying DNA identical to that of the probe can then be distinguished usmg stringent conditions By sequencing the individual clones thus identified with sequencing primers designed from the original sequence it is then possible to extend the sequence in both directions to determine the full gene sequence Conveniently, such sequencing is performed using denatured double stranded DNA prepared from a plasmid clone Suitable techniques are described by Maniatis, T . Fntsch, E F and Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989) (see in particular Screening By Hybridization 1 90 and Sequencing Denatured Double-Stranded DNA Templates 13 70) Illustrative of the mvention, the polynucleotide set out m Table 1 [SEQ ID NO 1, 3 or 7] was discovered m a DNA library denved from Streptococcus pneumoniae 0100993 The DNA sequence set out Table 1 [SEQ ID NO 1, 3 or 7] contains an open reading frame encoding a protem having about the number of ammo acid residues set forth m Table 1 [SEQ ID NO 2 or 4] with a deduced molecular weight that can be calculated usmg ammo acid residue molecular weight values well known m the art The polynucleotide of SEQ ID NO 1, between nucleottde number 1 and the stop codon which begins at nucleottde number 1237 of SEQ ID NO 1, encodes the polypepttde of SEQ ID NO 2 The nbA polypepttde of the mvention is structurally related to other proteins of the GTP cyclohydrolase II family The mvention provides a polynucleotide sequence identical over its entire length to a coding sequence in Table 1 [SEQ ID NO 1, 3 or 7] Also provided by the mvention is the coding sequence for the mature polypepttde or a fragment thereof, by itself as well as the coding sequence for the mature polypepttde or a fragment m reading frame with other coding sequence, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protem sequence The polynucleotide may also contain non-coding sequences, mcludmg for example, but not limited to non-coding 5' and 3' sequences, such as the transcnbed, non-translated sequences, termination signals, nbosome binding sites, sequences that stabilize mRNA. introns, polyadenylation signals, and additional coding sequence which encode additional ammo acids For example, a marker sequence that facilitates punficatton of the fused polypepttde can be encoded In certain embodiments of the mvention- the marker sequence is a hexa-histidine peptide, as provided m the pQE vector (Qiagen, Ine ) and descnbed m Gentz et al , Proc Natl Acad Sci , USA 86 821-824 (1989), or an HA tag (Wilson et al , Cell 37 767 (1984) Polynucleotides of the mvention also mclude. but are not limited to. polynucleotides compnsmg a structural gene and its naturally associated sequences that control gene expression A prefened embodiment of the mvention is a polynucleotide of compnsmg nucleottde 1 to the nucleottde immediately upstream of or mcludmg nucleottde 1237 set forth m SEQ ID NO 1 of Table 1, both of which encode the πbA polypepttde The mvention also mcludes polynucleotides of the formula X-(Rι) m -(R 2 )-(R 3 ) n -Y wherein, at the 5' end of the molecule. X is hydrogen, and at the 3 1 end of the molecule, Y is hydrogen or a metal. Ri and R3 is any nucleic acid residue, m is an mteger between 1 and 3000 or zero , n is an mteger between 1 and 3000 or zero, and R 2 is a nucleic acid sequence of the mvention, particularly a nucleic acid sequence selected from Table 1 In the polynucleotide formula above R 2 is onented so that its 5' end residue is at the left, bound to R and its 3' end residue is at the nght, bound to R3 Any stretch of nucleic acid residues denoted by either R group, where m and or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer hi a prefened embodiment m and/or n is an mteger between 1 and 1000. It is most prefened that the polynucleotides of the inventions are denved from Streptococcus pneumoniae, however, they may preferably be obtained from organisms of the same taxonomic genus They may also be obtained, for example, from orgamsims of the same taxonomic family or order The term "polynucleotide encoding a polypeptide" as used herem encompasses polynucleotides that mclude a sequence encoding a polypeptide of the mvention, particularly a bacteπal polypepttde and more particularly a polypepttde of the Streptococcus pneumoniae nbA having an ammo acid sequence set out m Table 1 [SEQ ID NO 2 or 4] The term also encompasses polynucleottdes that mclude a single continuous region or discontinuous regions encoding the polypeptide (for example, interrupted by integrated phage or an insertion sequence or editing) together with additional regions, that also may contain coding and/or non-coding sequences The mvention further relates to vanants of the polynucleotides descnbed herem that encode for vanants of the polypeptide having a deduced ammo acid sequence of Table 1 [SEQ ID NO 2 or 4] Vanants that are fragments of the polynucleottdes of the mvention may be used to synthesize full- length polynucleotides of the mvention Further particularly prefened embodiments are polynucleotides encoding nbA vanants. that have the ammo acid sequence of nbA polypeptide of Table 1 [SEQ ID NO 2 or 4] m which several, a few. 5 to 10. 1 to 5, 1 to 3, 2, 1 or no ammo acid residues are substituted, deleted or added, m any combination Especially prefened among these are silent substitutions, additions and deletions, that do not alter the properties and activities of nbA Further prefened embodiments of the mvention are polynucleottdes that are at least 70% identical over their entire length to a polynucleotide encoding nbA polypepttde having an ammo acid sequence set out m Table 1 [SEQ ID NO 2 or 4]. and polynucleottdes that are complementary to such polynucleotides Alternatively, most highly prefened are polynucleotides that compnse a region that is at least 80% identical over its entire length to a polynucleotide encoding nbA polypeptide and polynucleotides complementary thereto In this regard, polynucleotides at least 90% identical over their entire length to the same are particularly prefened. and among these particularly prefened polynucleotides, those with at least 95% are especially prefened Furthermore, those with at least 97% are highly prefened among those with at least 95%. and among these those with at least 98% and at least 99% are particularly highly prefened, with at least 99% bemg the more prefened Prefened embodiments are polynucleotides that encode polypeptides that retain substantially the same biological function or activity as the mature polypepttde encoded by a DNA of Table 1 [SEQ ID NO 1, 3 or 7] The mvention further relates to polynucleottdes that hybndize to the herem above-descnbed sequences In this regard, the mvention especially relates to polynucleotides that hybndize under strmgent conditions to the herem above-descnbed polynucleotides As herem used, the terms "stringent conditions" and "strmgent hybndizatton conditions" mean hybπdization will occur only if there is at least 95% and preferably at least 97% identity between the sequences An example of stringent hybridization conditions is overnight mcubation at 42°C m a solution compnsmg 50%) formamide, 5x SSC (150mM NaCl, 15mM tπsodium citrate), 50 mM sodium phosphate (pH7 6), 5x Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml denatured, sheared salmon sperm DNA, followed by washing the hybridization support in 0 lx SSC at about 65°C Hybridization and wash conditions are well known and exemplified in Sambrook, et al , Molecular Cloning A Laboratory Manual. Second Edition, Cold Spring Harbor. N Y . (1989), particularly Chapter 11 therein The mvention also provides a polynucleotide consisting essentially of a polynucleotide sequence obtainable by screening an appropnate library containing the complete gene for a polynucleotide sequence set forth in SEQ ID NO 1 under stringent hybndization conditions with a probe having the sequence of said polynucleotide sequence set forth m SEQ ID NO 1 or a fragment thereof, and isolating said DNA sequence Fragments useful for obtaining such a polynucleotide include, for example, probes and pnmers described elsewhere herem As discussed additionally herem regarding polynucleotide assays of the mvention, for instance, polynucleotides of the mvention as discussed above, may be used as a hybndization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding nbA and to isolate cDNA and genomic clones of other genes that have a high sequence siimlanty to the nbA gene Such probes generally will compnse at least 15 bases Preferably, such probes will have at least 30 bases and may have at least 50 bases Particularly prefened probes will have at least 30 bases and will have 50 bases or less For example, the coding region of the πbA gene may be isolated by screening usmg a DNA sequence provided m Table 1 [SEQ ID NO 1, 3 or 7] to synthesize an oligonucleottde probe A labeled oligonucleotide having a sequence complementary to that of a gene of the mvention is then used to screen a library of cDNA, genomic DNA or mRNA to determine which members of the library the probe hybπdizes to The polynucleotides and polypeptides of the mvention may be employed, for example, as research reagents and matenals for discovery of treatments of and diagnostics for disease, particularly human disease, as further discussed herem relatmg to polynucleotide assays Polynucleottdes of the invention that are ohgonucleotides derived from the sequences of Table 1 [SEQ ID NOS 1 or 2 or 3 or 4] may be used m the processes herem as descnbed, but preferably for PCR. to determine whether or not the polynucleotides identified herem in whole or m part are transcnbed in bactena in infected tissue It is recognized that such sequences will also have utility in diagnosis of the stage of infection and type of infection the pathogen has attained The mvention also provides polynucleotides that may encode a polypeptide that is the mature protem plus additional ammo or carboxyl-terminal ammo acids, or ammo acids intenor to the mature polypeptide (when the mature form has more than one polypepttde chain- for instance) Such sequences may play a role m processmg of a protem from precursor to a mature form, may allow protem transport, may lengthen or shorten protem half-life or may facilitate manipulation of a protem for assay or production, among other things As generally is the case in vivo, the additional ammo acids may be processed away from the mature protem by cellular enzymes A precursor protem, having the mature form of the polypeptide fused to one or more prosequences ma be an macttve form of the polypepttde When prosequences are removed such inactive precursors generally are activated Some or all of the prosequences ma} be removed before activation Generally, such precursors are called proproteins In addition to the standard A, G, C, T/U representations for nucleic acid bases, the term "N" may also be used in describing certain polynucleotides of the mvention "N" means that any of the four DNA or RNA bases may appear at such a designated position in the DNA or RNA sequence, except it is preferred that N is not a base that when taken in combmation with adjacent nucleotide positions, when read m the correct reading frame, would have the effect of generatmg a premature termination codon in such reading frame In sum. a polynucleotide of the mvention may encode a mature protein, a mature protem plus a leader sequence (which may be refened to as a preprotem), a precursor of a mature protem having one or more prosequences that are not the leader sequences of a preprotem. or a preproprotein, which is a precursor to a proprotem, having a leader sequence and one or more prosequences, which generally are removed dunng processmg steps that produce active and mature forms of the polypepttde Vectors, host cells, expression The mvention also relates to vectors that compnse a polynucleotide or polynucleottdes of the mvention. host cells that are genetically engineered with vectors of the mvention and the production of polypeptides of the mvention by recombinant techniques Cell-free translation systems can also be employed to produce such proteins usmg RNAs denved from the DNA constructs of the mvention For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the mvention Introduction of a polynucleotide mto the host cell can be effected by methods descnbed m many standard laboratory manuals, such as Davis et al , BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook et al , MOLECULAR CLONING A LABORATORY MANUAL, 2nd Ed , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic pid- mediated transfection. electroporation, transduction, scrape loading, ballistic introduction and infection Representative examples of appropnate hosts mclude bactenal cells, such as streptococci, staphylococci, enterococci E coli, streptomyces and Bacillus subtihs cells, fungal cells, such as yeast cells and Aspergillus cells, insect cells such as Drosophila S2 and Spodoptera Sf9 cells, animal cells such as CHO, COS, HeLa. C127, 3T3. BHK, 293 and Bowes melanoma cells, and plant cells A great vanety of expression systems can be used to produce the polypepttdes of the mvention Such vectors mclude. among others, chromosomal, episomal and us-denved vectors, e g , vectors denved from bactenal plasmids, from bactenophage, from ttansposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors denved from combinations thereof, such as those denved from plasrmd and bactenophage genetic elements, such as cosmids and phagemids The expression system constructs may contam control regions that regulate as well as engender expression Generally, any system or vector suitable to maintain, propagate or express polynucleottdes and/or to express a polypepttde m a host may be used for expression m this regard The appropnate DNA sequence may be inserted mto the expression system by any of a vanety of well-known and routme techniques, such as, for example, those set forth m Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL, (supra) For secretion of the translated protem mto the lumen of the endoplasmic rettculum, mto the penplasmic space or mto the extracellular environment, appropπate secretion signals may be incorporated mto the expressed polypeptide These signals may be endogenous to the polypeptide or they may be heterologous signals Polypeptides of the mvention can be recovered and punfied from recombinant cell cultures by well-known methods mcludmg ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification. Diagnostic Assays This invention is also related to the use of the ribA polynucleotides of the invention for use as diagnostic reagents. Detection of ribA in a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of a disease. Eukaryotes (herein also "individual(s)"). particularly mammals, and especially humans, particularly those infected or suspected to be infected with an organism comprising the ribA gene may be detected at the nucleic acid level by a variety of techniques. Nucleic acids for diagnosis may be obtained from an infected individual's cells and tissues, such as bone, blood, muscle, cartilage, and skin. Genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification technique prior to analysis, RNA, cDNA and genomic DNA may also be used in the same ways. Using amplification, characterization of the species and strain of prokaryote present in an individual, may be made by an analysis of the genotype of the prokaryote gene. Deletions and insertions can be detected by a change in size of the amplified product in comparison to the genotype of a reference sequence. Point mutations can be identified by hybridizing amplified DNA to labeled ribA polynucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures. DNA sequence differences may also be detected by alterations in the electrophoretic mobility of the DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing. See, e.g., Myers et al., Science, 230: 1242 (1985). Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase and SI protection or a chemical cleavage method. See, e.g., Cotton et al., Proc. Natl. Acad. Sci., USA, 85: 4397-4401 (1985). Cells carrying mutations or polymorphisms in the gene of the invention may also be detected at the DNA level by a variety of techniques, to allow for serotyping, for example. For example, RT- PCR can be used to detect mutations. It is particularly prefened to used RT-PCR in conjunction with automated detection systems, such as, for example, GeneScan. RNA, cDNA or genomic DNA may also be used for the same purpose, PCR or RT-PCR. As an example, PCR primers complementary to a nucleic acid encoding nbA can be used to identify and analyze mutations Examples of representative primers are shown below m Table 2 Table 2 Primers for amplification of ribA polynucleotides SEQ ID NO PRIMER SEQUENCE 5 5 ' -TCGCAGGCCAGGACACAT-3 ' 6 5 ' -CACAATCGCAACGTAAAGAGC-3 ' The mvention also mcludes primers of the formula X-(R 1 ) m -(R 2 )-(R 3 ) n -Y wherem, at the 5' end of the molecule, X is hydrogen, and at the 3' end of the molecule, Y is or a metal, Ri and R3 is any nucleic acid residue, m is an mteger between 1 and 20 or zero n is an mteger between 1 and 20 or zero, and R 2 is a pnmer sequence of the mvention. particularly a pnmer sequence selected from Table 2 In the polynucleotide formula above R 2 is onented so that its 5' end residue is at the left, bound to R\ and its 3' end residue is at the nght bound to R3 Any stretch of nucleic acid residues denoted by either R group, where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer bemg complementary to a region of a polynucleotide of Table 1 In a prefened embodiment m and/or n is an mteger between 1 and 10. The mvention further provides these primers with 1, 2, 3 or 4 nucleotides removed from the 5' and/or the 3' end These primers may be used for, among other things, amplifying πbA DNA isolated from a sample denved from an individual The primers may be used to amplify the gene isolated from an infected individual such that the gene may then be subject to vanous techniques for elucidation of the DNA sequence In this way, mutations m the DNA sequence may be detected and used to diagnose infection and to serotype and/or classify the infectious agent The mvention further provides a process for diagnosing, disease, preferably bacterial infections, more preferably infections by Streptococcus pneumoniae, compnsmg determining from a sample denved from an individual a mcreased level of expression of polynucleotide having a sequence of Table 1 [SEQ ID NO 1, 3 or 7] Increased or decreased expression of nbA polynucleotide can be measured using any on of the methods well known in the art for the quantatton of polynucleotides, such as, for example, amplification, PCR, RT-PCR. RNase protection, Northern blotting and other hybridization methods In addition, a diagnostic assay m accordance with the mvention for detecting over- expression of πbA protem compared to normal control tissue samples may be used to detect the presence of an infection, for example Assay techniques that can be used to determine levels of a nbA protein, m a sample denved from a host are well-known to those of skill m the art Such assay methods mclude radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays Antibodies The polypeptides of the mvention or vanants thereof, or cells expressmg them can be used as an immunogen to produce antibodies lmmunospecific for such polypepttdes "Antibodies" as used herem mcludes monoclonal and polyclonal antibodies, chimenc, smgle chain, simiamzed antibodies and humanized antibodies, as well as Fab fragments, mcludmg the products of an Fab lmmunolglobukn expression library Antibodies generated against the polypeptides of the mvention can be obtained by administering the polypeptides or epitope-beaπng fragments, analogues or cells to an animal, preferably a nonhuman, usmg routme protocols For preparation of monoclonal antibodies, any technique known m the art that provides antibodies produced by contmuous cell line cultures can be used Examples mclude vanous techniques, such as those m Kohler, G and Milstein, C , Nature 256 495-497 (1975). Kozbor et al , Immunology Today 4 72 (1983), Cole et al , pg 77-96 m MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R Liss, Ine (1985) Techniques for the production of smgle cham antibodies (U S Patent No 4,946,778) can be adapted to produce smgle chain antibodies to polypeptides of this mvention Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized antibodies Alternatively phage display technology may be utilized to select antibody genes with binding activities towards the polypeptide either from repertoires of PCR amplified v-genes of lymphocytes from humans screened for possessmg anti-nbA or from naive libraries (McCafferty, J et al , (1990), Nature 348, 552-554, Marks, J et al , (1992) Biotechnology 10, 779-783) The affinity of these antibodies can also be improved by chain shuffling (Clackson, T et al , (1991) Nature 352, 624-628) If two antigen binding domains are present each domain may be directed agamst a different epitope - termed 'bispecific' antibodies The above-descnbed antibodies may be employed to isolate or to identify clones expressmg the polypeptides to punfy the polypeptides by affinity chromatography Thus, among others, antibodies against nbA- polypeptide may be employed to treat infections, particularly bactenal infections Polypeptide vanants mclude antigenically, epitopically or l munologically equivalent vanants that form a particular aspect of this mvention The term "antigenically equivalent denvative" as used herem encompasses a polypeptide or its equivalent which will be specifically recognized by certain antibodies which, when raised to the protem or polypeptide according to the mvention, interfere with the immediate physical interaction between pathogen and mammalian host The term "lmmunologically equivalent derivative" as used herein encompasses a peptide or its equivalent which when used m a suitable formulation to raise antibodies in a vertebrate, the antibodies act to interfere with the immediate physical interaction between pathogen and mammalian host The polypeptide. such as an antigenically or lmmunologically equivalent derivative or a fusion protein thereof is used as an antigen to immunize a mouse or other animal such as a rat or chicken The fusion protem may provide stability to the polypeptide The antigen may be associated, for example by conjugation, with an immunogenic carrier prote for example bovine serum albumin (BSA) or keyhole limpet haemocyanm (KLH) Alternatively a multiple antigenic peptide comprising multiple copies of the protein or polypeptide. or an antigenically or lmmunologically equivalent polypeptide thereof may be sufficiently antigenic to improve immunogenicity so as to obviate the use of a earner Preferably, the antibody or variant thereof is modified to make it less immunogenic in the individual For example, if the individual is human the antibody may most preferably be "humanized", where the comphmentaπty determining regιon(s) of the hybπdoma-deπved antibody has been transplanted into a human monoclonal antibody , for example as descnbed m Jones, P et al (1986), Nature 321, 522-525 or Tempest et al , (1991) Biotechnology 9, 266-273 The use of a polynucleotide of the invention m genetic immunization will preferably employ a suitable delivery method such as direct injection of plasmid DNA mto muscles (Wolff et al , Hum Mol Genet 1992, 1 363, Manthorpe et al , Hum Gene Ther 1963 4, 419), delivery of DNA complexed with specific protem earners (Wu et al , J Biol Chem 1989 264.16985), coprecipitation of DNA with calcium phosphate (Benvemsty & Reshef, PNAS USA, 1986 83,9551). encapsulation of DNA in various forms of hposomes (Kaneda et al , Science 1989 243,375). particle bombardment (Tang et al , Nature 1992, 356 152, Eisenbraun et al , DNA Cell Biol 1993, 12 791) and in vivo infection using cloned retroviral vectors (Seeger et al , -P-V- S USA 1984 81,5849) Antagonists and agonists - assays and molecules Polypeptides of the mvention may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical hbranes, and natural product mixtures These substrates and ligands may be natural substrates and ligands or may be structural or functional mimettcs See, e g , Coligan et al , Current Protocols in Immunology 1(2) Chapter 5 (1991) The mvention also provides a method of screening compounds to identify those which enhance (agomst) or block (antagomst) the action of πbA polypeptides or polynucleotides, particularly those compounds that are bacteπostatic and/or bactenocidal The method of screening may mvolve high-throughput techniques For example, to screen for agomsts or antagoists. a synthetic reaction mix. a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, compnsmg nbA polypeptide and a labeled substrate or ligand of such polypepttde is mcubated m the absence or the presence of a candidate molecule that may be a nbA agomst or antagomst The ability of the candidate molecule to agonize or antagonize the nbA polypepttde is reflected m decreased binding of the labeled ligand or decreased production of product from such substrate Molecules that bmd gratuitously, i e , without mducmg the effects of nbA polypeptide are most likely to be good antagonists Molecules that bmd well and mcrease the rate of product production from substrate are agomsts Detection of the rate or level of production of product from substrate may be enhanced by usmg a reporter system Reporter systems that may be useful m this regard mclude but are not limited to colonmetnc labeled substrate converted mto product, a reporter gene that is responsive to changes m πbA polynucleotide or polypeptide activity, and binding assays known m the art Another example of an assay for nbA antagonists is a competitive assay that combmes nbA and a potential antagomst with nbA-binding molecules, recombinant nbA binding molecules, natural substrates or ligands, or substrate or ligand mimettcs, under appropnate conditions for a competitive inhibition assay The nbA molecule can be labeled, such as by radioactivity or a colonmetnc compound, such that the number of nbA molecules bound to a binding molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagomst Potential antagonists mclude small organic molecules, peptides, polypeptides and antibodies that bmd to a polynucleotide or polypeptide of the mvention and thereby inhibit or extinguish its activity Potential antagonists also may be small organic molecules, a peptide, a polypeptide such as a closely related protem or antibody that bmds the same sites on a binding molecule, such as a binding molecule, without mducmg πbA-mduced activities, thereby preventmg the action of nbA by excluding nbA from binding Potential antagonists mclude a small molecule that bmds to and occupies the binding site of the polypeptide thereby preventmg binding to cellular binding molecules, such that normal biological activity is prevented Examples of small molecules mclude but are not limited to small organic molecules, peptides or peptide-like molecules Other potential antagonists mclude antisense molecules (see Okano, J Neurochem 56 560 (1991), OLLGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION. CRC Press, Boca Raton. FL (1988). for a descnption of these molecules) Prefened potential antagonists mclude compounds related to and vanants of nbA Each of the DNA sequences provided herein may be used m the discovery and development of antibacterial compounds The encoded protem, upon expression, can be used as a target for the screemng of antibactenal drugs Additionally, the DNA sequences encodmg the ammo terminal regions of the encoded protein or Shine-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the coding sequence of interest The mvention also provides the use of the polypeptide, polynucleotide or inhibitor of the invention to interfere with the initial physical interaction between a pathogen and mammalian host responsible for sequelae of infection In particular the molecules of the invention ma} be used in the prevention of adhesion of bacteria, in particular gram positive bacteria, to mammalian extracellular matnx proteins on in-dwelling devices or to extracellular matrix protems m wounds, to block nbA protem-mediated mammalian cell invasion by, for example, initiating phosphorylation of mammalian tyrosme kmases (Rosenshtne et al . Infect Immun 60 2211 (1992), to block bacteπal adhesion between mammalian extracellular matnx protems and bactenal πbA protems that mediate tissue damage and, to block the normal progression of pathogenesis in infections initiated other than by the implantation of in-dwelling devices or by other surgical techniques The antagonists and agomsts of the mvention may be employed, for instance, to inhibit and treat diseases Helicobacter pylori (herein H pylori) bacteria infect the stomachs of over one-third of the world's population causing stomach cancer, ulcers, and gastritis (International Agency for Research on Cancer (1994) Schistosomes, Liver Flukes and Helicobacter Pylon (International Agency for Research on Cancer, Lyon, France, http //www uicc ch/ecp/ecp2904 htm) Moreover, the international Agency for Research on Cancer recently recognized a cause-and- effect relationship between H pylori and gastric adenocarcinoma, classifying the bacterium as a Group I (definite) carcinogen Preferred antimicrobial compounds of the mvention (agomsts and antagonists of nbA) found using screens provided by the mvention. particularly broad- spectrum antibiotics, should be useful in the treatment of H pylori infection Such treatment should decrease the advent of H #y/or. -induced cancers, such as gastrointestinal carcinoma Such treatment should also cure gastric ulcers and gastritis Vaccines Another aspect of the mvention relates to a method for mducing an lmmunological response in an mdividual, particularly a mammal which comprises inoculating the mdividual with nbA, or a fragment or variant thereof, adequate to produce antibody and or T cell immune response to protect said individual from infection, particularly bacterial mfection and most particularly Streptococcus pneumoniae mfection Also provided are methods whereby such lmmunological response slows bacterial replication Yet another aspect of the mvention relates to a method of inducing lmmunological response m an individual which compnses dehvermg to such mdividual a nucleic acid vector to direct expression of nbA, or a fragment or a variant thereof, for expressing nbA, or a fragment or a vanant thereof in vivo in order to mduce an lmmunological response, such as. to produce antibody and/ or T cell immune response, mcludmg. for example, cytokme-producing T cells or cytotoxic T cells, to protect said mdividual from disease, whether that disease is already established within the mdividual or not One wa} of administering the gene is by acceleratmg it mto the desired cells as a coatmg on particles or otherwise Such nucleic acid vector may comprise DNA, RNA, a modified nucleic acid, or a DNA/RNA hybrid A further aspect of the invention relates to an lmmunological composition which, when introduced mto an mdividual capable or havmg induced withm it an lmmunological response, induces an lmmunological response m such mdividual to a nbA or protem coded therefrom, wherein the composition comprises a recombinant nbA or protem coded therefrom compnsmg DNA which codes for and expresses an antigen of said πbA or protein coded therefrom The lmmunological response may be used therapeutically or prophylactically and may take the form of antibody immunity or cellular immunity such as that arising from CTL or CD4+ T cells A nbA polypeptide or a fragment thereof may be fused with co-protem which may not by itself produce antibodies, but is capable of stabilizing the first protem and producmg a fused protem which will have immunogenic and protective properties Thus fused recombinant protem, preferably further compnses an antigenic co-protem, such as hpoprotein D from Hemophilus influenzae. Glutathione-S-transferase (GST) or beta-galactosidase, relatively large co-protems which solubihze the protem and facilitate production and purification thereof Moreover, the co-protem may act as an adjuvant in the sense of providing a generalized stimulation of the immune system The co-protem may be attached to either the ammo or carboxy termmus of the first protem Provided by this mvention are compositions, particularly vaccine compositions, and methods compnsmg the polypeptides or polynucleotides of the invention and immunostimulatory DNA sequences, such as those described in Sato, Y et al Science 273 352 (1996) Also, provided by this invention are methods using the descnbed polynucleotide or particular fragments thereof which have been shown to encode non-variable regions of bactenal cell surface protems m DNA constructs used in such genetic immunization experiments m animal models of infection with Streptococcus pneumoniae will be particularly useful for identifying protem epitopes able to provoke a prophylactic or therapeutic immune response It is believed that this approach will allow for the subsequent preparation of monoclonal antibodies of particular value from the requisite organ of the animal successfully resisting or clearing infection for the development of prophylactic agents or therapeutic treatments of bactenal infection, particularly Streptococcus pneumoniae mfection, m mammals, particularly humans The polypeptide may be used as an antigen for vaccmation of a host to produce specific antibodies which protect against invasion of bactena, for example by blocking adherence of bactena to damaged tissue Examples of tissue damage include wounds in skm or connective tissue caused, e g , by mechamcal, chemical or thermal damage or by implantation of indwellmg devices, or wounds in the mucous membranes, such as the mouth, mammary glands, urethra or vagina The invention also mcludes a vaccine formulation which comprises an immunogenic recombmant protein of the invention together with a suitable carrier Since the protem may be broken down in the stomach, it is preferably administered parenterally, mcludmg, for example, administration that is subcutaneous, intramuscular, mtravenous, or mtradermal Formulations suitable for parenteral administration include aqueous and non-aqueous stenle mjection solutions which may contain anti-oxidants, buffers, bactenostats and solutes which render the formulation rnsotomc with the bodily fluid, preferably the blood, of the individual, and aqueous and non-aqueous sterile suspensions which may mclude suspending agents or thickening agents The formulations may be presented m unit-dose or multi-dose containers, for example, sealed ampules and vials and may be stored m a freeze-dned condition requinng only the addition of the sterile liquid carrier immediately prior to use The vaccine formulation may also mclude adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation While the invention has been described with reference to certain nbA protem, it is to be understood that this covers fragments of the naturally occurnng protem and similar protems with additions, deletions or substitutions which do not substantially affect the immunogenic properties of the recombmant protem Compositions, kits and administration The mvention also relates to compositions compnsmg the polynucleotide or the polypeptides discussed above or their agomsts or antagonists The polypeptides of the mvention may be employed m combination with a non-steπle or stenle earner or earners for use with cells, tissues or organisms, such as a pharmaceutical earner suitable for administration to a subject Such compositions compnse, for instance, a media additive or atherapeutically effective amount of a polypeptide of the mvention and a pharmaceutically acceptable earner or excipient Such earners may mclude. but are not limited to. saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof The formulation should suit the mode of administration The mvention further relates to diagnostic and pharmaceutical packs and kits compnsmg one or more containers filled with one or more of the ingredients of the aforementioned compositions of the mvention Polypeptides and other compounds of the mvention may be employed alone or m conjunction with other compounds, such as therapeutic compounds The pharmaceutical compositions may be administered m any effective, convement manner mcludmg. for instance, administration by topical, oral, anal, vaginal, mtravenous. lntrapentoneal, intramuscular, subcutaneous, mtranasal or mtradermal routes among others In therapy or as a prophylactic, the active agent may be administered to an mdividual as an mjectable composition, for example as a sterile aqueous dispersion, preferably isotomc Alternatively the composition may be formulated for topical application for example in the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, mouthwash, impregnated dressings and sutures and aerosols, and may contain appropnate conventional additives, mcludmg, for example, preservatives, solvents to assist drug penetration, and emollients m ointments and creams Such topical formulations may also contain compatible conventional earners, for example cream or ointment bases, and ethanol or oleyl alcohol for lotions Such carriers may constitute from about 1% to about 98% weight of the formulation, more usually they will constitute up to about 80% by weight of the formulation For administration to mammals, and particularly humans, it is expected that the daily dosage level of the active agent will be from 0 01 mg/kg to 10 mg kg, typically around 1 mg/kg The physician m any event will determine the actual dosage which will be most suitable for an individual and will vary with the age, weight and response of the particular mdividual The above dosages are exemplary of the average case There can, of course, be mdividual instances where higher or lower dosage ranges are merited, and such are withm the scope of this mvention In-dwelling devices mclude surgical implants, prosthetic devices and catheters, l e , devices that are introduced to the body of an individual and remain m position for an extended time Such devices mclude, for example, artificial jomts, heart valves, pacemakers, vascular grafts, vascular catheters, cerebrospmal fluid shunts, urmary catheters, contmuous ambulatory peritoneal dialysis (CAPD) catheters The composition of the invention may be administered by injection to achieve a systemic effect aga st relevant bacteria shortly before insertion of an m-dwelhng device Treatment may be contmued after surgery during the m-body time of the device In addition, the composition could also be used to broaden peπoperative cover for any surgical technique to prevent bactenal wound infections, especially Streptococcus pneumoniae wound infections Many orthopaedic surgeons consider that humans with prosthetic jomts should be considered for antibiotic prophylaxis before dental treatment that could produce a bacteremia Late deep mfection is a serious complication sometimes leading to loss of the prosthetic jomt and is accompanied by significant morbidity and mortality It may therefore be possible to extend the use of the active agent as a replacement for prophylactic antibiotics in this situation In addition to the therapy described above, the compositions of this mvention may be used generally as a wound treatment agent to prevent adhesion of bactena to matrix proteins exposed in wound tissue and for prophylactic use m dental treatment as an alternative to, or in conjunction with, antibiotic prophylaxis Alternatively, the composition of the invention may be used to bathe an indwelling device immediately before insertion The active agent will preferably be present at a concentration of 1 μg ml to 1 Omg/ml for bathing of wounds or indwelling devices A vaccine composition is conveniently m injectable form Conventional adjuvants may be employed to enhance the immune response A suitable unit dose for vaccmation is 0 5-5 microgram/kg of antigen, and such dose is preferably admimstered 1-3 times and with an interval of 1 -3 weeks With the mdicated dose range, no adverse toxicological effects will be observed with the compounds of the invention which would preclude their administration to suitable individuals Each reference disclosed herein is incorporated by reference herem m its entirety Any patent application to which this application claims priority is also incorporated by reference herem in its entirety GLOSSARY The following definitions are provided to facilitate understanding of certain terms used frequently herem "Dιsease(s)" means and disease caused by or related to infection by a bactena, mcludmg otttis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example mfection of cerebrospmal fluid "Host cell" is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous polynucleotide sequence "Identity," as known m the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences hi the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences "Identity" and "similarity" can be readily calculated by known methods, mcludmg but not limited to those described in (Computational Molecular Biology, Lesk, A M , ed , Oxford University Press, New York, 1988, Biocomputing Informatics and Genome Projects. Smith, D W , ed , Academic Press, New York, 1993, Computer Analysis of Sequence Data. Part I, Gnffin, A M , and Griffin, H G , eds , Humana Press, New Jersey, 1994, Sequence Analysis in Molecular Biology, von Heinje, G , Academic Press, 1987, and Sequence Analysis Primer, Gnbskov. M and Devereux, J , eds , M Stockton Press, New York, 1991, and Caπllo, H , and Lipman. D , SLAM J Applied Math , 48 1073 (1988) Preferred methods to determine identity are designed to give the largest match between the sequences tested Methods to determine identity and similarity are codified m publicly available computer programs Preferred computer program methods to determine identity and similanty between two sequences include, but are not limited to, the GCG program package (Devereux, J . et al , Nucleic Acids Research 12(1) 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S F et al , J Molec Biol 215 403-410 (1990) The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul. S , et al , NCBI NLM NM Bethesda. MD 20894, Altschul. S , et al , J Mol Biol 215 403-410 (1990) As an illustration, by a polynucleotide having a nucleottde sequence having at least, for example, 95% "identity" to a reference nucleotide sequence of SEQ ID NO 1 it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence of SEQ ID NO 1 In other words, to obtam a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides m the reference sequence may be inserted into the reference sequence These mutations of the reference sequence may occur at the 5 or 3 terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence Analogously , by a polypeptide having an amino acid sequence having at least, for example, 95% identity to a reference ammo acid sequence of SEQ ID NO 2 is intended that the ammo acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may mclude up to five ammo acid alterations per each 100 ammo acids of the reference amino acid of SEQ ID NO 2 In other words, to obta a polypepttde having an ammo acid sequence at least 95% identical to a reference ammo acid sequence, up to 5% of the ammo acid residues in the reference sequence may be deleted or substituted with another ammo acid, or a number of ammo acids up to 5% of the total ammo acid residues in the reference sequence may be inserted mto the reference sequence These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups withm the reference sequence "Isolated" means altered "by the hand of man" from its natural state, i e , if it occurs m nature, it has been changed or removed from its ongmal environment or both For example, a polynucleotide or a polypeptide naturally present m a living organism is not "isolated," but the same polynucleotide or polypepttde separated from the coexisting matenals of its natural state is "isolated", as the term is employed herem Moreover, a polynucleotide or polypepttde that is introduced mto an organism by transformation, genetic manipulation or by any other recombinant method is "isolated" even if it is still present m said organism, which organism may be living or nonliving "Polynucleotide(s)" generally refers to any polyπbonucleotide or polydeoxnbonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA "Polynucleotide(s)" mclude, without limitation, smgle- and double-stranded DNA, DNA that is a mixture of smgle- and double- stranded regions or smgle-, double- and tnple-stranded regions, smgle- and double-stranded RNA, and RNA that is mixture of smgle- and double-stranded regions, hybπd molecules compnsmg DNA and RNA that may be single-stranded or, more typically, double-stranded, or tnple-stranded regions, or a mixture of smgle- and double-stranded regions In addition, "polynucleotide" as used herem refers to tnple-stranded regions compnsmg RNA or DNA or both RNA and DNA The strands m such regions may be from the same molecule or from different molecules The regions may mclude all of one or more of the molecules, but more typically mvolve only a region of some of the molecules One of the molecules of a tπple-helical region often is an oligonucleottde As used herem, the term "polynucleottde(s)" also mcludes DNAs or RNAs as descnbed above that contam one or more modified bases Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleottde(s)" as that term is mtended herem Moreover, DNAs or RNAs compnsing unusual bases, such as mosme, or modified bases, such as tntylated bases, to name just two examples, are polynucleottdes as the term is used herem It will be appreciated that a great vanety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill m the art The term "polynucleottde(s)" as it is employed herem embraces such chemically, enzymatically or metabohcally modified forms of polynucleotides, as well as the chemical foims of DNA and RNA charactenstic of viruses and cells, mcludmg, for example, simple and complex cells "Polynucleotide(s)" also embraces short polynucleotides often referred to as olιgonucleotide(s) "Polvpeptide(s)" refers to any peptide or protem compnsmg two or more ammo acids jomed to each other by peptide bonds or modified pepttde bonds "Polypeptιde(s)" refers to both short chains, commonly refened to as peptides. oligopepttdes and ohgomers and to longer chains generally refened to as proteins Polypepttdes may contam ammo acids other than the 20 gene encoded ammo acids "Pohpepttde(s)" mclude those modified either by natural processes, such as processmg and other post-translattonal modifications, but also by chemical modification techniques Such modifications are well descnbed m basic texts and m more detailed monographs, as well as m a voluminous research literature, and they are well known to those of skill m the art It will be appreciated that the same type of modification may be present m the same or varying degree at several sites m a given polypeptide Also, a given polypeptide may contam many types of modifications Modifications can occur anywhere m a polypeptide, mcludmg the peptide backbone, the ammo acid side-chains, and the ammo or carboxyl termini Modifications mclude, for example, acetylation. acylatton, ADP-nbosylatton, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleottde deπvattve. covalent attachment of a hpid or lipid denvattve, covalent attachment of phosphotidy nositol. cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteme, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylatton, GPI anchor formation, lodination, methylation, mynstoylation, oxidation, proteolytic processmg, phosphorylation, prenylation. racemization, glycosylation, hpid attachment, sulfation, gamma- carboxylation of glutamic acid residues, hydroxylation and ADP-πbosylation, selenoylation, sulfation, transfer-RNA mediated addition of ammo acids to protems, such as arginylatton. and ubiquitination See. for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed , T E Creighton, W H Freeman and Company, New York (1993) and Wold, F , Posttranslational Protem Modifications Perspectives and Prospects, pgs 1-12 m POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS. B C Johnson, Ed , Academic Press, New York (1983), Seifter et al , Meth Enzymol 182 626-646 (1990) and Rattan et al , Protein Synthesis Posttranslational Modifications and Aging, Ann N Y Acad Sci 663 48- 62 (1992) Polypeptides may be branched or cyclic, with or without branching Cyclic, branched and branched circular polypepttdes may result from post-translational natural processes and may be made by entirely synthetic methods, as well 'Nanant(s)" as the term is used herem, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide Changes m the nucleotide sequence of the variant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result in ammo acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below A typical variant of a polypeptide differs in ammo acid sequence from another, reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the vanant are closely similar overall and, in many regions, identical A vanant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combmation A substituted or inserted ammo acid residue may or may not be one encoded by the genetic code A variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic vanant, or it may be a variant that is not known to occur naturally Non- naturally occurring vanants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans EXAMPLES The examples below are earned out usmg standard techniques, which are well known and routme to those of skill m the art, except where otherwise descnbed m detail The examples are illustrative, but do not limit the mvention Example 1 Strain selection, Library Production and Sequencing The polynucleotide havmg a DNA sequence given m Table 1 [SEQ ID NO 1, 3 or 7] was obtained from a library of clones of chromosomal DNA of Streptococcus pneumoniae m E coli The sequencing data from two or more clones containing overlapping Streptococcus pneumoniae DNAs was used to construct the contiguous DNA sequence in SEQ ID NO 1 Libraries mav be prepared bv routme methods, for example Methods 1 and 2 below Total cellular DNA is isolated from Streptococcus pneumoniae 0100993 according to standard procedures and size-fractionated by either of two methods Method 1 Total cellular DNA is mechanically sheared by passage through a needle in order to size-fractionate accordmg to standard procedures DNA fragments of up to 1 lkbp m size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added Fragments are ligated mto the vector Lambda ZapII that has been cut with EcoRI, the library packaged by standard procedures and E coli infected with the packaged library The library is amplified by standard procedures Method 2 Total cellular DNA is partially hydrolyzed with a one or a combination of restriction enzymes appropnate to generate a senes of fragments for cloning mto library vectors (e g , Rsal, Pall, Alul, Bshl235I), and such fragments are size-fractionated accordmg to standard procedures EcoRI linkers are ligated to the DNA and the fragments then ligated mto the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E coli infected with the packaged library The library is amplified by standard procedures Example 2 ribA Characterization Gene expression in vivo Recently several novel approaches have been described which purport to follow global gene expression dunng mfection (Chuang, S et al , (1993), Mahan, M J et al , Science 259 686-688 (1993), Hensel, M et al , Science 269 400-403 (1995) These new techniques have so far been demonstrated with gram negative pathogen infections but not with infections with gram positives presumably because the much slower development of global transposon mutagenesis and suitable vectors needed for these strategies m these orgamsms, and m the case of that process described by Chuang, S et al , J Bactenol 775 2026-2036 (1993) the difficulty of isolating suitable quantities of bacterial RNA free of mammalian RNA denved from the infected tissue to furnish bacteπal RNA labelled to sufficiently high specific activity The present invention employs a novel technology to determine gene expression m the pathogen at different stages of infection of the mammalian host Use of the technology of the present mvention enables identification of bactenal genes transcribed during infection, inhibitors of which would have utility in anti-bacterial therapy Specific inhibitors of such gene transcription or of the subsequent translation of the resultant niRNA or of the function of the corresponding expressed protems would have utility in antibacterial therapy The determination of expression during infection of a gene from Streptococcus pneumoniae Lung tissue from a 48 hr pneumonia infection of Streptococcus pneumomae #100993 m the mouse is efficiently disrupted and processed in the presence of acid phenol and detergent to provide a mixture of animal and bacterial RNA By freezing the tissue immediately in liquid nitrogen, and processmg the tissue samples while still frozen, changes m the population of bacterial mRNA is minimized The resultant total RNA is free of DNA and protein (mcludmg RNAases and DNAases) The optimal conditions for disruption and processmg to give high yields of bacterial mRNA with transcripts of long length are followed by reverse transcribing the resultmg mRNA to cDNA and amplified with ORF-specific primers for a bactenal gene known to be expressed constitutively and at low copy number Aspects of this example II, part b, are modifications of a published protocol (Cheung, et al , Anal Biochem (1994) 222 511- 514) a) Isolation of lung tissue infected with Streptococcus pneumoniae #0100993 from a mouse respiratory tract infection model. Streptococcus pneumoniae #100993 was seeded onto TSA (Tryptic Soy Agar, BBL) plates containing 5% horse blood and allowed to grow overnight at 37°C in a C0 2 incubator. Bacterial growth was scraped into 5 ml of phosphate-buffered saline (PBS) and adjusted to an Agoo ~ 0-6 (4 x lOfyml). Mice (male CBA/J-1 mice, approximately 20g) were anaesthetized with isoflurane and 50 microliters of the prepared bacterial inoculum was delivered by intranasal instillation. Animals were allowed to recover and observed twice daily for signs of moribundancy. Forty-eight hours after infection the animals were euthanized by carbon dioxide overdose and their torsos swabbed with ethanol and then RNAZap. The torso was then opened, and the lungs were aseptically removed.. Half of each pair of lungs was placed in a cryovial and immediately frozen in liquid nitrogen; the other half was used for bacterial enumeration after homogenization of the tissue in 1 ml of PBS . b) Isolation of Streptococcus pneumoniae #0100993 RNA from infected tissue samples Infected tissue samples, in 2-ml cryo-strorage tubes, are removed from liquid nitrogen storage for immediate processing of the frozen tissue. In a microbiological safety cabinet the samples are disrupted up to eight at a time. To disrupt the bacteria within the tissue sample, 50-100 mg of the tissue is transfered to a FastRNA tube containing a silica/ceramic matrix (BIO 101). Immediately, 1 ml of extraction reagents (FastRNA reagents, BIO 101) are added to give a sample to reagent volume ratio of approximately 1 to 20. The tubes are shaken in a reciprocating shaker (FastPrep FP120, BIO101) ata setting of 5.5 to 6 for 20-120 sec. The crude RNA preparation is extracted with chloroform/isoamyl alcohol, and precipitated with DEPC-treated/Isopropanol Precipitation Solution (BIO101). RNA preparations are stored in this isopropanol solution at -80°C if necessary. The RNA is pelleted (12,000g for 10 min.), washed with 75% ethanol (v/v in DEPC-treated water), air-dried for 5-10 min, and resuspended in 0.1 ml of DEPC-treated water. Quality of the RNA isolated is assessed by the ability to detect bacterial transcripts up to 2 kb in length by RT-PCR (as described below in section). To demonstrate the isolation of bacterial RNA from the infected tissue, samples of RNA are reverse transcribed, and the presence of a constitutively expressed gene is detected through the use of quantitative PCR in the presence of a TaqMan probe (as described below). c) The removal of DNA from Streptococcus pneumoniae (0100993)-derived RNA DNA was removed from 50 microgram samples of RNA by a 30 minute treatment at 37°C with 10 units of RNAase-free DNAasel (GeneHunter) m the buffer supplied m a final volume of 57 microliters The DNAase was inactivated and removed by phenol chloroform extraction RNA was precipitated with 5 microliters of 3 M NaOAc and 200 microliters 100% EtOH, and pelleted by centnfugation at 12,000g for 10 minutes The RNA is pelleted (12,000g for 10 mm ), washed with 75% ethanol (v/v in DEPC-treated water), air-dried for 5-10 mm, and resuspended m 10-20 microliters of DEPC-treated water RNA yield is quantitated by OD 2 6Q after 1 1000 dilution of the cleaned RNA sample RNA is stored at -80°C if necessary and reverse-transcribed withm one week d) The preparation of cDNA from RNA samples derived from infected tissue 10 microhter samples of DNAase treated RNA are reverse transcribed using a Superscript Preamphfication System for First Strand cDNA Synthesis kit (Gibco BRL, Life Technologies) according to the manufacturers instructions 1 nanogram of random hexamers is used to prime each reaction Controls without the addition of SuperScnptll reverse transcnptase are also run Both +/-RT samples are treated with RNaseH before proceeding to the PCR reaction e) The use of PCR to determine the quality of bacterial RNA derived from infected tissue Long transcnpts, which are expected to be of low copy number withm the bactenal cell, such as penicillin-b ding protem 2 (PBP2), are reverse transcribed with random primers as descnbed above and amplified by the following PCR method using ORF-specific primers, in order to ascertain the quality, represented by length amplified, of the mRNA obtamed during extraction and punfication PCR reactions are set up on ice m 0 2 ml tubes m a total volume of 50 ul by adding the following components [final concentration] AmphTaq PCR Buffer II (IX), 1 5mM MgCL 1 mM dNTPs, 0 5uM forward pnmer, 0 5uM reverse pnmer, and 2 ul reverse-transcribed RNA PCR reactions are run on a PE GeneAmp PCR System 9600 with an initial step of 94°C for 2mιn, followed by 35 cycles of 94 °C for 30sec, 42 °C for 30sec and 72 °C for 30sec, followed by a final extensison at 72 °C for 7mm f) The use of PCR to determine the presence of a bacterial cDNA species PCR reactions are set up as described above using 0.5 microM each of the ORF specific forward and reverse primers. PCR product in 20 microliter aliquots are separated by electrophoresis through 1 to 1.5% 1 x TBE agarose gels or 10% lx TBE acrylamide gels. PCR product is visualized by staining the gel with ethidium bromide. Size estimates are made by comparison to a 100 bp DNA Ladder (Gibco BRL. Life Technologies). Alternatively, if the PCR products are conveniently labelled by the use of a labelled PCR primer (e.g. labelled at the 5'end with a dye) a suitable aliquot of the PCR product is run out on a polyacrylamide sequencing gel and its presence and quantity detected using a suitable gel scanning system (e.g. ABI Prism^M 377 Sequencer using GeneScan^M software as supplied by Perkin Elmer). RT/PCR controls may include +/- reverse transcriptase reactions, 16s rRNA primers or DNA specific primer pairs designed to produce PCR products from non-transcribed Streptococcus pneumoniae 0100993 genomic sequences. To test the efficiency of the primer pairs they are used in DNA PCR with Streptococcus pneumomae 0100993 total DNA. PCR reactions are set up and run as described above using approx. 1 microgram of DNA in place of the cDNA and 35 cycles of PCR. Primer pairs which fail to give the predicted sized product in either DNA PCR or RT/PCR are PCR failures and as such are uninformative. Of those which give the correct size product with DNA PCR two classes are distinguished in RT/PCR: 1. Genes which are not transcribed in vivo reproducibly fail to give a product in RT/PCR; and 2. Genes which are transcribed in vivo reproducibly give the correct size product in RT/PCR and show a stronger signal in the +RT samples than the signal (if at all present) in -RT controls. g) The use of PCR and fluorogenic probes to determine the presence of a bacterial cDNA species Specific sequence detection occurs by amplification of target sequences in the PE Applied Biosystems 7700 Sequence Detection System in the presence of an oligonucleotide probe labeled at the 5' and 3' ends with a reporter and quencher fluorescent dye, respectively (FQ probe), which anneals between the two PCR primers. Only specific product will be detected when the probe is bound between the primers. As PCR amplification proceeds, the 5'- nuclease activity of Taq polymerase initially cleaves the reporter dye from the probe. The signal generated when the reporter dye is physically separated from the quencher dye is detected by measuring the signal with an attached CCD camera. Each signal generated equals one probe cleaved which corresponds to amplification of one target strand PCR reactions are set up using the PE Applied Biosystem TaqMan PCR Core Reagent Kit accordmg to the instructions supplied such that each reaction contains 5 microliters 10X PCR Buffer II, 7 microliters 25 mM MgCl 2 , 5 microliters 300 nM forward pnmer, 5 microliters reverse pnmer, 5 microliters specific FQ probe, 1 microhter each 10 mM dATP, 10 mM dCTP, 10 mM dGTP and 20 mM dUTP, 13.25 microliters distilled water, 0.5 microliters AmpErase UNG, and 0 25 microliters Amp Taq DNA polymerase to give a total volume of 45 microliters Amplification proceeds under the following thermal cycling conditions 50°C hold for 2 minutes, 95°C hold for 10 minutes. 40 cycles of 95°C for 15 seconds and 60°C for 1 minute, followed by a 25 °C hold until sample is retrieved Detection occurs real-time Data is collected at the end of the reaction Based on these analyses it was discovered that the Streptococcus pneumoniae ribA gene was transcribed in vivo Riboflavin Biosynthesis Operon This ORF is part of an operon which encodes genes ribG, ribB, nbA and nbH Gene ribG starts at nucleotide 1 and ends at nucleotide 1101 Gene nbB starts at nucleotide 1086 and ends at nucleotide 1721 Gene nbA starts at nucleotide 1711 and ends at nucleotide 2949 Gene nbH starts at nucleottde 2950 and ends at nucleotide 3417 This operon [SEQ ID NO 7] is as follows ATGAGCGATTCAAAATATATGAAATTAGCAATAAAACTGGCACAAAAAGGGGCTGGTTACGTCAATCCC AATCCTATGGTTGGCGCAATTATTGTAAAAGATAATCACATTATCGGACAAGGTTATCATGAGTTTTTT GGTGGCCCACATGCTGAGAGAAATGCTCTTAAAAACTGTAGAAAATCCCCTGTCGGAGCGACGCTTTAT GTAACACTTGAACCCTGTTGTCACTTCGGGAAAACACCTCCCTGTATAGATGCTATAATCGATAGTGGT ATTACAAGAGTAGTCATTGGAAGCCTAGACTGTAATCCTATTGTATCTGGAAAAGGAGTAAAGATACTT GAGGAAAATAATCTTCAAGTTACTGTTGGAATTTTAGAAAATGAGTGTCTTAACTTAATAAAAAGTTTT AGAAAGTATATTACCCAGCATGTACCCTATGTTTTTATGAAATATGCAATGTCAATGGATGGAAAAATA GCCACTAAAACAAATCAATCCAAATGGATTACTGAAGAAGAAGCAAGAAAGCATGTGCATCAGTTACGA CACTATGTTAGTGCAATTATGGTGGGAGTCAATACTGTTATTCAAGACGATCCTTTGCTGACATGTAGA TTGGAGGAAGGGAAAAATCCTATCCGTATCATATGCGATACACATTTACGAACTCCTCTTACCTCTAAA ATCGTAAAAACAGCAAATGATATTAAAACTTACATTGCCACTTCCTCTGAAGACAAAAATAAAATGAAG CTATATCAAAATCATGGCTGTGAAATACTTTCCATAAAGAAAAAAGGCAATCATATAGACTTATCGAGT TTAATGCAACATCTAGGAAACATGCAGATTGATAGCCTAGTTCTAGAGGGGGGCAGTCTAATGAATTGG AGTGCTTTGGAACAACAAATTGTTGATGAGCTGAAAATATATATTGCACCAAAAATTTTTGGAGGCAGT GCCAAGTTTCCTGTCGGAGGTGAAGGCATTTCTTTGCCAAATGACGCTATTAGATTGAAACCTTATGCT TTTTCTCAAATAGGAAATGACTATCTCATAGAAAGTGAAGTGATTTATCCATGTTCACAGGAATAATTG AAGAAATCGGAAAAGTTGAAAGAATACAGAAAGACTCTCGTAATTGTAAACTATCAATTAAAGCCTCAA AAATATTAACGGATATCCATTTAGGCGATAGTATAGCAGTAAATGGTATCTGTCTTACAGTTACTCATT TCAATCATCAATCCTTTACAGTTGATGTAATGAATGAAACATGGAGTCGAACAGCTCTTACTCTATTAA AACATGGAAGTGAGGTGAATCTAGAAAGAGCCTTATCTGTCAACGGTCGACTTGGGGGTCACGTCGTTA CAGGACACATTGATGGTACAGGAAAAATCTCGTCAATAAAAAAAGATGATAATGCTGTATGGTATCAAA TCAACACACAAAAAGAAATTTTAGATTTAATAGTTGAAAAAGGATCTATTACAATTGACGGCATTAGTC TGACTGTCGCTAAAGTCTCCAAAGTAAACTTTTCAGTATCTGTTATCCCTCATACCTTGAAACAAACCA TTCTTAAGAGTAAACAAGTCGGGAGTACAGTAAATCTTGAAAATGATATCTTAGGTAAATATGTGCAAA AACTGATGGATAACTCTCCAAAATCAGAAATATCGAAGGAACTATTATATCAAAATGGATTTTAGCAGA AAGGATAATCAGTCAATGGAATATCGAAAAATGACAAGAAGCATTGAGGAAGCATTGCAGAAGGGACGA CTTGTTCTTGTTATAGACGACAAGGATAGAGAAAATGAAGGAGACTTAATTTGTTCTGCACAAGCAGCT ACAACAGAAAATGTTAATTTTATGGCTACTTATGCCAAAGGATTAATTTGTATGCCTATGAGCGAAAGT TTAGCTAATCAATTAATGCTTTCACCTATGGTTGAAAACAATACAGATAATCATAAGACTGCTTTTACA GTTTCAATTGATTATAAAGAAACGACCACAGGTATTTCTGCCGAGGAAAGAGGACTGACCGCACGTATG TGTGTAGCTGAAGATATAACACCCTCTGATTTTCGCAGGCCAGGACACATGTTTCCTTTAATTGCAAAA AAAGGTGGTGTTCTAGAAAGAAATGGACACACAGAAGCAACTGTTGATTTATTAAAATTAGCTGGACTA AAAGAGTGTGGCCTATGTTGTGAAATAATGAATCATGATGGCAAAATGATGAGAACAGATGATTTAATT CAGTTCTCGAAGAAACACAACATTCCACTAATTACCATCAAAGAATTACAAGAATATAGAAAAGTATAT GATCAGCTGGTAGAACGAGTTTCAACTGTCAATATGCCTACTAGATACGGTAATTTCAAAGCAATTAGC TATATAGATAACTAAATGGGGAACATCATCTTGCTCTTATTATGGGAAACATAGAGGATGAAGCCAAT GTATTATGTCGGGTCCACTCCGAATGTTTAACAGGAGATGTTTTAGGCTCTTTACGTTGCGATTGTGGA CAGCAATTCGATAAAGCTATGAAAATGATTGTTGAGAATGGTTCGGGTGTCTTACTTTACTTGCGACAG GAGGGACAAGGAATTGGACTTATCAATAAATTAAAAGCCTATCATTTACAAAATCAAGGCATGGATACG CTTGATGCCAATCTTGCATTAGGCTTTGAAGGTGATTTAAGAGAATATCATATTGGAGCACAAATGCTT AAAGATCTGGGACTTCAGTCACTTCATTTACTGACAAATAATCCTGACAAGGTTGAACAGTTAGAAAAA TATGGAATTACCATTTCCAGTAGAATATCAATCGAAATAGAAGCCAATCCTTACGATAGTTTTTATTTA GAAACAAAGAAAAATCGAATGGGTCACATTTTAAATATGGAGGAAAAATAAATGAACACTTATGAAGGT AATTTAGTAGCAAACAATATTAAAATAGGTATTGTTGTAGCGAGATTTAATGAATTTATAACTTCAAAA TTATTATCTGGAGCACTAGATAATCTCAAAAGAGAGAATGTAAACGAGAAAGATATCGAGGTAGCCTGG GTTCCAGGAGCTTTTGAAATACCACTGATTGCATCAAAAATGGCAAAAAGTAAAAAATATGATGCAATT ATCTGCTTGGGAGCTGTCATTAGAGGGAATACAAGTCATTATGATTATGTATGTAGCGAGGTATCTAAA GGAATCGCCCAAATCAGTTTAAATAGCGAAATTCCTGTTATGTTTGGTGTGCTAACGACAGATACAATT GAACAAGCCATAGAACGAGCTGGTACTAAAGCAGGAAATAAGGGTTCTGAGTGTGCACAAGGAGCTATT GAAATGGTCAACCTAATTCGTACATTAGACGCATAG

Claims: What is claimed is: 1 An isolated polynucleottde compnsmg a polynucleotide having at least a 70% identity to a polynucleottde encodmg a polypepttde compnsmg the ammo acid sequence of SEQ ID NO 2 2 An isolated polynucleottde compnsing a polynucleottde having at least a 70% identity to a polynucleottde encodmg the same mature polypepttde expressed by the nbA gene contained m the Streptococcus pneumoniae of the deposited strain 3 An isolated polynucleotide compnsmg a polynucleotide encodmg a polypeptide compnsmg an ammo acid sequence which is at least 70% identical to the ammo acid sequence of SEQ ID NO 2 4 An isolated polynucleotide that is complementary to the polynucleotide of claim 1 5 The polynucleotide of Claim 1 wherem the polynucleotide is DNA or RNA 6 The polynucleotide of Claim 1 compnsmg the nucleic acid sequence set forth m SEQ ID NO 1 7 The polynucleottde of Claim 1 compnsmg nucleottde 1 to the stop codon which begms at nucleotide number 1237 set forth m SEQ ID NO 1 8 The polynucleottde of Claim 1 which encodes a polypepttde compnsmg the ammo acid sequence of SEQ ID NO 2 9 A vector compnsmg the polynucleotide of Claim 1 10 A host cell compnsmg the vector of Claim 9 11 A process for producmg a polypeptide compnsmg expressmg from the host cell of Claim 10 a polypeptide encoded by said DNA 12 A process for producmg a nbA polypepttde or fragment comprising cultunng a host of claim 10 under conditions sufficient for the production of said polypeptide or fragment 13 A polypeptide compnsmg an ammo acid sequence which is at least 70% identical to the ammo acid sequence of SEQ ID NO 2 14 A polypeptide compnsmg an ammo acid sequence as set forth m SEQ ID NO 2 15 An antibody against the polypeptide of claim 14 16 An antagomst which inhibits the activity or expression of the polypeptide of claim 14 17 A method for the treatment of an mdividual m need of nbA polypeptide compnsmg administering to the mdividual a therapeuttcally effective amoimt of the polypepttde of claim 14 18 A method for the treatment of an mdividual having need to inhibit nbA polypepttde compnsmg admimstenng to the mdividual a therapeutically effective amount of the antagomst of Claim 14 19 A process for diagnosing a disease related to expression or activity of the polypeptide of claim 14 m an mdividual compnsmg (a) determining a nucleic acid sequence encodmg said polypepttde. and/or (b) analyzing for the presence or amount of said polypeptide m a sample denved from the mdividual 20 A method for identifying compounds which mteract with and inhibit or activate an activity of the polypeptide of claim 14 compnsmg contactmg a composition compnsmg the polypeptide with the compound to be screened under conditions to permit mteraction between the compound and the polypeptide to assess the mteraction of a compound, such mteraction bemg associated with a second component capable of providing a detectable signal m response to the mteraction of the polypepttde with the compound, and determining whether the compound interacts with and activates or inhibits an activity of the polypepttde by detectmg the presence or absence of a signal generated from the mteractton of the compound with the polypeptide 21 A method for mducmg an lmmunological response in a mammal which compnses inoculating the mammal with nbA polypeptide of claim 14, or a fragment or vanant thereof, adequate to produce antibody and/or T cell immune response to protect said animal from disease 22 A method of mducmg lmmunological response in a mammal which compnses dehvenng a nucleic acid vector to direct expression of nbA polypeptide of claim 14, or fragment or a variant thereof, for expressing said nbA polypeptide, or a fragment or a vanant thereof in vivo in order to induce an lmmunological response to produce antibody and/ or T cell immune response to protect said animal from disease 23 An isolated polynucleotide compnsmg a polynucleotide having at least a 70% identity to a polynucleotide encodmg a polypeptide compnsmg the ammo acid sequence of SEQ ID NO 4 24. An isolated polynucleotide comprising a polynucleotide having at least a 70% identity to the polynucleotide sequence of SEQ ID NO: 3.

Inventors: BLACK MICHAEL T, FEDON JASON C, HODGSON JOHN E, KNOWLES DAVID J C, KOSMATKA ANNA L, LONETTO MICHAEL A, NICHOLAS RICHARD O, PALMER LESLIE M, SHILLING LISA K, STODOLA ROBERT K, WARREN RICHARD L, BLACK, MICHAEL, T., FEDON, JASON, C., HODGSON, JOHN, E., KNOWLES, DAVID, J., C., KOSMATKA, ANNA, L., LONETTO, MICHAEL, A., NICHOLAS, RICHARD, O., PALMER, LESLIE, M., SHILLING, LISA, K., STODOLA, ROBERT, K., WARREN, RICHARD, L.
Applicants: SMITHKLINE BEECHAM CORP, SMITHKLINE BEECHAM CORPORATION
Application year: 1998
Main classification: C12P21
IPC codes: A61K31, A61K38, A61K39, A61K45, A61K48, A61P31, A61P37, C07K14, C07K16, C12N1, C12N9, C12N15, C12P21, G01N33