WO2001032022A1 - YtgP - Google Patents

Abstract

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

Description

YtgP

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 hi particular, the invention relates to polynucleotides and polypeptides of the ytgP (membrane protein) family, as well as their vanants, herein referred to as "ytgP," "ytgP polynucleotιde(s)," and "ytgP polypeptιde(s)" as the case may be

BACKGROUND OF THE INVENTION It is particularly preferred to employ Staphylococcal genes and gene products as targets for the development of antibiotics The Staphylococci make up a medically important genera of microbes They are known to produce two types of disease, invasive and toxigenic Invasive infections are charactenzed generally by abscess formation effecting both skin surfaces and deep tissues S aureus is the second leading cause of bacteremia in cancer patients Osteomyelitis, septic arthntis. septic thrombophlebitis and acute bacterial endocarditis are also relatively common There are at least three clinical conditions resulting from the toxigenic properties of Staphylococci The manifestation of these diseases result from the actions of exotoxins as opposed to tissue invasion and bacteremia These conditions include Staphylococcal food poisoning, scalded skin syndrome and toxic shock syndrome

The frequency of Staphylococcus aureus infections has nsen dramatically in the past few decades This has been attributed to the emergence of multiply antibiotic resistant strams and an increasing population of people with weakened immune systems It is no longer uncommon to isolate Staphylococcus aureus strains that are resistant to some or all of the standard antibiotics This phenomenon has created an unmet medical need and demand for new anti-microbial agents, vaccines, drug screening methods, and diagnostic tests for this organism Moreover, the drug discovery process is currently undergoing a fundamental revolution as it embraces "functional genomics," that is, high throughput genome- or gene-based biolog) This approach is rapidly superseding earlier approaches based on "positional cloning" and other methods Functional genomics relies heavily on the vanous tools of bioinformatics to identify gene sequences of potential interest from the many molecular biology databases now available as well as from other sources There is a continuing and significant need to identify and characterize further genes and other polynucleotides sequences and their related polypeptides, as targets for drug discovery

Clearly, there exists a need for polynucleotides and polypeptides. such as the ytgP embodiments of the invention, that have a present benefit of, among other tilings, being useful to screen compounds for antimicrobial 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

SUMMARY OF THE INVENTION The present invention relates to ytgP. in particular ytgP polypeptides and ytgP polynucleotides. recombinant matenals and methods for their production In another aspect, the mvention relates to methods for usmg such polypeptides and polynucleotides. including treatment of microbial diseases, amongst others In a further aspect, the invention relates to methods for identifying agonists and antagonists using the materials provided by the invention, and for treating microbial infections and conditions associated with such infections with the identified agonist or antagonist compounds In a still further aspect, the mvention relates to diagnostic assays for detectmg diseases associated with microbial infections and conditions associated with such infections, such as assays for detecting ytgP expression or activity

Vanous changes and modifications within the spint and scope of the disclosed mvention will become readily apparent to those skilled in the art from reading the following descnptions and from reading the other parts of the present disclosure

DESCRIPTION OF THE INVENTION

The mvention relates to ytgP polypeptides and polynucleotides as descnbed in greater detail below In particular, the mvention relates to polypeptides and polynucleotides of a ytgP of Staphylococcus aureus that is related by ammo acid sequence homology to B subtihs ytgP polypeptide The mvention relates especially to ytgP havmg a nucleotide and ammo acid sequences set out m Table 1 as SEQ ID NO 1 and SEQ ID NO 2 respectively Note that sequences recited in the Sequence Listing below as "DNA" represent an exemplification of the invention, since those of ordinary skill will recognize that such sequences can be usefully employed in polynucleotides in general, including πbopolynucleotides

TABLE 1 YtgP Polynucleotide and Polypeptide Sequences

(A) Staphylococcus aureus ytgP polynucleotide sequence [SEQ ID NO 1] 5 ' -

ATGAGTGAAAGTAAAGAAATGGTGCGTGGAACCTTTTTAATTACCATTAGTATATTAATTACCAAGGTATTGGGC GTACTTTTTATCATTCCATTCAACTATTTAATTGGTGGACAAGAAAATATGGCGCCGTTCACATACGCTTATGCA CCATATAATATTGCAATTGCAGTTGCGACAGCAGGTGTACCATTAGCAGCTTCGAAATATGTTGCGAAATATAAT GCTATTGGGGCATAT AAAGTCAGTCAGAAATTTTATAAATCGAGTTTTATTGTAATGAGTATTACCGGTGTTTTAGGTTTCTTGGTACTG TATTTCTTAGCACCTTATATTTCTGAATTAACATTAGCTAGAAACATTCATGATAAAAACGGTTGGTCAGTCGAT GATATTACTTGGATTATAAGAATCATTAGTATGGTTGTTATCTTTATTCCTGTATTAGCGACATGGAGGGGGATA TTCCAAGGTTATAAA TCTATGGGGCCAACAGCAGTGTCAGAAGTAACTGAGCAAATTGCACGCGTGATTTTCATATTAATCGGAAGTTAT TTAGTACTAAATGTCTTTGATGGCTCTATTTTATTAGCCAATGGTATTGCTACTTTTGCAGCAGCAGTTGGTGCG ATTATTGGTATATTCACACTCTGGTATTACTGGAGAAAGCGTAAGCATAATATTGATCGTATGGTCGAATCTGAT TACACAGATATAGAT GTGTCATATGGCAAGATGTATAAAGAAATTATTGCTTATAGTATACCGTTCGTTATTGTAAGTTTGAATTATCCA TTATTTAACTTAGTAGATCAATTTACACATAACGGTGCTTTATCTTTAGTTGGTATACCTTCACAGTTACAAGAT ATATTCTTTAATATGCTAAATATGTCAACTAATAAAATTGTAATGATTCCGACATCATTAAGTGCTGGTTTTGCA GTGAGTTTAATTCCT

TATATTACAAAAACATTTGCAGAAGGTCGCTTACATGAAATGCATCATCAAATTAGAACATCTATTGGTGTATTA ATGTTTATAACTGTTCCAGCTAGTATTGGAATTATGGCCTTGGCACAACCATTATTCACAGTTTTCTATGGTTAC GATCCAATTGTTTTAGGTCATGACCCTAACCATGATGGTAGTCGCTTATTATTCTATTATGCGCCTGTAGCAATA CTAATTTCACTATTA

AGTGTAACGGCATCAATGTTACAAGGTATAGATAAACAGAAATTAACAGTTTATGTTATTTTAGCTTCTGTAGTT ATCAAGTTAGCGTTAAACTATCCATTAATTATGTTATTCCATACACCTGGTGCAATTTTAAGTACTAGTATCGCA TTATTATTTGCAATTGGATGTAATTTCTATATTCTTAAAAAATATGCGAAGTTTAAATTCAGCTATAGTTGGATT CATTTTGCAAAAATA

TTCTTGTATTCATTTATTATGATGCTAGGTGTAGAATTAGTATTTTTCCTTGCAAATCTATTCTTAGAACCTACT AAACTTGGTTACTTAATCATTATTATTTTAGGTGTTACAGTAGGTATTCTAATTTACGGTACAATCACAATTAAA ACGCGTCTTGCGGACGAATTCTTAGGTGAAATTCCTGAAAAATTAAGACGTAGAGTTAGGTTTTTACGATGA-3'

(B) Staphylococcus aureus ytgP polypeptide sequence deduced from a polynucleotide sequence in this table [SEQ ID NO 2]

NH₂-

VSYGKMYKEI IAYS I PFVIVSLNYPLFNLVDQFTHNGALSLVGI PSQLQDI FFNMLNMSTNKIVMI PTSLSAGFA VSLI PYITKTFAEGRLHEMHHQI RTS I GVLMFITVPAS I GIMALAQPLFTVFYGYDPIVLGHDPNHDGSRL FYY APVAI LI SLLSV ASMLQGI DKQKLTVYVILASWI KLALNYPLIMLFHTPGAILSTS IALLFAI GCNFYILKKY AKFKFSYSWIHFAKI

FLYS FIMMLGVELVFFLANLFLEPTKLGYLI I I I LGV VGILIYGTITI KTRLADEFLGEI PEKLRRRVRFLR- COOH

Deposited materials

A deposit compnsmg a Staphylococcus aureus WCUH 29 stram has been deposited with the National Collections of Industnal and Marine Bactena Ltd (herem "NCIMB"). 23 St Machar Dnve. Aberdeen AB2 IRY, Scotland on 11 September 1995 and assigned NCIMB Deposit No 40771, and referred to as Staphylococcus aureus CUH29 on deposit The Staphylococcus aureus stram deposit is referred to herem as "the deposited strain" or as "the DNA of the deposited stram " The deposited stram compnses a full length ytgP gene The sequence of the polynucleotides compnsed m the deposited strain, as well as the ammo acid sequence of any polypeptide encoded thereby, are controlling m the event of any conflict with any descnption of sequences herem

The deposit of tire deposited stram 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 deposited stram will be irrevocably and without restriction or condition released to the public upon the issuance of a patent The deposited stram is provided merely as convenience 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 stram, and compounds denved therefrom, and no such license is hereby granted

In one aspect of the mvention there is provided an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Staphylococcus aureus WCUH 29 strain, which polypeptide is compnsed in the deposited stram Further provided by the mvention are ytgP polynucleotide sequences m the deposited strain, such as DNA and RNA, and ammo acid sequences encoded thereby Also provided by the mvention are ytgP polypeptide and polynucleotide sequences isolated from the deposited stram

Polypeptides

YtgP polypeptide of the mvention is substantially phylogenetically related to other proteins of the ytgP (membrane protem) family

In one aspect of the mvention there are provided polypeptides of Staphylococcus aureus referred to herem as "ytgP" and "ytgP polypeptides" as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same

Among the particularly preferred embodiments of the mvention are vanants of ytgP polypeptide encoded by naturally occurring alleles of a ytgP gene

The present mvention further provides for an isolated polypeptide that (a) comprises or consists of an ammo acid sequence that has at least 95% identity, most preferably at least 97-99% or exact identity, to that of SEQ ID NO 2 over the entire length of SEQ ID NO 2, (b) a polypeptide encoded by an isolated polynucleotide comprising or consisting of a polynucleotide sequence that has at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID NO 1 over the entire length of SEQ ID NO 1, (c) a polypeptide encoded by an isolated polynucleotide comprising or consistmg of a polynucleotide sequence encoding a polypeptide that has at least 95% identity, even more preferably at least 97-99% or exact identity, to the ammo acid sequence of SEQ ID NO 2, over the entire length of SEQ ID N02

The polypeptides of the mvention mclude a polypeptide of Table 1 [SEQ ID NO 2] (m particular a mature polypeptide) as well as polypeptides and fragments, particularly those that has a biological activity of ytgP, and also those that have at least 95% identity to a polypeptide of Table 1 [SEQ ID NO:2] and also include portions of such polypeptides with such portion of the polypeptide generally comprising at least 30 amino acids and more preferably at least 50 amino acids.

The invention also includes a polypeptide consisting of or comprising a polypeptide of the formula: X-(R₁)_m-(R₂)-(R₃)_n-Y wherein, at the amino terminus, X is hydrogen, a metal or any other moiety described herein for modified polypeptides, and at the carboxyl terminus, Y is hydrogen, a metal or any other moiety described herein for modified polypeptides, Ri and R3 are any amino acid residue or modified amino acid residue, m is an integer between 1 and 1000 or zero, n is an integer between 1 and 1000 or zero, and R₂ is an amino acid sequence of the invention, particularly an amino acid sequence selected from Table 1 or modified fonns tliereof. hi the formula above, R₂ i^s oriented so that its amino terminal amino acid residue is at the left, covalently bound to R\ and its carboxy terminal amino acid residue is at the right, covalently bound to R3. Any stretch of amino acid residues denoted by either R_} or R3, where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer. Other preferred embodiments of the invention are provided where m is an integer between 1 and 50, 100 or 500, and n is an integer between 1 and 50, 100, or 500.

It is most preferred that a polypeptide of the invention is derived from Staphylococcus aureus, however, it may preferably be obtained from other organisms of the same taxonomic genus. A polypeptide of the invention may also be obtained, for example, from organisms of the same taxonomic family or order.

A fragment is a variant polypeptide having an amino acid sequence that is entirely the same as part but not all of any amino acid sequence of any polypeptide of the invention. As with ytgP polypeptides, fragments may be "free-standing," or comprised within a larger polypeptide of which they form a part or region, most preferably as a single continuous region in a single larger polypeptide.

Preferred fragments include, for example, truncation polypeptides having a portion of an amino acid sequence of Table 1 [SEQ ID NO:2], or of variants tliereof. such as a continuous series of residues that includes an amino- and/or carboxyl-terminal amino acid sequence. Degradation forms of the polypeptides of the invention produced by or in a host cell, particularly a Staphylococcus aureus, are also preferred. Further preferred are fragments characterized by structural or functional attributes such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and tiim-forming regions, coil and coil-forming regions, hydrophihc regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions.

Further preferred fragments include an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids from the amino acid sequence of SEQ ID NO:2, or an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated or deleted from the amino acid sequence of SEQ ID NO:2.

Fragments of the polypeptides of the mvention may be employed for producing the corresponding full-length polypeptide by peptide synthesis, therefore, these vanants may be employed as intermediates for producmg the full-length polypeptides of the mvention Polynucleotides It is an object of the mvention to provide polynucleotides that encode \tgP polypeptides, particularly polynucleotides that encode a polypeptide herem designated ytgP

In a particularly preferred embodiment of the mvention the polynucleotide compnses a region encodmg ytgP polypeptides compnsmg a sequence set out in Table 1 [SEQ ID NO 1] that mcludes a full length gene, or a variant thereof This mvention provides that this full length gene is essential to the growth and or survival of an organism that possesses it, such as Staphylococcus aureus

As a further aspect of the mvention there are provided isolated nucleic acid molecules encoding and/or expressmg ytgP polypeptides and polynucleotides, particularly Staphylococcus aureus ytgP polypeptides and polynucleotides, including, for example, unprocessed RNAs, nbozyme RNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs Further embodiments of the mvention mclude biologically, diagnostically, prophylactically, clinically or therapeutically useful polynucleotides and polypeptides, and vanants thereof, and compositions compnsmg the same

Another aspect of the mvention relates to isolated polynucleotides, including at least one full length gene, that encodes a ytgP polypeptide havmg a deduced ammo acid sequence of Table 1 [SEQ ID NO 2] and polynucleotides closely related thereto and vanants thereof

In another particularly preferred embodiment of the mvention there is a ytgP polypeptide from Staphylococcus aureus comprising or consisting of an ammo acid sequence of Table 1 [SEQ ID NO 2], or a variant thereof Usmg the information provided herein, such as a polynucleotide sequence set out in Table 1 [SEQ ID

NO 1], a polynucleotide of the mvention encodmg ytgP polypeptide may be obtained usmg standard cloning and screening methods, such as those for cloning and sequencmg chromosomal DNA fragments from bacteπa usmg Staphylococcus aureus WCUH 29 cells as starting matenal, followed b\ obtaining a full length clone For example, to obtain a polynucleotide sequence of the mvention. such as a polynucleotide sequence given in Table 1 [SEQ ID NO 1], typically a library of clones of chromosomal DNA of Staphylococcus aureus WCUH 29 in E coh or some other suitable host is probed with a radiolabeled ohgonucleotide. preferably a 17-mer or longer, derived from a partial sequence Clones carrying DNA identical to that of the probe can then be distinguished using stringent hybridization conditions By sequencing the individual clones thus identified by hybridization with sequencing primers designed from the oπgmal polypeptide or polynucleotide sequence it is then possible to extend the polynucleotide sequence m both directions to determine a full length gene sequence Conveniently, such sequencing is performed, for example, using denatured double stranded DNA prepared from a plasmid clone Suitable techniques are described by Mamatis, T , Fπtsch. 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) Direct genomic DNA sequencing may also be performed to obtain a full length gene sequence Illustrative of the mvention. each polynucleotide set out m Table 1 [SEQ ID NO 1] was discovered m a DNA library denved from Staphylococcus aureus WCUH 29 Moreover, each DNA sequence set out m Table 1 [SEQ ID NO 1] contains an open readmg frame encodmg a protem havmg about the number of ammo acid residues set forth in Table 1 [SEQ ID NO 2] with a deduced molecular weight that can be calculated usmg ammo acid residue molecular weight values well known to those skilled in the art The polynucleotide of SEQ ID NO 1, between nucleotide number 721 and the stop codon that begins at nucleotide number 1660 of SEQ ID NO 1, encodes the polypeptide of SEQ ID NO 2

In a further aspect, the present mvention provides for an isolated polynucleotide comprising or consistmg of (a) a polynucleotide sequence that has at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID NO 1 over the entire length of SEQ ID NO 1, or the entire length of that portion of SEQ ID NO 1 that encodes SEQ ID NO 2, (b) a polynucleotide sequence encoding a polypeptide that has at least 95% identity, even more preferably at least 97-99% or 100% exact, to the ammo acid sequence of SEQ ID NO 2, over the entire length of SEQ ID NO 2

A polynucleotide encodmg a polypeptide of the present mvention, mcludmg homologs and orthologs from species other than Staphylococcus aureus. may be obtained by a process that compπses the steps of screening an appropπate library under stringent hybπdization conditions with a labeled or detectable probe consistmg of or compπsmg the sequence of SEQ ID NO 1 or a fragment thereof, and isolatmg a full-length gene and/or genomic clones compπsmg said polynucleotide sequence

The mvention provides a polynucleotide sequence identical over its entire length to a codmg sequence (open reading frame) m Table 1 [SEQ ID NO 1] Also provided by the mvention is a codmg sequence for a mature polypeptide or a fragment thereof, by itself as well as a codmg sequence for a mature polypeptide or a fragment m readmg frame with another codmg sequence, such as a sequence encodmg a leader or secretoiy sequence, a pre-, or pro- or prepro-protem sequence The polynucleotide of the mvention may also compnse at least one non-coding sequence, mcludmg for example, but not limited to at least one non-coding 5^" and 3' sequence, such as the transcπbed but non-translated sequences, termination signals (such as rho-dependent and rho-mdependent termination signals), πbosome binding sites, Kozak sequences, sequences that stabilize mRNA mtrons, and polyadenylation signals The polynucleotide sequence may also compnse additional codmg sequence encoding additional ammo acids For example, a marker sequence that facilitates punfication of a fused polypeptide can be encoded In certain embodiments of the mvention, the marker sequence is a hexa-histidine peptide, as provided m the pQE vector (Qiagen, Inc ) and descnbed m Gentz et al , Proc Natl Acad Set , USA 86 821-824 (1989), or an HA peptide tag (Wilson et al , Cell 37 161 (1984), both of that may be useful m punfying polypeptide sequence fused to them 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 preferred embodiment of the mvention is a polynucleotide consistmg of or compπsmg nucleotide 721 to the nucleotide immediately upstream of or mcludmg nucleotide 1660 set fortli m SEQ ID NO 1 of Table 1, both of which encode a ytgP polypeptide

The mvention also mcludes a polynucleotide consistmg of or compπsmg a polynucleotide of the formula

X-(R₁)_m-(R₂)-(R₃)_n-Y wherein, at the 5' end of the molecule, X is hydrogen, a metal or a modified nucleotide residue, or together with Y defines a covalent bond, and at the 3' end of the molecule, Y is hydrogen, a metal, or a modified nucleotide residue, or together with X defines the covalent bond, each occurrence of Ri and R3 is independently any nucleic acid residue or modified nucleic acid residue, m is an integer between 1 and 3000 or zero , n is an integer between 1 and 3000 or zero, and R₂ is a nucleic acid sequence or modified nucleic acid sequence of the invention, particularly a nucleic acid sequence selected from Table 1 or a modified nucleic acid sequence thereof In the polynucleotide formula above, R₂ is oriented so that its 5' end nucleic acid residue is at the left, bound to Ri and its 3' end nucleic acid residue is at the right, bound to R3 Any stretch of nucleic acid residues denoted by either Rj and/or R₂, where m and or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer Where, in a preferred embodiment, X and Y together define a covalent bond, the polynucleotide of the above formula is a closed, circular polynucleotide, that can be a double-stranded polynucleotide wherein the formula shows a first strand to which the second strand is complementary In another preferred embodiment m and/or n is an integer between 1 and 1000. Other prefened embodiments of the mvention are provided where m is an mteger between 1 and 50, 100 or 500, and n is an mteger between 1 and 50, 100, or 500

It is most prefened that a polynucleotide of the mvention is deπved from Staphylococcus aureus, however, it may preferably be obtained from other organisms of the same taxonomic genus A polynucleotide of the mvention may also be obtained, for example, from organisms of the same taxonomic family or order The term "polynucleotide encoding a polypeptide" as used herem encompasses polynucleotides that mclude a sequence encodmg a polypeptide of the mvention, particularly a bactenal polypeptide and more particularly a polypeptide of the Staphylococcus aureus ytgP havmg an ammo acid sequence set out m Table 1 [SEQ ED NO 2] The tenn also encompasses polynucleotides that mclude a smgle contmuous region or discontinuous regions encoding the polypeptide (for example, polynucleotides interrupted by integrated phage, an mtegrated insertion sequence, an integrated vector sequence, an integrated transposon sequence, or due to RNA editing or genomic DNA reorganization) together with additional regions, that also may compnse codmg and or non-coding sequences

The mvention further relates to vanants of the polynucleotides descnbed herem that encode vanants of a polypeptide havmg a deduced ammo acid sequence of Table 1 [SEQ ID NO 2] Fragments of polynucleotides of the mvention may be used, for example, to synthesize full-length polynucleotides of the mvention

Further particularly prefened embodiments are polynucleotides encodmg ytgP vanants. that have the ammo acid sequence of ytgP polypeptide of Table 1 [SEQ ID NO 2] m which several, a few, 5 to 10, 1 to 5, 1 to 3, 2. 1 or no ammo acid residues are substituted, modified, deleted and/or added, m any combmation

Especially preferred among these are silent substitutions, additions and deletions, that do not alter the properties and activities of ytgP polypeptide

Preferred isolated polynucleotide embodiments also mclude polynucleotide fragments, such as a polynucleotide comprising a nuclic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids from the polynucleotide sequence of SEQ ID NO: l, or an polynucleotide comprising a nucleic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids truncated or deleted from the 5' and/or 3' end of the polynucleotide sequence of SEQ ID NO. l

Further prefened embodiments of the mvention are polynucleotides that are at least 95% or 97% identical over their entire lengtli to a polynucleotide encodmg ytgP polypeptide having an ammo acid sequence set out m Table 1 [SEQ ID NO 2], and polynucleotides that are complementary to such polynucleotides Most highly prefened are polynucleotides that compnse a region that is 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 preferred, with at least 99% bemg the more prefened

Preferred embodiments are polynucleotides encoding polypeptides that retain substantially the same biological function or activity as a mature polypeptide encoded by a DNA of Table 1 [SEQ ID NO 1] In accordance with certain prefened embodiments of this mvention there are provided polynucleotides that hybndize, particularly under stringent conditions, to ytgP polynucleotide sequences, such as those polynucleotides m Table 1

The mvention further relates to polynucleotides that hybndize to the polynucleotide sequences provided herem In this regard, the mvention especially relates to polynucleotides that hybndize under stringent conditions to the polynucleotides descnbed herem A specific example of stringent hybridization conditions is overnight incubation at 42°C m a solution compnsmg 50% formamide, 5x SSC (150mM NaCl, 15mM tnsodium citrate), 50 mM sodium phosphate (pH7 6), 5x Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml of 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 Spnng Harbor, N Y , (1989), particularly Chapter 11 therein Solution hybridization may also be used with the polynucleotide sequences provided by the invention

The invention also provides a polynucleotide consistmg of or comprising a polynucleotide sequence obtained by screening an appropriate library comprising a complete gene for a polynucleotide sequence set forth m SEQ ID NO 1 under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence set forth SEQ ID NO 1 or a fragment thereof, and isolating said polynucleotide sequence Fragments useful for obtaining such a polynucleotide include, for example, probes and pnmers fully described elsewhere herein As discussed elsewhere herem regarding polynucleotide assays of the mvention. for instance, the polynucleotides of the mvention, may be used as a hybndization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encodmg ytgP and to isolate cDNA and genomic clones of other genes that have a high identity, particularly high sequence identity, to a ytgP gene Such probes generally will compnse at least 15 nucleotide residues or base pairs Preferably, such probes will have at least 30 nucleotide residues or base parrs and may have at least 50 nucleotide residues or base pairs Particularly prefened probes will have at least 20 nucleotide residues or base pairs and will have lee than 30 nucleotide residues or base pairs

A codmg region of a ytgP gene may be isolated by screening usmg a DNA sequence provided m Table 1 [SEQ ID NO 1] to synthesize an oligonucleotide probe A labeled ohgonucleotide havmg 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 hybndizes to

There are several methods available and well known to those skilled m the art to obtain full- length DNAs, or extend short DNAs, for example those based on the method of Rapid Amplification of cDNA ends (RACE) (see, for example, Frohman. et al , PNAS USA 85 8998-9002, 1988) Recent modifications of the techmque, exemplified by the Marathon™ technology (Clontech Laboratories Inc ) for example, have significantly simplified the search for longer cDNAs In the Marathon™ technology, cDNAs have been prepared from mRNA extracted from a chosen tissue and an 'adaptor' sequence gated onto each end Nucleic acid amplification (PCR) is then carried out to amplify the "missing" 5' end of the DNA using a combination of gene specific and adaptor specific ohgonucleotide primers The PCR reaction is then repeated using "nested" primers, that is, primers designed to anneal withm the amplified product (typically an adaptor specific primer that anneals further 3' in the adaptor sequence and a gene specific primer that anneals further 5' in the selected gene sequence) The products of this reaction can then be analyzed by DNA sequencing and a full-length DNA constructed either by joining the product directly to the existing DNA to give a complete sequence, or carrying out a separate full- length PCR usmg the new sequence information for the design of the 5' primer

The polynucleotides and polypeptides of the mvention may be employed, for example, as research reagents and mateπals for discovery of treatments of and diagnostics for diseases, particularly human diseases, as further discussed herem relatmg to polynucleotide assays The polynucleotides of the invention that are ohgonucleotides derived from a sequence of Table

1 [SEQ ID NOS 1 or 2] may be used in the processes herein as described, but preferably for PCR, to determine whether or not the polynucleotides identified herein in whole or in part are transcribed in bacteria in mfected tissue It is recognized that such sequences will also have utility m diagnosis of the stage of infection and type of infection the pathogen has attained The mvention also provides polynucleotides that encode a polypeptide that is a mature protem plus additional ammo or carboxyl-termmal ammo acids, or ammo acids mtenor to a mature polypeptide (when a mature form has more than one polypeptide 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-hfe or may facilitate manipulation of a protem for assay or production, among other thmgs As generally is the case in vivo, the additional ammo acids may be processed awav from a mature protem by cellular enzymes

For each and every polynucleotide of the mvention there is provided a polynucleotide complementary to it It is prefened that these complementary polynucleotides are fully complementary to each polynucleotide with which they are complementary A precursor protem, havmg a mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide When prosequences are removed such mactive precursors generally are activated Some or all of the prosequences may be removed before activation Generalh . such precursors are called proproterns As will be recognized, the entire polypeptide encoded by an open readmg frame is often not required for activity Accordingly, it has become routme m molecular biology to map the boundanes of the primary structure required for activity with N-teπninal and C-terminal deletion experiments These experiments utilize exonuclease digestion or convenient restπction sites to cleave codmg nucleic acid sequence For example, Promega (Madison, WT) sell an Erase-a-base™ system that uses Exonuclease III designed to facihtate analysis of the deletion products (protocol available at www promega com) The digested endpomts can be repaired (e g , by ligation to synthetic linkers) to the extent necessary to preserve an open reading frame In this way, the nucleic acid of SEQ ID NO 1 readily provides contiguous fragments of SEQ ID NO 2 sufficient to provide an activity, such as an enzymatic, binding or antibody-inducing activity Nucleic acid sequences encoding such fragments of SEQ ID NO 2 and vanants thereof as descnbed herem are within the mvention. as are polypeptides so encoded

As is known m the art, portions of the N-termmal and/or C-terminal sequence of a protein can generally be removed without serious consequence to the function of the protem The amount of sequence that can be removed is often quite substantial The nucleic acid cutting and deletion methods used for creating such deletion variants are now quite routine Accordingly, anv contiguous fragment of SEQ ID NO 2 which retams at least 20%, preferably at least 50%. of an activity of the polypeptide encoded by the gene for SEQ ID NO 2 is within the invention, as are corresponding fragment which are 70%, 80%, 90%, 95%,97%, 98% or 99% identical to such contiguous fragments In one embodiment, the contiguous fragment compnses at least 70% of the ammo acid residues of SEQ ID NO 2, preferably at least 80%. 90% or 95% of the residues

In sum, a polynucleotide of the mvention may encode a mature protem, a mature protem plus a leader sequence (that may be refened to as a preprotem), a precursor of a mature protem havmg one or more prosequences that are not the leader sequences of a preprotem, or a preproprotem, that is a precursor to a proprotern, havmg a leader sequence and one or more prosequences, that generally are removed dunng processmg steps that produce active and mature forms of the polypeptide

Vectors, Host Cells, Expression Systems

The mvention also relates to vectors that compnse a polynucleotide or polynucleotides 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 deπved from the DNA constructs of the mvention

Recombinant polypeptides of the present mvention may be prepared by processes well known m those skilled m the art from genetically engineered host cells compnsmg expression systems Accordingly, m a further aspect, the present mvention relates to expression systems that compnse a polynucleotide or polynucleotides of the present mvention, to host cells that are genetically engineered with such expression systems, and to the production of polypeptides of the mvention by recombinant techniques

For recombinant production of the polypeptides of the mvention, 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, N Y (1989). such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection. micromjection, catiomc hpid-mediated transfection, electroporation, transduction. scrape loading, ballistic mtroduction and infection

Representative examples of appropnate hosts mclude bacteπal cells, such as cells of streptococci, staphylococci, enterococci E col , streptomyces, cyanobactena, Bacillus subtihs, and Staphylococcus aureus, fungal cells, such as cells of a yeast, Kluveromyces, Saccharomyces, a basidiomycete, Candida albicans and Aspergillus, insect cells such as cells of Drosophύa S2 and Spodoptera Sf9, animal cells such as CHO, COS, HeLa. C127, 3T3, BHK, 293, CV-1 and Bowes melanoma cells, and plant cells, such as cells of a gymnosperm or angiosperm

A great vaπety of expression systems can be used to produce the polypeptides of the mvention Such vectors mclude, among others, chromosomal-, episomal- and virus-denved vectors, for example, vectors denved from bacteπal plasmids, from bacteπophage, from transposons, 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, picornaviruses and retroviruses, and vectors denved from combinations thereof, such as those denved from plasmid and bactenophage genetic elements, such as cosmids and phagemids The expression system constructs may compnse control regions that regulate as well as engender expression Generally, any system or vector suitable to mamtam, propagate or express polynucleotides and/or to express a polypeptide 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)

In recombinant expression systems m eukaryotes. for secretion of a translated protem mto the lumen of the endoplasmic reticulum, mto the penplasmic space or mto the extracellular environment, appropnate secretion signals may be mcorporated 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 mteraction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography Most preferably, high performance liquid chromatography is employed for punfication Well known techniques for refolding protem may be employed to regenerate active conformation when the polypeptide is denatured dunng isolation and or punfication

Diagnostic, Prognostic, Serotyping and Mutation Assays

This mvention is also related to the use of ytgP polynucleotides and polypeptides of the mvention for use as diagnostic reagents Detection of ytgP polynucleotides and or polypeptides m a eukaryote, particularly a mammal, and especially a human will provide a diagnostic method for diagnosis of disease staging of disease or response of an infectious organism to drugs Eukaryotes, particularly mammals, and especially humans, particularly those infected or suspected to be infected with an organism compnsmg the ytgP gene or protem, may be detected at the nucleic acid or ammo acid level by a vaπety of well known techniques as well as by methods provided herem

Polypeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtained from a putatively infected and/or infected individual's bodily matenals Polynucleotides from any of these sources, particularly DNA or RNA, may be used directly for detection or may be amplified enzymatically by usmg PCR or any other amplification techmque pnor to analysis RNA. particularly mRNA, cDNA and genomic DNA may also be used in the same ways Usmg amplification, charactenzation of the species and stram of infectious or resident organism present m an individual, may be made by an analysis of the genotype of a selected polynucleotide of the organism Deletions and msertions can be detected by a change m size of the amplified product m companson to a genotype of a reference sequence selected from a related organism, preferably a different species of the same genus or a different stram of the same species Pomt mutations can be identified by hybπdizmg amplified DNA to labeled ytgP polynucleotide sequences Perfectlj or significantly matched sequences can be distinguished from imperfectly or more significantly mismatched duplexes by DNase or RNase digestion, for DNA or RNA respectively, or by detectmg differences in meltmg temperatures or renaturation kinetics Polynucleotide sequence differences may also be detected by alterations in the electrophoretic mobility of polynucleotide fragments in gels as compared to a reference sequence This may be earned out with or without denaturing agents Polynucleotide differences may also be detected by direct DNA or RNA sequencmg See, for example, Myers et al , Science, 230 1242 (1985) Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase, VI and SI protection assay or a chemical cleavage method See, for example, Cotton et al . Proc Natl Acad Set , USA 85 4397-4401 (1985)

In another embodiment, an anay of oligonucleotides probes compπsmg ytgP nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of, for example, genetic mutations, serotype, taxonomic classification or identification Array technology methods are well known and have general applicability and can be used to address a vanety of questions m molecular genetics mcludmg gene expression, genetic linkage, and genetic vanabi ty (see, for example, Chee et al , Science, 274 610 (1996))

Thus in another aspect, the present invention relates to a diagnostic kit that comprises (a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NO 1, or a fragment thereof , (b) a nucleotide sequence complementary to that of (a), (c) a polypeptide of the present invention, preferably the polypeptide of SEQ ID NO 2 or a fragment thereof, or (d) an antibod) to a polypeptide of the present invention, preferably to the polypeptide of SEQ ID NO 2 It will be appreciated that in any such kit, (a), (b). (c) or (d) may comprise a substantial component Such a kit will be of use in diagnosing a disease or susceptibility to a Disease, among others

This mvention also relates to the use of polynucleotides of the present mvention as diagnostic reagents Detection of a mutated form of a polynucleotide of the mvention, preferable. SEQ ID NO 1, that is associated with a disease or pathogenicity will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, a prognosis of a course of disease, a determination of a stage of disease, or a susceptibility to a disease, that results from under-expression, over-expression or altered expression of the polynucleotide

Organisms, particularly infectious organisms, carrying mutations m such polynucleotide may be detected at the polynucleotide level by a vaπety of techniques, such as those descnbed elsewhere herem

The differences in a polynucleotide and/or polypeptide sequence between organisms possessing a first phenotype and organisms possessing a different, second different phenotype can also be determined If a mutation is observed m some or all organisms possessing the first phenotype but not in any orgamsms possessing the second phenotype, then the mutation is likely to be the causative agent of the first phenotype

Cells from an organism carrying mutations or polymorphisms (alle c vanations) m a polynucleotide and/or polypeptide of the mvention may also be detected at the polynucleotide or polypeptide level by a vanety of techniques, to allow for serotypmg, for example For example, RT-PCR can be used to detect mutations m the RNA It is particularly prefened to use RT-PCR m conjunction with automated detection systems, such as, for example, GeneScan RNA, cDNA or genomic DNA may also be used for the same purpose. PCR As an example, PCR primers complementary to a polynucleotide encodmg ytgP polypeptide can be used to identify and analyze mutations The mvention further provides these pπmers with 1, 2, 3 or 4 nucleotides removed from the 5' and/or the 3' end These primers may be used for, among other thmgs, amphfymg ytgP DNA and/or RNA isolated from a sample denved from an individual, such as a bodily matenal The primers may be used to amplify a polynucleotide isolated from an infected individual, such that the polynucleotide may then be subject to vanous techmques for elucidation of the polynucleotide sequence In this way, mutations in the polynucleotide sequence may be detected and used to diagnose and/or prognose the infection or its stage or course, or to serotype and or classify the infectious agent

The mvention further provides a process for diagnosmg, disease, preferably bacterial infections, more preferably infections caused by Staphylococcus aureus, comprising determmmg from a sample denved from an mdividual, such as a bodily material, an increased level of expression of polynucleotide havmg a sequence of Table 1 [SEQ ID NO 1] Increased or decreased expression of a ytgP polynucleotide can be measured using any on of the methods well known in the art for the quantitation of polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNase protection, Northern blotting, spectrometry and other hybridization methods In addition, a diagnostic assay m accordance with the mvention for detectmg over-expression of ytgP polypeptide compared to noπnal control tissue samples may be used to detect the presence of an infection, for example Assay techmques that can be used to determine levels of a ytgP polypeptide, m a sample denved from a host, such as a bodily matenal, are well-known to those of skill m the art Such assay methods mclude radiormmunoassays, competitive-binding assays, Western Blot analysis, antibody sandwich assays, antibody detection and ELISA assays

Antagonists and Agonists - Assays and Molecules

Polypeptides and polynucleotides 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 braπes, and natural product mixtures These substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics See, e g , Coligan et al , Current Protocols in Immunology 1(2) Chapter 5 (1991)

Polypeptides and polynucleotides of the present mvention are responsible for many biological functions, mcludmg many disease states, m particular the Diseases herem mentioned It is therefore desirable to devise screening methods to identify compounds that agonize (e g , stimulate) or that antagonize (e g ,ιnhιbιt) the function of the polypeptide or polynucleotide Accordmgly, m a further aspect, the present mvention provides for a method of screening compounds to identify those that agomze or that antagonize the function of a polypeptide or polynucleotide of the mvention, as well as related polypeptides and polynucleotides In general, agonists or antagonists (e g , inhibitors) may be employed for therapeutic and prophylactic purposes for such Diseases as herem mentioned Compounds may be identified from a vaπety of sources, for example, cells, cell-free preparations, chemical braπes, and natural product mixtures Such agonists and antagonists so-identified may be natural or modified substrates, ligands, receptors, enzymes, etc , as the case may be, of ytgP polypeptides and polynucleotides, or may be structural or functional mimetics thereof (see Coligan et al , Current Protocols in Immunology 1(2) Chapter 5 (1991)) The screemng methods may simply measure the binding of a candidate compound to the polypeptide or polynucleotide, or to cells or membranes bearing the polypeptide or polynucleotide, or a fusion protein of the polypeptide by means of a label directly or indirectly associated with the candidate compound Alternatively, the screening method may involve competition with a labeled competitor Further, these screemng methods may test whether the candidate compound results m a signal generated by activation or inhibition of the polypeptide or polynucleotide, usmg detection systems appropriate to the cells comprising the polypeptide or polynucleotide Inhibitors of activation are generally assayed in the presence of a known agomst and the effect on activation by the agomst by the presence of the candidate compound is observed Constitutively active polypeptide and or constitutively expressed polypeptides and polynucleotides may be employed in screening methods for inverse agonists, in the absence of an agonist or antagonist, by testing whether the candidate compound results m inhibition of activation of the polypeptide or polynucleotide, as the case may be Further, the screening methods may simply comprise the steps of mixing a candidate compound with a solution comprising a polypeptide or polynucleotide of the present invention, to form a mixture, measuring ytgP polypeptide and/or polynucleotide activity in the mixture, and comparing the ytgP polypeptide and/or polynucleotide activity of the mixture to a standard Fusion proteins, such as those made from Fc portion and ytgP polypeptide, as herein described, can also be used for high-throughput screening assays to identify antagonists of the polypeptide of the present invention, as well as of phylogenetically and and or functionally related polypeptides (see D Bennett et al , J Mol Recognition, 8 52-58 (1995), and K Johanson et al , J Biol Chem, 270(16) 9459-9471 (1995))

The polynucleotides, polypeptides and antibodies that bind to and/or interact with a polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and/or polypeptide in cells For example, an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide usmg monoclonal and polyclonal antibodies by standard methods known m the art This can be used to discover agents that may inhibit or enhance the production of polypeptide (also called antagonist or agomst, respectively) from suitably manipulated cells or tissues

The mvention also provides a method of screemng compounds to identify those that enhance (agonist) or block (antagonist) the action of ytgP polypeptides or polynucleotides, particularly those compounds that are bactenstatic and/or bactencidal The method of screening may mvolve high-throughput techniques For example, to screen for agonists or antagonists, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, compnsmg ytgP polypeptide and a labeled substrate or gand of such polypeptide is mcubated m the absence or the presence of a candidate molecule that may be a ytgP agomst or antagonist The ability of the candidate molecule to agonize or antagonize the ytgP polypeptide is reflected in decreased binding of the labeled ligand or decreased production of product from such substrate Molecules that bmd gratuitously, i e , without inducing the effects of ytgP polypeptide are most likely to be good antagonists Molecules that bmd well and, as the case may be, mcrease the rate of product production from substrate, mcrease signal transduction, or mcrease chemical channel activity are agomsts Detection of the rate or level of, as the case may be, production of product from substrate, signal transduction, or chemical channel activity may be enhanced by usmg a reporter system Reporter systems that may be useful m this regard mclude but are not limited to colonmetπc, labeled substrate converted mto product, a reporter gene that is responsive to changes m ytgP polynucleotide or polypeptide activity, and binding assays known m the art Polypeptides of the invention may be used to identify membrane bound or soluble receptors, if any, for such polypeptide, through standard receptor bindmg techniques known in the art These techniques include, but are not limited to, ligand binding and crosslinking assays m which the polypeptide is labeled with a radioactive isotope (for instance, I), chemically modified (for instance, biotinylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (e g , cells, cell membranes, cell supematants, tissue extracts, bodily materials) Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy These screening methods may also be used to identify agonists and antagonists of the polypeptide that compete with the binding of the polypeptide to its receptor(s), if any Standard methods for conducting such assays are well understood in the art The fluorescence polarization value for a fluorescently-tagged molecule depends on the rotational correlation time or tumbling rate Protem complexes, such as formed by ytgP polypeptide associating with another ytgP polypeptide or other polypeptide, labeled to comprise a fluorescently- labeled molecule will have higher polarization values than a fluorescently labeled monomeπc protem It is preferred that this method be used to characterize small molecules that disrupt polypeptide complexes

Fluorescence energy transfer may also be used characterize small molecules that interfere with the formation of ytgP polypeptide dimers, tπmers, tetramers or higher order structures, or structures formed by ytgP polypeptide bound to another polypeptide YtgP polypeptide can be labeled with both a donor and acceptor fluorophore Upon mixing of the two labeled species and excitation of the donor fluorophore. fluorescence energy transfer can be detected by observing fluorescence of the acceptor Compounds that block dimeπzation will inhibit fluorescence energy transfer

Surface plasmon resonance can be used to monitor the effect of small molecules on ytgP polypeptide self-association as well as an association of ytgP polypeptide and another polypeptide or small molecule YtgP polypeptide can be coupled to a sensor chip at low site density such that covalently bound molecules will be monomeπc Solution protein can then passed over the ytgP polypeptide -coated surface and specific bindmg can be detected in real-time by monitoring the change in resonance angle caused by a change in local refractive index This technique can be used to characterize the effect of small molecules on kinetic rates and equilibrium binding constants for ytgP polypeptide self-association as well as an association of ytgP polypeptide and another polypeptide or small molecule

A scintillation proximity assay may be used to characterize the interaction between an association of ytgP polypeptide with another ytgP polypeptide or a different polypeptide YtgP polypeptide can be coupled to a scmtillation-filled bead Addition of radio-labeled ytgP polypeptide results in bmdmg where the radioactive source molecule is in close proximity to the scintillation fluid Thus, signal is emitted upon ytgP polypeptide bindmg and compounds that prevent ytgP polypeptide self-association or an association of ytgP polypeptide and another polypeptide or small molecule will dimmish signal

In other embodiments of the mvention there are provided methods for identifying compounds that bmd to or otherwise interact with and inhibit or activate an activity or expression of a polypeptide and/or polynucleotide of the mvention compnsmg contacting a polypeptide and/or polynucleotide of the mvention with a compound to be screened under conditions to permit bmdmg to or other mteraction between the compound and the polypeptide and or polynucleotide to assess the bmdmg to or other mteraction with the compound, such bmdmg or mteraction preferably bemg associated with a second component capable of providmg a detectable signal m response to the bmdmg or mteraction of the polypeptide and/or polynucleotide with the compound, and determining whether the compound bmds to or otherwise mteracts with and activates or inhibits an activity or expression of the polypeptide and/or polynucleotide by detectmg the presence or absence of a signal generated from the bmdmg or mteraction of the compound with the polypeptide and/or polynucleotide

Another example of an assay for ytgP agonists is a competitive assaj that combines ytgP and a potential agomst with ytgP-bmdmg molecules, recombinant ytgP bmdmg molecules, natural substrates or ligands, or substrate or ligand mimetics, under appropnate conditions for a competitive inhibition assay YtgP can be labeled, such as by radioactivity or a colonmetnc compound, such that the number of ytgP molecules bound to a bmdmg molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist It will be readily appreciated by the skilled artisan that a polypeptide and/or polynucleotide of the present invention may also be used in a method for the structure-based design of an agonist or antagonist of the polypeptide and or polynucleotide, by (a) determining in the first instance the three- dimensional structure of the polypeptide and or polynucleotide, or complexes thereof, (b) deducing the three-dimensional structure for the likely reactive sιte(s), bmdmg sιte(s) or motιf(s) of an agonist or antagonist, (c) synthesizing candidate compounds that are predicted to bmd to or react with the deduced bmdmg sιte(s), reactive sιte(s), and/or motιf(s), and

(d) testing whether the candidate compounds are indeed agomsts or antagonists It will be further appreciated that this will normally be an iterative process, and this iterative process may be performed using automated and computer-controlled steps hi a further aspect, the present mvention provides methods of treatmg abnormal conditions such as. for instance, a Disease, related to either an excess of, an under-expression of, an elevated activity of. or a decreased activity of ytgP polypeptide and/or polynucleotide

If the expression and/or activity of the polypeptide and or polynucleotide is m excess, several approaches are available One approach compπses administering to an mdividual m need thereof an inhibitor compound (antagonist) as herem descnbed, optionally m combmation with a pharmaceutically acceptable earner, in an amount effective to inhibit the function and/or expression of the polypeptide and/or polynucleotide, such as, for example, by blockmg the bmdmg of ligands, substrates, receptors, enzymes, etc , or by inhibiting a second signal, and thereby alleviating the abnormal condition In another approach, soluble forms of the polypeptides still capable of binding the ligand, substrate, enzymes, receptors, etc m competition with endogenous polypeptide and or polynucleotide may be administered Typical examples of such competitors include fragments of the ytgP polypeptide and or polypeptide

In still another approach, expression of the gene encoding endogenous ytgP polypeptide can be inhibited usmg expression blockmg techmques This blockmg may be targeted against any step in gene expression, but is preferably targeted against transcription and/or translation An examples of a known techmque of this sort mvolve the use of antisense sequences, either internally generated or separately administered (see, for example, O'Connor, J Neurochem (1991) 56 560 m Ohgodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)) Alternatively, ohgonucleotides that form tπple helices with the gene can be supplied (see, for example, Lee et al , Nucleic Acids Res (1979) 6 3073, Cooney et al , Science (1988) 241 456, Dervan et al , Science (1991) 251 1360) These o gomers can be administered per se or the relevant ohgomers can be expressed in vivo Each of the polynucleotide sequences provided herein may be used in the discovery and development of antibacterial compounds The encoded protem, upon expression, can be used as a target for the screening of antibacterial drugs Additionally, the polynucleotide sequences encoding the amino terminal regions of the encoded protem 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 invention also provides the use of the polypeptide, polynucleotide. agonist or antagonist of the invention to interfere with the initial physical interaction between a pathogen or pathogens and a eukaryotic, preferably mammalian, host responsible for sequelae of infection In particular, the molecules of the invention may be used in the prevention of adhesion of bacteria, in particular gram positive and or gram negative bacteria, to eukaryotic, preferably mammalian, extracellular matrix proteins on m-dwelling devices or to extracellular matrix proteins in wounds, to block bacterial adhesion between eukaryotic, preferably mammalian, extracellular matrix proteins and bacteπal ytgP protems that mediate tissue damage and/or. to block the normal progression of pathogenesis in mfections initiated other than by the implantation of in-dwelling devices or b\ other surgical techniques In accordance with yet another aspect of the mvention, there are provided ytgP agomsts and antagomsts, preferably bactenstatic or bactencidal agomsts and antagomsts

The antagomsts and agomsts of the mvention may be employed, for instance, to prevent, inhibit and or treat diseases Antagomsts of the mvention mclude, among others, small organic molecules, peptides, polypeptides and antibodies that bmd to a polynucleotide and/or polypeptide of the mvention and thereby inhibit or extinguish its activity or expression Antagomsts 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 bmdmg molecule, such as a bmdmg molecule, without mducmg ytgP-mduced activities, thereby preventing the action or expression of ytgP polypeptides and/or polynucleotides by excludmg ytgP polypeptides and/or polynucleotides from bmdmg

Antagomsts of the mvention also mclude a small molecule that bmds to and occupies the bmdmg site of the polypeptide thereby preventmg bmdmg to cellular bmdmg 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 antagomsts mclude antisense molecules (see Okano. J Neurochem 56 560 (1991), OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION. CRC Press, Boca Raton, FL (1988), for a descnption of these molecules) Prefened antagomsts mclude compounds related to and vanants of ytgP Other examples of polypeptide antagomsts mclude antibodies or, m some cases, ohgonucleotides or proteins that are closely related to the ligands, substrates, receptors, enzymes, etc , as the case may be. of the polypeptide. e g . a fragment of the ligands, substrates, receptors, enzymes, etc , or small molecules that bmd to the polypeptide of the present mvention but do not elicit a response, so that the activity of the polypeptide is prevented

Small molecules of the invention preferably have a molecular weight below 2,000 daltons. more preferably between 300 and 1,000 daltons, and most preferably between 400 and 700 daltons It is preferred that these small molecules are organic molecules

Hehcobacter pylori (herein "H pylori") bacteria mfect 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 Hehcobacter Pylori (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 pylon and gastric adenocarcinoma, classifying the bacterium as a Group I (definite) carcinogen Preferred antimicrobial compounds of the invention (agonists and antagonists of ytgP polypeptides and/or polynucleotides) found usmg screens provided by the invention, or known in the art, particularly narrow-spectrum antibiotics, should be useful m the treatment of H pylon infection Such treatment should decrease the advent of H pylori -induced cancers, such as gastrointestinal carcinoma Such treatment should also prevent, inhibit and/or cure gastric ulcers and gastritis

All publications and references, including but not limited to patents and patent applications, cited m this specification are herein incorporated by reference m their entirety as if each individual publication or reference were specifically and individually indicated to be incorporated by reference herein as being fully set forth Any patent application to which this application claims pπorits is also incorporated by reference herein in its entirety m the manner described above for publications and references

GLOSSARY

The following definitions are provided to facilitate understanding of certain terms used frequently, herem

"Bodily mateπal(s) means any matenal denved from an mdividual or from an organism infecting, infesting or inhabiting an mdividual, mcludmg but not limited to, cells, tissues and waste, such as, bone, blood, serum, cerebrospmal fluid, semen, saliva, muscle, cartilage, organ tissue, skm urine, stool or autopsy matenals "Dιsease(s)" means any disease caused by or related to mfection by a bacteπa. mcludmg . for example, disease, such as, infections of the upper respiratory tract (e g , otitis media, bactenal tracheitis, acute epiglottitis, thyroiditis), lower respiratory (e g , empyema, lung abscess), cardiac (e g , infective endocarditis), gastrointestinal (e g . secretory diarrhoea, splenic absces, retropentoneal abscess), CNS (e g , cerebral abscess), eye (e g . blephantis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital cellu tis, darcryocystitis), kidney and urinary tract (e g , epididymitis, rntrarenal and pennephπc absces, toxic shock syndrome), skin (e g , impetigo, folhculitis, cutaneous abscesses, celluhtis, wound infection, bactenal myositis) bone and jomt (e g , septic arthritis, osteomyelitis)

"Host cell(s)" is a cell that has been introduced (e g , transformed or transfected) or is capable of introduction (e g , 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 the case may be, as determined by comparing the sequences In 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" can be readily calculated by known methods, including but not limited to those described in

(Computational Molecular Biology. Lesk, A M , ed , Oxford University Press, New York, 1988. Bwcomputing Informatics and Genome Projects, Smith, D W , ed , Academic Press, New York, 1993, Computer Analysis of Sequence Data, Part I. Griffin, 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, Gribskov, M and Devereux, J , eds , M Stockton Press, New York, 1991, and Caπllo, H , and Lipman, D , SIAM J Applied Math , 48 1073 (1988) Methods to determine identity are designed to give the largest match between the sequences tested Moreover, methods to determine identity are codified in publicly available computer programs Computer program methods to determine identity 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 (Altschul, S F et al , J Molec Bwl 215 403-410 (1990) The BLAST X program is publicly available from NCBI and other sources (BLAST Manual. Altschul, S , et al . NCBI NLM NIH Bethesda, MD 20894, Altschul, S . et al . J Mol Bwl 215 403-410 (1990) The well known Smith Waterman algorithm may also be used to detennine identity Parameters for polypeptide sequence comparison include the following Algorithm Needleman and Wunsch, J Mol Biol 48 443-453 (1970)

Comparison matrix BLOSSUM62 from Hentikoff and Hentikoff, Proc Natl Acad Sci USA 89 10915-10919 (1992) Gap Penalty 12 Gap Length Penalty 4

A program useful with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison WI The aforementioned parameters are the default parameters for peptide comparisons (along with no penalty for end gaps) Parameters for polynucleotide comparison include the following Algorithm Needleman and

Wunsch, J Mol Biol 48 443-453 (1970) Comparison matrix matches = +10, mismatch = 0 Gap Penalty 50 Gap Length Penalty 3 Available as The "gap" program from Genetics Computer Group, Madison WI These are the default parameters for nucleic acid comparisons

A preferred meaning for "identity" for polynucleotides and polypeptides, as the case may be, are provided in (1) and (2) below

(1) Polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide sequence having at least a 95, 97 or 100% identity to the reference sequence of SEQ ID NO 1, wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO 1 or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, mcludmg transition and transversion. or insertion, and wherem said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides m the reference sequence or in one or more contiguous groups within the reference sequence, and wherem said number of nucleotide alterations is determined by multiplying the total number of nucleotides m SEQ ID NO 1 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleotides in SEQ ID NO 1, or

n_n < x_n - (x_n • y),

wherein n_n is the number of nucleotide alterations, x_n is the total number of nucleotides in SEQ ID NO 1. y is 0 95 for 95%, 0 97 for 97% or 1 00 for 100%, and • is the symbol for the multiplication operator, and wherein any non-integer product of x_n and y is rounded down to the nearest mteger prior to subtracting it from x_n Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO 2 may create nonsense, missense or frameshift mutations m this codmg sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations (2) Polypeptide embodiments further include an isolated polypeptide comprising a polypeptide having at least a 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO 2, wherem said polypeptide sequence may be identical to the reference sequence of SEQ ID NO 2 or may include up to a certain integer number of ammo acid alterations as compared to the reference sequence, wherem said alterations are selected from the group consisting of at least one ammo acid deletion, substitution, mcludmg conservative and non-conservative substitution, or insertion, and wherem said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the ammo acids m the reference sequence or m one or more contiguous groups within the reference sequence, and wherem said number of ammo acid alterations is determined by multiplying the total number of ammo acids in SEQ ID NO 2 by the integer defining the percent identity divided b\ 100 and then subtracting that product from said total number of amino acids in SEQ ID NO 2. or

ⁿa ^{≤ x}a (^χ _a • y)»

wherein n_a is the number of am o acid alterations, x_a is the total number of amino acids m SEQ ID NO 2, y is 0 95 for 95%, 0 97 for 97% or 1 00 for 100%, and • is the symbol for the multiplication operator, and wherein any non-mteger product of x_a and y is rounded down to the nearest integer prior to subtracting it from x_a "Indιvιdual(s)" means a multicellular eukaryote, mcludmg, but not limited to a metazoan, a mammal, an ovid, a bovid, a simian, a primate, and a human

"Isolated" means altered "by the hand of man" from its natural state, z e . if it occurs m nature, it has been changed or removed from its oπgmal environment, or both For example, a polynucleotide or a polypeptide naturally present m a Irving organism is not "isolated," but the same polynucleotide or polypeptide separated from the coexistmg matenals of its natural state is "isolated", as the term is employed herem Moreover, a polynucleotide or polypeptide that is mtroduced mto an organism by transfonnation. 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 non-living

"Organιsm(s)" means a (I) prokaryote. mcludmg but not limited to. a member of the genus Streptococcus, Staphylococcus, Bordetella, Corynebacterium, Mycobactenum, Neissena, Haemophilus Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella Pasturella, Moraxella, Acmetobacter, Erysψelothnx, Branhamella, Actinobacύlus, Streptobacillus, Listena, Calymmatobacterium, Brucella, Bacillus, Clostndium, Treponema, Eschenchia, Salmonella, Kleibsiella, Vibno, Proteus, Erwinia, Borreha, Leptospira, Spirillum, Campylobacter, Shigella, Legwnella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borreha and Mycoplasma, and further mcludmg, but not limited to, a member of the species or group, Group A Streptococcus, Group B Streptococcus, Group C Streptococcus, Group D Streptococcus, Group G Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus faecahs Streptococcus faecium Streptococcus chtram Neissena gonorrheae, Neissena meningiUdis, Staphylococcus aureus, Staphylococcus epidermidis, Corynebactenum dipthenae, Gardnerella vaginahs, Mycobactenum tuberculosis, Mycobactenum bovis, Mycobactenum ulcerans, Mycobactenum leprae, Actinomyctes israelu, Listena monocytogenes, Bordetella pertusis, Bordatella parapertusis, Bordetella bronc septica, Eschenchia coll, Sh gella dysentenae, Haemophilus influenzae, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonella typhi, Citrobacter freundii, Proteus mirabilis, Proteus vulgans, Yersinia pestis, Kleibsiella pneumoniae, Serratia marcessens Serratta hquefacien Vtbno cholera Shigella dysentem Shigella flexnen, Pseudomonas aerugmosa, Franscisella tularensis, Bmcella abortis, Bacillus anthracis, Bacillus cereus, Clostndium perfnngens, Clostndium tetani, Clostndium botulmum, Treponema palhdum, Rickettsia rickettsu and Chlamydia trachomitis, (n) an archaeon, mcludmg but not hmited to Archaebacter, and (in) a umcellular or filamentous eukaryote, mcludmg but not limited to, a protozoan, a fungus, a member of the genus Saccharomyces, Kluveromyces, or Candida, and a member of the species Saccharomyces cenviseae, Kluveromyces lactis, or Candida albicans

"Polynucleotide(s)" generally refers to any polynbonucleotide or polydeox)τιbonucleotιde. that ma) 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 single-, double- and tnple-stranded regions, smgle- and double-stranded RNA, and RNA that is mixture of smgle- and double-stranded regions, hybnd 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 hi addition, "polynucleotide" as used herem refers to tnple-stranded regions compπsmg 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 tnple-helical region often is an ohgonucleotide As used herem, the term "polynucleotide(s)" also mcludes DNAs or RNAs as descnbed above that compnse one or more modified bases Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotide(s)" as that term is mtended herem Moreover, DNAs or RNAs compnsmg unusual bases, such as mosme, or modified bases, such as tntylated bases, to name just two examples, are polynucleotides as the term is used herem It will be appreciated that a great vaπety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill m the art The term "pol)iιucleotιde(s)" as it is employed herem embraces such chemically, enzymatically or metabo cally modified forms of polynucleotides, as well as the chemical forms of DNA and RNA charactenstic of viruses and cells, mcludmg, for example, simple and complex cells "Polynucleotιde(s)" also embraces short polvnucleotides often referred to as ohgonucleotιde(s)

"Polypeptιde(s)" refers to any peptide or protem compnsmg two or more ammo acids jomed to each other by peptide bonds or modified peptide bonds "Polypeptide(s)" refers to both short chains, commonly refened to as peptides, o gopeptides and ohgomers and to longer chains generally refened to as proteins Polypeptides may compnse ammo acids other than the 20 gene encoded ammo acids "Polypeptιde(s)" mclude those modified either by natural processes, such as processmg and other post-translational modifications, but also by chemical modification techmques Such modifications are well descnbed m basic texts and m more detailed monographs, as well as in a volummous 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 compnse 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, acylation, ADP-nbosylation. amidation, covalent attachment of flavm, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide deπvative, covalent attachment of a hpid or pid deπvative, covalent attachment of phosphotidylmositol, cross-lmkmg. cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteme, formation of pyroglutamate, formylation, gamma-carboxylation. GPI anchor formation, hydroxylation, lodmation, methylation. myπstoylation, oxidation, proteolytic processmg, phosphorylation, prenylation, racemizauon, glycosylation, hpid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-nbosylation, selenoylation. sulfation, transfer-RNA mediated addition of ammo acids to protems. such as arginylation. 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). Serfter et al , Meth Enzymol 182 626-646 (1990) and Rattan et al , Protem 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 polypeptides may result from post- translational natural processes and may be made by entirely synthetic methods, as well

"Recombinant expression system(s)" refers to expression systems or portions tliereof or polynucleotides of the mvention mtroduced or transformed mto a host cell or host cell lysate for the production of the polynucleotides and polypeptides of the mvention "Vaπant(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 in 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 amino acid substitutions, additions, deletions, fusion proteins and truncations m the polypeptide encoded by the reference sequence, as discussed below A typical variant of a polypeptide differs m ammo acid sequence from another, reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in man} regions identical A \ aπant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions any combination A substituted or inserted amino acid residue may or may not be one encoded by the genetic code The present mvention also mcludes mclude vanants of each of the polypeptides of the mvention, that is polypeptides that vary from the referents by conservative ammo acid substitutions, whereby a residue is substituted by another with like charactenstics Typical such substitutions are among Ala, Val. Leu and He, among Ser and Thr, among the acidic residues Asp and Glu, among Asn and Gin, and among the basic residues Lys and Arg. or aromatic residues Phe and Tyr Particularly prefened are vanants m which several, 5-10, 1-5, 1-3, 1-2 or 1 ammo acids are substituted, deleted, or added m any combmation A variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturall} Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis teclimques, by direct synthesis, and by other recombinant methods known to skilled artisans EXAMPLES

The examples below are earned out usmg standard teclimques, that 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 having a DNA sequence given in Table 1 [SEQ ID NO 1] was obtained from a library of clones of chromosomal DNA of Staphylococcus aureus in E cob The sequencing data from two or more clones comprising overlapping Staphylococcus aureus DNAs was used to construct the contiguous DNA sequence m SEQ ID NO 1 Libraries may be prepared by routme methods, for example Methods 1 and 2 below

Total cellular DNA is isolated from Staphylococcus aureus WCUH 29 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 m order to size- fractionate according to standard procedures DNA fragments of up to 1 lkbp in size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added Fragments are hgated mto the vector Lambda ZapII that has been cut with EcoRI, the library packaged by standard procedures and E co infected with the packaged hbrar) The library is amplified by standard procedures

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combmation of restriction enzymes appropriate to generate a series of fragments for cloning into library vectors (e g , Rsal. Pall. Alul. Bshl235I), and such fragments are size-fractionated according to standard procedures EcoRI linkers are hgated to the DNA and the fragments then hgated into the vector Lambda ZapII that have been cut with EcoRJ. the library packaged by standard procedures, and E co infected with the packaged hbrarv The library is amplified by standard procedures Example 2 ytgP Characterization

The ytgP gene is expressed during infection of Staphylococcus aureus WCUH29 in a pyelonephritis infection model

Necrotic fatty tissue from a kidney from a seven day pyelonephritis infection of Staphylococcus aureus WCUH29 in the mouse is efficiently disrupted and processed m the presence of acid phenol and detergent to provide a mixture of animal and bacterial RNA By freezing the tissue immediately m liquid nitrogen, and processing the tissue samples while still frozen changes m the population of bacterial mRNA is minimized The resultant total RNA is free of DNA and protem (including RNAases and DNAases) The optimal conditions for disruption and processing to give high yields of bacterial mRNA with transcripts of long length are followed by reverse transcribing the resulting mRNA to cDNA and amplified with ORF-specific primers for a bacterial gene known to be expressed constitutively and at low copy number in Staphylococcus aureus WCUH29

a) Isolation of tissue mfected with Staphylococcus aureus WCUH29 from a muπne model of hematogenous pyelonephritis Overnight cultures of S aureus WCUH29 were started from single colonies in 5 ml of tryptic soy broth (TSB) and grown at 37C with shaking The cultures were then washed twice m sterile phosphate-buffered saline (PBS) and diluted to an A600 = 0 3 Male CD-I mice (18 - 20g) were infected with 0 2 ml of this suspension by tail vein inoculation using a 30g needle attached to a tuberculin syringe Each mouse receives approximately 4 x 107 bacteria in this fashion Mice are monitored daily for signs of illness, and usually withm 48 hours show signs of lethargy, ruffled fur, sluggishness, animals which appear moribund are euthanized prior to the end of the experiment All animals are euthanized via carbon dioxide overdose seven days post-mfection The animal is placed on its back and swabbed with ethanol, and then with RNAZap, and instruments are swabbed as well The abdominal cavity is opened and the kidneys aseptically removed, cut mto four pieces, and placed in cryovials which are immediately frozen m liquid nitrogen

b) Isolation of Staphylococcus aureus WCUH29 RNA from infected tissue samples

Infected tissue samples, in 2-ml cyro-strorage tubes, are removed from -80°C storage into a dry ice ethanol bath In a microbiological safety cabinet the samples are disrupted up to eight at a time while the remaining samples are kept frozen m the dry ice ethanol bath To disrupt the bacteria within the tissue sample, 50-100 mg of the tissue is transfered to a FastRNA tube containing a sihca/ceramic matrix (BIO 101) Immediately, 1 ml of extraction reagents (FastRNA reagents, BIOIOI) 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, BIOIOI) at 6000 rpm for 20-120 sec The crude RNA preparation is extracted with chloroform/isoaπvs 1 alcohol and precipitated with DEPC-treated/Isopropanol Precipitation Solution (BIOIOI) RNA preparations are stored in this isopropanol solution at -80°C if necessary 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 in 0 1 ml of DEPC-treated water

c) The removal of DNA from Staphylococcus aureus WCUH29-deπved RNA DNA was removed from 50 microgram samples of RNA by a 30 minute treatment at 37°C with

10 umts of RNAase-free DNAasel (GeneHunter) in the buffer supplied in a final volume of 57 microhters

The DNAase was inactivated and removed by phenol chloroform extraction RNA was precipitated with 5 microhters of 3 M NaOAc and 200 microhters 100% EtOH, and pelleted by centrifiigation 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 in 10-20 microhters of DEPC-treated water RNA yield is quantitated by OD260 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 SuperScπpt 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 SuperScπptll reverse transcπptase are also run Both +/- RT samples are treated with RNaseH before proceeding to the PCR reaction

e) The use of PCR to detennine the presence of a bacterial cDNA species PCR reactions are set up on ice in 0 2ml tubes by addmg the following components 43 microhtres PCR Master Mix (Advanced Biotechnologies Ltd ), 1 microhtre PCR primers (optimally 18-25 basepairs m length and designed to possess similar annealing temperatures), each primer at 1 OmM initial concentration, and 5 microhtres cDNA PCR reactions are run on a Perkm Elmer GeneAmp PCR System 9600 as follows 2 minutes at

94 oC, then 50 cycles of 30 seconds each at 94 oC, 50 oC and 72 oC followed by 7 minutes at 72 oC and then a hold temperature of 20 oC (the number of cycles is optimally 30-50 to determine the appearance or lack of a PCR product and optimally 8-30 cycles if an estimation of the starting quantity of cDNA from the RT reaction is to be made), 10 microhtre ahquots are then run out on 1% 1 x TBE gels stained with ethidium bromide, with PCR product, if present, sizes estimated b> comparison to a 100 bp DNA Ladder (Gibco BRL, Life Technologies) Alternativelv 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 PnsmTM 377 Sequencer using GeneScanTM software as supplied by Perkm Elmer)

RT/PCR controls may include +/- reverse transcπptase 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 m DNA PCR with Streptococcus pneumoniae 0100993 total DNA PCR reactions are set up and run as described above using approx 1 microgram of DNA in place of the cDNA

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 reproducib fail to give a product in RT/PCR, and 2 Genes which are transcribed m 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

Claims

What is claimed is:

1 An isolated polypeptide selected from the group consisting of

(1) an isolated polypeptide comprising an amino acid having at least 95% identity to the amino acid sequence of SEQ ID NO 2 over the entire length of SEQ ID NO 2, (n) an isolated polypeptide compnsmg the amino acid sequence of SEQ ID NO 2, (in) an isolated polypeptide that is the ammo acid sequence of SEQ ID NO 2, and (iv) a polypeptide that is encoded by a recombinant polynucleotide compnsmg the polyncleotide sequence of SEQ ID NO 1

2 An isolated polynucleotide selected from the group consisting of

(I) an isolated polynucleotide compnsmg a polynucleotide sequence encoding a polypeptide that has at least 95% identity to the ammo acid sequence of SEQ ID NO 2, over the entire length of SEQ ID NO 2,

(n) an isolated polynucleotide compnsmg a polynucleotide sequence that has at least 95% identity over its entire lengtli to a polynucleotide sequence encoding the polypeptide of SEQ ID

NO 2,

(m) an isolated polynucleotide compπsmg a nucleotide sequence that has at least 95%o identity to that of SEQ ID NO 1 over the entire length of SEQ ID NO 1,

(iv) an isolated polynucleotide compnsmg a nucleotide sequence encoding the polypeptide of SEQ ID

NO 2,

(v) an isolated polynucleotide that is the polynucleotide of SEQ ID NO 1.

(vi) an isolated polynucleotide of at least 30 nucleotides in lengtli obtainable b\ screening an appropnate library under stringent hybπdization conditions with a probe havmg the sequence of SEQ

ID NO 1 or a fragment thereof of of at least 30 nucleotides m length,

(vn) an isolated polynucleotide encodmg a mature polypeptide expressed by the ytgP gene compnsed m the Staphylococcus aureus, and

(vni) a polynucleotide sequence complementary to said isolated polynucleotide of (l), (n). (m), (iv).

3 A method for the treatment of an individual

(l) m need of enhanced activity or expression of or lmmunological response to the polypeptide of claim 1 comprising the step of administering to the individual a therapeutically effective amount of an antagonist to said polypeptide, or (n) having need to inhibit activity or expression of the polypeptide of claim 1 comprising

(a) administering to the mdividual a therapeutically effective amount of an antagonist to said polypeptide, or

(b) administering to the mdividual a nucleic acid molecule that inhibits the expression of a polynucleotide sequence encoding said polypeptide,

(c) administering to the individual a therapeutically effective amount of a polypeptide that competes with said polypeptide for its ligand, substrate. or receptor, or

(d) administering to the individual an amount of a polypeptide that induces an lmmunological response to said poh eptide in said individual

4 A process for diagnosing or prognosing a disease or a susceptibility to a disease m an mdividual related to expression or activity of the polypeptide of claim 1 m an mdividual comprising the step of

(a) determining the presence or absence of a mutation in the nucleotide sequence encoding said polypeptide m an organism in said individual, or

(b) analyzing for the presence or amount of said polypeptide expression in a sample derived from said individual

5 A process for producing a polypeptide selected from the group consisting of

(l) an isolated polypeptide comprising an ammo acid sequence selected from the group havmg at least 95% identity to the ammo acid sequence of SEQ ID NO 2 over the entire length of SEQ ID NO 2. (u) an isolated polypeptide compnsmg the amino acid sequence of SEQ ID NO 2, (m) an isolated polypeptide that is the ammo acid sequence of SEQ ID NO 2, and (iv) a polypeptide that is encoded by a recombinant polynucleotide comprising the polynucleotide sequence of SEQ ID NO 1, comprising the step of cultuπng a host cell under conditions sufficient for the production of the polypeptide

6 A process for producmg a host cell comprising an expression system or a membrane thereof expressing a polypeptide selected from the group consisting of (1) an isolated polypeptide comprising an amino acid sequence selected from the group having at least 95% identity to the amino acid sequence of SEQ ID NO 2 over the entire length of SEQ ID NO 2,

(n) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO 2.

(in) an isolated polypeptide that is the ammo acid sequence of SEQ ID NO 2, and

(iv) a polypeptide that is encoded by a recombmant polynucleotide comprising the polynucleotide sequence of SEQ ID NO 1, said process comprising the step of transforming or transfecting a cell with an expression system compnsmg a polynucleotide capable of producing said polypeptide of (I), (u), (m) or (iv) when said expression system is present in a compatible host cell such the host cell, under appropriate culture conditions, produces said polypeptide of (l), (n), (in) or (iv)

7 A host cell or a membrane expressing a polypeptide selected from the group consistmg of (I) an isolated polypeptide comprising an ammo acid sequence selected from the group having at least 95% identity to the amino acid sequence of SEQ ID NO 2 over the entire length of SEQ ID

NO 2,

(n) an isolated polypeptide comprising the ammo acid sequence of SEQ ID NO 2, (m) an isolated polypeptide that is the amino acid sequence of SEQ ID NO 2. and (iv) a polypeptide that is encoded by a recombinant polynucleotide comprising the polynucleotide sequence of SEQ ID NO 1

8 An antibody lmmunospecific for the polypeptide of claim 1

9 A method for screening to identify compounds that agonize or that mhibit the function of the polypeptide of claim 1 that compnses a method selected from the group consistmg of

(a) measurmg the bmdmg of a candidate compound to the polypeptide (or to the cells or membranes bearing the polypeptide) or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound,

(b) measuring the bmdmg of a candidate compound to the polypeptide (or to the cells or membranes bearing the polypeptide) or a fusion protein thereof in the presence of a labeled competitor.

(c) testing whether the candidate compound results m a signal generated by activation or inhibition of the polypeptide, using detection systems appropriate to the cells or cell membranes beanng the polypeptide, (d) mixing a candidate compound with a solution comprising a polypeptide of claim 1 , to form a mixture, measuring activity of the polypeptide in the mixture, and comparing the activity of the mixture to a standard; or

(e) detecting the effect of a candidate compound on the production of mRNA encoding said polypeptide and said polypeptide in cells, using for instance, an ELISA assay.

10. An agonist or antagonist to the polypeptide of claim 1.