WO2000056865A1 - Histidine kinase - Google Patents

Abstract

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

Description

histidine kinase

FIELD OF THE INVENTION

This invention relates to ne lv identified polvnucleotides and polvpeptides. and their production and uses, as well as their variants agonists and antagonists and dieir uses hi particular tin- -mention n-lαtes to polynucleotides and polypeptides of the histidine kinase familv. as well as their \anants herein referred to as

"histidine kinase." "histidine kinase polynucleotιde(s)," and "histidine kinase polypeptιde(s)" as the case ma\ 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 Thev are known to produce two types of disease, invasive and toxigenic Invasive infections are characterized generally by abscess formation effecting both skin surfaces and deep tissues S aureus is the second leading cause of bacteremia in cancer patents Osteomyelitis, septic arthntis. septic tl rombophlebitis and acute bactenal 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 strains 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 biology Tins approach is rapidly superseding earlier approaches based on "positional cloning" and other methods Functional genomics rehes heavily on the vanous tools of bioinfoimatics 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 polvnucleotides sequences and their related polypeptides, as targets for drug discovery

Clearly, there exists a need for polynucleotides and pohpeptides. such as the histidine kinase embodiments of the invention, that have a present benefit of. among odier things 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 wa\s to prevent, ameliorate or correct such infection. d\sfunctιon and disease

SUMMARY OF THE INVENTION The present invention relates to histidine kinase in particular histidine kinase poKpeptides and histidine kinase polynucleotides. recombinant matenals and methods for their production In another aspect the invention relates to methods for using 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 invention relates to diagnostic assays for detecting diseases associated with microbial infections and conditions associated with such infections, such as assays for detecting histidine kinase expression or activity

Vanous changes and modifications wit in the spmt and scope of the disclosed invention 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 invention relates to histidine kinase polypeptides and polynucleotides as descnbed in greater detail below In particular, the invention relates to polypeptides and polynucleotides of a histidine kinase of Staphylococcus aureus. that is related by amino acid sequence homology to DegS protein from B subtύis polypeptide The invention relates especially to histidine kinase having a nucleotide and amino acid sequences set out in Table 1 as SEQ ID NO 1 and SEQ ID NO 2 respectrv eh Note that sequences recited in the Sequence Listing below as "DNA" represent an exemplification of the invention, since those of ordinary skill will recogmze that such sequences can be usefully employed in polynucleotides in general, including πbopolynucleotides

TABLE 1 histidine kinase Polynucleotide and Polypeptide Sequences

(A) Staphylococcus aureus histidine kinase polynucleotide sequence [SEQ ID NO 1]

5 ' - 1 AGCTGGAGCT CCACCGCGGT GGCGGCCGCT CTAGAACTAG TGGATCCCCC

51 GGGCTGCAGG AATTCGGCAC GAGCTGCTTT TTATGGAAGT ACTTACGTGA

101 ATGAAAAATC ATATAAAGTT GAAAATAACA ATATTCATAT TGAAGAAATG 151 ATGAAACCGG ATAACTATAC AGTTAATATC TACGTATCAA CGTTTATCGG 201 AGACGTAGAG GTGATTTATC GATGAACCAC TACATTAGAA CAATTGGTTC

251 AATGCTCATC TTAGTATATA GCATGCTAGC TGCATTTCTA TTCATCGATA

301 AAGTTTTTGT AAATATCATC TATTTTCAAG GTATGTTTTA TACACAAATA.

351 TTCGGAATAC CAGTCTTTTT ATTTTTAAAT CTCATCATCA TATTATTGTG 401 TATTATTGTT GGTTCGGTAC TCGCATACAA AATCAATCAG CAAAATGATT

451 GGATTAAGAC GCAAATTGAG CGTTCAATGG AAGGCGAAAC AGTTGGCATT

501 AATGATCAAA ATATAGAAAT ATATAGTGAA ACGTTAGATT TATACCATAC

551 ACTCGTACCT TTAAATCAAG AATTGCATAA GTTGCGACTT AAAACTCAAA

601 ACTTAACCAA TGAAAATTAT AATATTAATG ATGTGAAAGT TAAAAAGATT 651 ATTGAAGATG AACGTCAAAG ACTAGCTCGA GAACTTCACG ATTCTGTTAG

701 TCAGCAACTT TTTGCGGCAA GTATGATGCT TTCTGCTATC AAAGAAACGA

751 AGTTAGAACC ACCATACCAA CAAATTCCGA TTTTAGAGAA AATGGTTCAA

801 GATTCGCAGT TAGAAATGCG TGCTTTGCTG TTACATTTAA GACCGCTTGG

851 TTTAAAAGAC AAATCTTTAG GTGAGGGTAT TAAAGATTTA GTTATTGATT 901 TACAAAAAAA AGTGCCAATG AAAGTTGTGC ATGAAATACA AGATTTTAAA

951 GTGCCTAAAG GTATTGAAGA TCATTTGTTC AGAATTACAC AGGAAGCAAT

1001 TTCGAATACA TTGCGTCATT CAAACGGTAC AAAAGTGACA GTAGAATTGT

1051 TTAATAAAGA CGATTATTTA TTGTTGAGAA TTCAAGATAA TGGTAAAGGT

1101 TTTAATGTTG ATGAAAAATT AGAACAAAGT TATGGACTTA AAAATATGCG 1151 TGAAAGAGCT TTGGAAATTG GTGCAACGTT CCATATTGTA TCATTGCCAG

1201 ATTCAGGTAC ACGTATCGAG GTGAAAGCAC CTTTAAATAA GGAGGATTCG

1251 TATGACGATT AAAGTATTGT TTGTGGATGA TCATGAAATG GTACGTATAG

1301 GAATTTCAAG TTATCTATCA ACGCAAAGTG ATATTGAAGT AGTTGGTGAA 1351 GGCGCTTCTG GTAAAGAAGC AATTGCCAAA GCCCATGAGT TGAAGCCAGA 1401 TTTAATTTTA ATGGATTTAC TTATGGAAGA CATGGATGGT GTAGAAGCGA

1451 CGACTCAGAT TAAAAAAGAT TTACCGCAAA TTAAAGTATT AATGTTAACT-3 '

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

NH . -

1 MNHYIRTI GS MLILVYSMLA AFLFIDKVFV NI IYFQGMFY TQI FGI PVFL

51 FLNLI I ILLC IIVGSVLAYK INQQND IKT QIERSMEGET VGINDQNIEI

101 YSETLDLYHT LVPLNQELHK LRLKTQNLTN ENYNINDVKV KKI IEDERQR 151 LARELHDSVS QQLFAASMML SAIKETKLEP PYQQI PI LEK MVQDSQLEMR

201 ALLLHLRPLG LKDKSLGEGI KDLVIDLQKK VPMKWHEIQ DFKVPKGIED

251 HLFRITQEAI SNTLRHSNGT KVTVELFNKD DYLLLRIQDN GKGFNVDEKL

301 EQSYGLKNMR ERALEI GATF HIVS LPDSGT RI EVKAP NK EDSYDD-COOH

Deposited materials A deposit compnsing a Staphylococcus aureus WCUH 29 strain has been deposited witii die

National Collections of Industnal and Marine Bactena Ltd (herein "NCIMB"). 23 St Machar Dnve

Aberdeen AB2 1RY. Scotland on 11 September 1995 and assigned NCIMB Deposit No 40771 and referred to as Staphylococcus aureus WCUH29 on deposit The Staphylococcus aureus strain deposit is referred to herein as "d e deposited strain" or as "die DNA of the deposited strain "

The deposited stram compnses a full length histidme kinase gene The sequence of the polvnucleotides compπsed m the deposited strain, as well as the ammo acid sequence of any polypeptide encoded therebv. are controlling in the event of any conflict with any descnption of sequences herein

The deposit of the deposited strain has been made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for Purposes of Patent Procedure The deposited stram will be irrevocably and without restnction or condition released to the public upon the issuance of a patent The deposited strain is provided merely as convemence to those of skill in the art and is not an admission that a deposit is required for enablement. such as that required under 35 U S C §112 A license may be required to make, use or sell the deposited strain, and compounds denved therefrom, and no such license is hereby granted

In one aspect of the invention 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 histidme kinase polynucleotide sequences m the deposited strain, such as DNA and RNA. and ammo acid sequences encoded thereby Also provided by the mvention are histidme kinase polypeptide and polynucleotide sequences isolated from the deposited stram

Polypeptides

Histidme kinase polypeptide of the mvention is substantially phylogeneticallv related to other proteins of the histidme kinase family

In one aspect of the mvention there are provided polypeptides of Staphylococcus aureus referred to herem as "histidme kinase" and "histidme kinase polypeptides" as well as biologically, diagnosticallv.

clinically or therapeutically useful vaπants thereof, and compositions compnsmg the same

Among the particularly preferred embodiments of the mvention are vanants of histidme kinase polypeptide encoded by naturally occurring alleles of a histidine kinase 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 identrtΛ to that of SEQ ID NO 2 over the entire length of SEQ ID NO 2, (b) a polypeptide encoded bv an isolated pohnucleotide compnsmg or consisting of a polvnucleotide sequence that has at least 95% identiu e\en more preferablv at least 97-99% or exact identi to SEQ ID NO 1 o\er the enure length of SEQ ID NO 1. (c) a polypeptide encoded by an isolated polynucleotide comprising or consisting of a polynucleotide sequence encoding a polypeptide that has at least 95% identity, even more preferabl . at least 97-99% or exact identity, to the ammo acid sequence of SEQ ID NO 2. over the entire length of SEQ ID NO 2

The polypeptides of die mvention mclude a polypeptide of Table 1 [SEQ ID NO 2] (m particular a mature polypeptide) as well as polypeptides and fragments, particularly those tiiat has a biological actiλ it. of histidme kinase, and also those that have at least 95% identity to a polypeptide of Table 1 [SEQ ID NO 2] and also mclude portions of such polypeptides with such portion of the polypeptide generally compnsmg at least 30 ammo acids and more preferably at least 0 ammo acids

The mvention also mcludes a polypeptide consisting of or compnsmg a polypeptide of the formula X-(R₁)_rn-(R₂)-(R₃)_n-Y wherein, at the ammo terminus. X is hydrogen, a metal or any other moiety descnbed herem for modified polypeptides. and at the carboxyl terminus, Y is hydrogen, a metal or any other moiety descnbed herem for modified polypeptides. Ri and R3 are any ammo acid residue or modified ammo acid residue, m is an integer between 1 and 1000 or zero, n is an mteger between 1 and 1000 or zero, and R is an ammo acid sequence of the mvention, particularly an ammo acid sequence selected from Table 1 or modified forms thereof In the formula above, R is onented so that its ammo terminal ammo acid residue is at the left, covalently bound to Ri and its carboxy terminal ammo acid residue is at the πght, covalently bound to R3 Any stretch of ammo acid residues denoted by either Ri or R3, where m and/or n is greater than 1. may be either a heteropo-ymer or a homopolymer. preferably a heteropolymer Other preferred 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 preferred that a polypeptide of the mvention is deπved from Staphylococcus aureus, however, it may preferably be obtained from other organisms of the same taxononuc genus A polypeptide of the mvention may also be obtained, for example, from organisms of the same taxonomic family or order

A fragment is a vanant polypeptide having an ammo acid sequence that is entirely the same as part but not all of any ammo acid sequence of any polypeptide of the mvention As with histidme kinase polypeptides. fragments may be "free-standing," or compπsed within a larger polypeptide of which they form a part or region, most preferably as a smgle continuous region in a single larger polypeptide

Preferred fragments mclude, for example, truncation polypeptides having a portion of an ammo acid sequence of Table 1 [SEQ ID NO 2], or of vanants thereof, such as a continuous senes of residues that mcludes an ammo- and/or carboxyl-termmal ammo acid sequence Degradation forms of the polypeptides of the mvention produced bv or m a host cell, particularly a Staphylococcus aureus. are also preferred Further preferred are fragments charactenzed bv structural or functional attributes such as fragments Uiat compnse alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-formmg regions, turn and turn- forming regions, coil and coil-forming regions.

regions, alpha amphipathic regions, beta amphipathic regions, flexible regions surface-forming regions, substrate binding region, and high antigenic index regions

Further preferred fragments mclude 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 oϊ 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 ma\ be employed for producmg the corresponding full-length polypeptide by peptide synthesis, therefore, these vanants mav 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 histidme kinase polypeptides, particularly polynucleotides that encode a polypeptide herem designated histidine kinase

In a particularly preferred embodiment of the mvention die polynucleotide compnses a region encodmg histidme kinase polypeptides compnsmg a sequence set out in Table 1 [SEQ ID NO 1] that mcludes a full length gene, or a variant thereof The Applicants believe 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 encodmg and/or expressmg histidme kinase polypeptides and polynucleotides, particularly Staphylococcus aureus histidme kinase 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, prophvlacticalh . clinically or therapeuticallv useful polvnucleotides 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 histidme kinase 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 invention there is a histidme kinase polypeptide from Staphylococcus aureus compnsmg or consistmg of an ammo acid sequence of Table 1 [SEQ ID NO 2], or a vaπant thereof Using the information provided herein, such as a polynucleotide sequence set out m Table 1 [SEQ

ID NO 1], a polynucleotide of the mvention encodmg histidme kinase pohpeptide mav be obtained usmg standard cloning and screening methods, such as those for cloning and sequencing chromosomal DNA fragments from bactena usmg Stapnylococcus aureus WCUH 29 cells as starting matenal followed b\ obtainmg a full length clone For example, to obtain a polynucleotide sequence of the invention such as a polynucleotide sequence given m Table 1 [SEQ ID NO 1], t\pιcallv a library of clones of chromosomal DNA of Staphylococcus aureus WCUH 29 m E colt or some other suitable host is probed with a radiolabeled o gonucleotide. preferably a 17-mer or longer, denved from a partial sequence Clones carrying DNA identical to that of the probe can then be distinguished using stringent hvbndization conditions B\ sequencing the individual clones thus identified b\ hybridization with sequencing primers designed from the original polypeptide or poh nucleotide sequence it is then possible to extend the polynucleotide sequence in both directions to determine a full length gene sequence Conveniently such sequencing is performed, for example, usmg denatured double stranded DNA prepared from a plasmid clone Suitable techniques are descnbed by Maniatis. 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 obtam 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 reading frame encodmg a protem havmg about the number of ammo acid residues set forth m 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 m the art The polynucleotide of SEQ ID NO 1, between nucleotide number 222 and the stop codon that begins at nucleotide number 1259 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 consisting 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 which encodes SEQ ID NO 2, (b) a polynucleotide sequence encodmg 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, including homologs and orthologs from species other than Staphylococcus aureus. may be obtained by a process that compnses the steps of screening an appropnate library under stringent h\ bndization conditions with a labeled or detectable probe consisting of or compnsmg die sequence of SEQ ID NO 1 or a fragment thereof, and isolating a full-length gene and/or genomic clones compnsmg said polynucleotide sequence

The mvention provides a pohnucleotide sequence identical over its entire lengtii to a coding sequence (open reading frame) in Table 1 [SEQ ID NO 1] Also provided by the mvention is a coding sequence for a mature polypeptide or a fragment thereof, by itself as well as a coding sequence for a mature polypeptide or a fragment m reading frame with another coding sequence, such as a sequence encodmg a leader or secretory sequence, a pre-, or pro- or prepro-protem sequence The polynucleotide of die invention mav also compnse at least one non-coding sequence including for example but not limited to at least one non-coding 5 and 3^' sequence such as the transcnbed but non-translated sequences termination signals (such as rho-dependent and rho-mdependent termination signals), nbosome binding sites kozak sequences sequences that stabilize mRNA. mtrons. and polyadenylation signals The polynucleotide sequence m \ also compnse additional coding sequence encodmg additional ammo acids. For example, a marker sequence that facilitates purification 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 el al . Proc Natl Acad Set USA 86 821-824 (1989). or an HA peptide tag (Wilson et al . Cell 37 767 (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 of consisting of or compnsmg nucleotide 222 to the nucleotide immediately upstream of or including nucleotide 1259 set forth in SEQ ID NO 1 of Table 1. both of that encode a histidme kinase polypeptide

The mvention also mcludes a polynucleotide consisting of or compnsmg a polynucleotide of die formula X-(R₁)_m-(R₂)-(R₃)n-Y wherein, at the 5¹ 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 R\ and R3 is independently any nucleic acid residue or modified nucleic acid residue, m is an mteger 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 mvention, 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 nght, 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 mteger between 1 and 1000. Otiier preferred 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 preferred that a polynucleotide of the mvention is denved from Staphylococcus aureus however, it mav preferably be obtained from other organisms of the same taxonomic genus A. polynucleotide of the invention mav also be obtained, for example from organisms of die same taxonomic family or order

The term "polynucleotide encodmg a polypeptide" as used herem encompasses polvnucleotides that mclude a sequence encodmg a polypeptide of the mvention. particularly a bactenal polypeptide and more particularly a polypeptide of the Staphylococcus aureus histidme kinase havmg an ammo acid sequence set out m Table 1 [SEQ ID NO 2] The term also encompasses polynucleotides that mclude a smgle contmuous region or discontinuous regions encoding the polypeptide (for example, polynucleotides interrupted bv integrated phage. an integrated 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 coding 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 preferred embodiments are polynucleotides encodmg histidme kinase vanants. that have the ammo acid sequence of histidme kinase polypeptide of Table 1 [SEQ ID NO 2] in 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 combination Especially preferred among these are silent substitutions, additions and deletions, that do not alter the properties and activities of histidme kinase 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 havmg 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 preferred embodiments of the mvention are polynucleotides that are at least 95% or 97% identical over their entire length to a polynucleotide encodmg histidme kinase polypeptide havmg an ammo acid sequence set out m Table 1 [SEQ ID NO 2], and polynucleotides that are complementary to such polvnucleotides Most highly preferred are polvnucleotides that compnse a region tiiat is at least 95% are especially preferred Furthermore, those with at least 97% are highly preferred among those with at least 95%o. and among these those with at least 98% and at least 99% are particularly highly preferred, with at least 99% bemg the more preferred

Preferred embodiments are polvnucleotides encodmg polypeptides that retain substantially the same biological function or activit as a mature polvpeptide encoded bv a D A. of Table 1 [SEQ ID NO 1 1 In accordance witii certain preferred embodiments of this invention there arc provided polynucleotides that hybndize. particularly under strmgent conditions to histidme kinase 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 strmgent conditions to the polynucleotides descnbed herem As herem used, the terms "strmgent conditions" and "strmgent hybndization conditions" mean hvbπdization occurring only if there is at least 95% and preferably at least 97% identity between the sequences A specific example of strmgent hybndization conditions is overnight incubation at 42°C m a solution compnsmg 50% formamide. 5x SSC (150mM NaCl, 15mM tπsodium citrate), 50 mM sodium phosphate (pH7 6). 5x Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml of denatured, sheared salmon sperm DNA, followed by washmg the hybndization support m 0 lx SSC at about 65°C Hybndization and wash conditions are well known and exemplified in Sambrook. et al . Molecular Cloning A Laboratory Manual. Second Edition, Cold Spring Harbor, N Y , (1989), particularly Chapter 11 therem Solution hybridization may also be used with the polynucleotide sequences provided by the mvention The invention also provides a polynucleotide consisting of or comprising a polynucleotide sequence obtamed by screemng an appropnate library compnsmg a complete gene for a polynucleotide sequence set forth in SEQ ID NO 1 under strmgent hybridization conditions with a probe having the sequence of said polynucleotide sequence set forth m SEQ ID NO 1 or a fragment thereof, and isolating said polynucleotide sequence Fragments useful for obtaimng such a polynucleotide include. for example, probes and primers fully descnbed elsewhere herein

As discussed elsewhere herem regarding polynucleotide assays of the mvention. for instance, die 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 histidme kinase and to isolate cDNA and genomic clones of other genes that have a high identity, particularly high sequence identity, to a histidme kinase 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 preferred probes will have at least 20 nucleotide residues or base pairs and will have lee than 30 nucleotide residues or base pairs A coding region of a histidme kinase gene may be isolated by screening usmg a DNA sequence provided m Table 1 [SEQ ID NO 1] to synthesize an o gonucleotide probe A labeled oligonucleotide 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 hbrarv die probe hvbndizes to There are several methods available and well known to those skilled in 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 technique, 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 hgated onto each end Nucleic acid amplification (PCR) is then carried out to amplify the "missing" 5' end of the DNA usmg a combmation of gene specific and adaptor specific ohgonucleotide primers The PCR reaction is then repeated using "nested" primers, that is. primers designed to anneal within 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 existmg 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 matenals 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 denved from a sequence of Table 1 [SEQ ID NOS 1 or 2] may be used in the processes herem as descnbed. but preferably for PCR. to determine whether or not the polynucleotides identified herem in whole or m part are transcribed in bactena m infected tissue It is recogmzed that such sequences will also have utility in diagnosis of the stage of infection and type of infection the pathogen has attained

The mvention also provides polynucleotides that encode a polypeptide that is a mature protem plus additional ammo or carboxyl-terminal ammo acids, or ammo acids tenor 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-life or may facilitate manipulation of a protem for assay or production, among other things As generally is the case in vivo, the additional ammo acids may be processed awav from a mature protem bv cellular enzymes For each and every polynucleotide of the mvention there is provided a polynucleotide complementarv to it It is preferred that these complementary polynucleotides are fullv complementarv to each polynucleotide with which they are complementary

A precursor protem havmg a mature form of the polvpeptide fused to one or more prosequences may be an mactive form of the polypeptide When prosequences are removed such mactive precursors generallv are activated Some or all of the prosequences mav be remo ed before activation Generally such precursors are called proproterns

As will be recognized, the entire polvpeptide encoded by an open reading frame is often not required for activity Accordingly, it has become routine m molecular biology to map the boundanes of the primary structure required for activity with N-termmal and C-terminal deletion experiments Tliese experiments utilize exonuclease digestion or convement restnction sites to cleave coding nucleic acid sequence For example. Promega (Madison- WI) sell an Erase-a-base™ system that uses Exonuclease III designed to facilitate analysis of the deletion products (protocol available at www promega com) The digested endpoints can be repaired (e g , by gation 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

In sum, a polynucleotide of the mvention may encode a mature protem, a mature protem plus a leader sequence (which may be referred 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 preproprotein. that is a precursor to a proprotein. having a leader sequence and one or more prosequences. that generally are removed during processing 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 engmeered with vectors of the mvention and the production of polypeptides of the mvention by recombinant techniques Cell-free translation systems can also be employed to produce such proteins usmg RNAs denved from the DNA constructs of the mvention

Recombinant polypeptides of the present mvention may be prepared by processes well known m those skilled m the art from genetically engmeered 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 engmeered with such expression systems, and to the production of polypeptides of the mvention bv recombinant techniques For recombinant production of the polypeptides of the mvention. host cells can be genetically engmeered 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 in 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. microinjection. cationic lipid-mediated transfection. electroporation. transduction. scrape loading, ballistic mtroduction and infection

Representative examples of appropnate hosts mclude bactenal cells, such as cells of streptococci. staphylococci. enterococci E colt, streptomyces. cyanobactena. Bacillus subtilis. 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 Drosophtla 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 vanety of expression systems can be used to produce the polypeptides of the mvention

Such vectors mclude, among others, chromosomal-, episomal- and vmis-denved vectors, for example, vectors denved from bactenal plasmids, from bactenophage, 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 maintain, 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 techmques, 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 reuculum. mto the penplasmic space or mto the extracellular environment, appropnate secretion signals may be incorporated mto the expressed polypeptide These signals may be endogenous to the polypeptide or they may be heterologous signals

Polypeptides of the mvention can be recovered and punfied from recombinant cell cultures by well- known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography. phosphocellulose chromatographv hvdrophobic interaction chromatographv affinity chromatography. hydroxylapatite chromatography. and lectm chromatographv Most preferably, high performance liquid chromatography is employed for purification Well known techmques for refolding protem mav be employed to regenerate active conformation when the polvpeptide is denatured during isolation and or purification

Diagnostic, Prognostic, Serotyping and Mutation Assays This mvention is also related to the use of histidme kinase polvnucleotides and polypeptides of the mvention for use as diagnostic reagents Detection of histidme kinase polvnucleotides and/or polypeptides in 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 Eukarvotes particularly mammals, and especially humans, particularly those infected or suspected to be infected with an organism compnsmg the histidme kinase gene or protein, may be detected at the nucleic acid or ammo acid level by a vanety of well known techmques as well as by methods provided herem

Polypeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtained from a putative-y 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 technique pnor to analysis RNA, particularly mRNA, cDNA and genomic DNA may also be used m the same ways Using amplification, characterization 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 insertions 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 hybndizmg amplified DNA to labeled histidme kinase polynucleotide sequences Perfectly 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 detecting differences m meltmg temperatures or renaturation kinetics Polynucleotide sequence differences may also be detected bv alterations m the electrophoretic mobility of polynucleotide fragments m 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 Nail Acad Sci , USA, 85 4397-4401 (1985)

In another embodiment, an array of oligonucleotides probes compnsmg histidme kinase nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of. for example genetic mutations, serotype. taxonomic classification or identification Array technology' metiiods are w ell known and have general applicability and can be used to address a vanetv of questions m molecular genetics including gene expression, genetic linkage, and genetic vanabihty (see. for example, Chee et al . Science 274 610 (1996))

Thus m another aspect, the present mvention relates to a diagnostic kit that comprises (a) a polynucleotide of the present mvention. preferably the nucleotide sequence of SEQ ID NO 1. or a fragment thereof . (b) a nucleotide sequence complementary to that of (a) (c) a poh peptide of the present invention, preferably the polypeptide of SEQ ID NO 2 or a fragment thereof or (d) an antibody to a polypeptide of the present invention, preferably to the polvpeptide of SEQ ID NO 2 It will be appreciated that in any such kit, (a), (b). (c) or (d) may compnse a substantial component Such a kit will be of use m 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 orgamsms. carrying mutations m such polynucleotide may be detected at the polynucleotide level by a vanety of techmques, such as those descnbed elsewhere herem

The differences m a polynucleotide and/or polypeptide sequence between orgamsms possessmg a first phenotype and organisms possessmg a different, second different phenotype can also be determined If a mutation is observed m some or all orgamsms possessing the first phenotype but not in any orgamsms possessmg 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 (allehc vanations) m a polynucleotide and/or polypeptide of the mvention may also be detected at the polynucleotide or polypeptide level by a vanety of techmques, to allow for serotyping, for example For example, RT-PCR can be used to detect mutations m the RNA It is particularly preferred 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 histidme kinase polypeptide can be used to identify and analyze mutations. The mvention further provides these primers with 1, 2, 3 or 4 nucleotides removed from the 5' and/or the 3' end These primers may be used for. among other dungs, amplifying histidme kinase DNA and/or RNA isolated from a sample denved from an individual, such as a bodύy 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 tins vvav. mutations m the polynucleotide sequence mav 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 diagnosing, disease, preferably bacterial infections, more preferably infections caused by Staphylococcus aureus. comprising determining from a sample derived from an individual 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 histidme kinase polynucleotide can be measured using any on of the methods ell knovvn in the art tor 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 histidme kinase polypeptide compared to normal 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 histidme kinase polypeptide. m a sample denved from a host, such as a boddy matenal. are well-known to those of skill m the art Such assay methods mclude radioimmunoassays, 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 hgands in. for example, cells, cell-free preparations, chemical hbranes. and natural product mixtures These substrates and gands may be natural substrates and hgands 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, including 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 Accordingly, m a further aspect, the present mvention provides for a method of screening compounds to identify those that agonize 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 vanety of sources, for example, cells, cell-free preparations, chemical hbranes. and natural product mixtures Such agonists and antagonists so-identified may be natural or modified substrates, hgands. receptors, enzymes, etc , as the case mav be. of histidme kinase polypeptides and polynucleotides. or mav be structural or functional mimetics thereof (see Coligan et al . Current Protocols in Immunology 1 (2) Chapter 5 ( 1991 ))

The screemng methods mav simply measure the binding of a candidate compound to the polypeptide or polynucleotide. or to cells or membranes bearmg the polypeptide or polynucleotide. or a fusion protem of the polypeptide bv means of a label directly or indirectly associated with the candidate compound Alternativ elv the screenmg method may involve competition with a labeled competitor Further, these screenmg methods mav test whether the candidate compound results in a signal generated by activation or inhibition of the polvpeptide or polynucleotide. using detection systems appropnate to the cells compnsmg the polypeptide or polynucleotide Inhibitors of activation are generally assayed m the presence of a known ago st and the effect on activation by the agonist bv the presence of the candidate compound is observed Constitutively active polypeptide and/or constitutively expressed polypeptides and polynucleotides may be employed in screemng methods for inverse agomsts, m the absence of an agonist or antagonist, by testing whether the candidate compound results in inhibition of activation of the polypeptide or polynucleotide, as the case may be Further, the screemng methods may simply compnse the steps of mixing a candidate compound with a solution compnsmg a polypeptide or polynucleotide of the present invention, to form a mixture, measuring histidme kinase polvpeptide and/or polynucleotide activity in the mixture, and comparing the histidme kinase polypeptide and/or polynucleotide activity of the mixture to a standard Fusion protems. such as those made from Fc portion and histidme kinase polypeptide. as herein descnbed. can also be used for high-throughput screemng 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 bmd to and/or mteract with a polypeptide of the present mvention may also be used to configure screemng methods for detectmg the effect of added compounds on the production of mRNA and/or polypeptide m cells For example, an ELISA assay may be constructed for measuπng secreted or cell associated levels of polypeptide usmg monoclonal and polvclonal antibodies bv standard methods known in the art This can be used to discover agents that may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues

The mvention also provides a method of screenmg compounds to identify those that enhance (agomst) or block (antagonist) the action of histidme kinase polypeptides or polvnucleotides particularly those compounds that are bacteπstatic and/or bacteπcidal The method of screening mav mvolve high- throughput techmques For example, to screen for agomsts or antagonists, a synthetic reaction mix a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of anv thereof compnsmg histidme kinase polypeptide and a labeled substrate or ligand of such polypeptide is mcubated m the absence or the presence of a candidate molecule that may be a histidme kinase agomst or antagonist The ability of the candidate molecule to agonize or antagonize the histidme kinase polypeptide is reflected in decreased binding of the labeled hgand or decreased production of product from such substrate Molecules that bmd gratuitously'. ; e . widiout inducing the effects of histidme kinase polvpeptide are most likelv 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 activ ιtv 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 tiiat mav be useflil in this regard include but are not limited to colonmetric. labeled substrate converted into product, a reporter gene that is responsive to changes in histidine kinase 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 binding techniques known in the art. These techniques include, but are not limited to, ligand binding and crosslinking assays in which the polypeptide is labeled with a radioactive isotope (for instance, ^5^ 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 supernatants. tissue extracts, bodily materials) Other methods include biophy sical techniques such as surface plasmon resonance and spectroscopy These screening methods

also be used to identify agonists and antagonists of the polypeptide that compete with the binding of the polvpeptide 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 histidme kinase polypeptide associating with another histidine kinase polypeptide or other polypeptide. labeled to compnse a fluorescently-labeled molecule will have higher polarization values than a fluorescently labeled monomeric protein. It is preferred that this method be used to characteπze small molecules that disrupt polypeptide complexes. Fluorescence energy transfer may also be used characteπze small molecules that interfere with the formation of histidine kinase polypeptide dimers. trimers, tetramers or higher order structures, or structures formed by histidme kinase polypeptide bound to another polypeptide. histidme kmase polvpeptide can be labeled w ith both a donor and acceptor fluorophore pon mixmg 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 dimenzation will inhibit fluorescence energy transfer

Surface plasmon resonance can be used to monitor the effect of small molecules on histidine kmase polvpeptide self-association as ell as an association of histidine kmase polypeptide and another polypeptide or small molecule Histidme kinase 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 ov er the histidme kinase polypeptide -coated surface and specific binding can be detected in real-time bv monitoπng the change m resonance angle caused bv a change in local refractive index This technique can be used to characteπze the effect of small molecules on kmetic rates and equihbπum binding constants for histidine kinase polvpeptide self-association as well as an association of histidine kinase polvpeptide and another polv peptide or bmall molecu

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

In other embodiments of the mvention diere are provided methods for identifying compounds that bmd to or otherwise mteract 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 binding to or other interaction between the compound and the polypeptide and/or polynucleotide to assess the binding to or other interaction with the compound, such binding or interaction preferably bemg associated with a second component capable of providing a detectable signal m response to the binding or interaction of the polypeptide and/or polynucleotide with the compound, and determining whether the compound bmds to or otherwise interacts with and activates or inhibits an activity or expression of the polypeptide and/or polynucleotide by detecting the presence or absence of a signal generated from the binding or mteraction of the compound with the polypeptide and/or polynucleotide Another example of an assay for histidme kinase agomsts is a competitive assav that combmes histidme kinase and a potential agomst with histidme kinase-binding molecules, recombinant histidine kinase binding molecules, natural substrates or hgands. or substrate or hgand mimetics. under appropnate conditions for a competitive inhibition assay Histidme kinase can be labeled, such as by radioactivity or a colonmetnc compound, such that the number of histidme kinase molecules bound to a binding molecule or converted to product can be determmed accurately to assess the effectiveness of the potential antagomst

It will be readily appreciated by the skilled artisan that a polypeptide and/or polynucleotide of the present mv ention may also be used m 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). binding sιte(s) or motιf(s) of an agonist or antagomst. (c) synthesizing candidate compounds that are predicted to bind to or react with the deduced binding sιte(s). reactive sιte(s), and/or moti 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 rterativ e process may be performed using automated and computer-controlled steps

In a further aspect, the present mvention provides methods of treating 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 histidme kinase 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 comprises administering to an individual m need thereof an inhibitor compound (antagomst) as herem descnbed. optionally m combination with a pharmaceutically acceptable earner, m an amount effective to inhibit the function and or expression of the polypeptide and/or polynucleotide. such as. for example, by blockmg the binding of hgands. 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 bmding the gand. substrate, enzymes, receptors, etc in competition with endogenous polypeptide and/or polynucleotide may be administered Typical examples of such competitors include fragments of the histidme kinase polypeptide and/or polypeptide In still another approach, expression of the gene encoding endogenous histidme kinase polypeptide can be inhibited usmg expression blockmg techniques This blocking may be targeted against any step m gene expression, but is preferably targeted agamst transcription and/or translation An examples of a known technique 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 in Ohgodeoxv nucleotides as Antisense Inhibitors of Gene Expression CRC Press Boca Raton FL (1988)) Alternatively , oligonucleotides that form triple helices with the gene can be supplied (see toi example. Lee et al Nucleic Acids Res (1979) 6 3073, Coonev et al Science ( 1988) 241 456 Dervan et al , Science (1991) 251 1360) These ohgomers can be administered per se or the relevant ohgomers can be expressed in vivo

Each of the polynucleotide sequences provided herem may be used in the discovery and development of antibacterial compounds The encoded protein, 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 protein or Shme-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the codmg sequence of interest The invention also provides the use of the polypeptide, polynucleotide. agonist or antagonist of the invention to interfere with the initial phy sical interaction between a pathogen or pathogens and a eukaryotic. preferably mammalian, host responsible for sequelae of infection In particular, the molecules of the mvention may be used' in the prevention of adhesion of bacteria, in particular gram positive and/or gram negative bacteπa. to eukaryotic. preferably' mammalian, extracellular matrix proteins on in-dwelling devices or to extracellular matrix proteins in wounds, to block bacterial adhesion between eukaryotic, preferably mammalian, extracellular matrix proteins and bacterial histidine kinase proteins that mediate tissue damage and/or. to block the normal progression of pathogenesis in infections initiated other than by the implantation of in-dwelling devices or by other surgical techniques .

In accordance with yet another aspect of the mvention. there are provided histidine kinase agonists and antagonists, preferably bacteristatic or bactericidal agonists and antagonists.

The antagonists and agomsts of the invention may be employed, for instance, to prevent, inhibit and/or treat diseases. Hehcobacter pylon (herein "H pylon") bacteria infect the stomachs of over one-third of the world's population causing stomach cancer, ulcers, and gastritis (International Agency for Research on

Cancer (1994) Schistosυmes; Liver Flukes and Hehcobacter P\ lon (International Agency for

Research on Cancer. Lyon. France. http://vwvvv uιcc.ch/ecp/ecp2904 htm) Moreov er, the International

Agency for Research on Cancer recently recogmzed a cause-and-effect relationship between H. pylori and gastric adenocarcinoma, classifying the bacterium as a Group I (definite) carcinogen Preferred antimicrobial compounds of the invention (agonists and antagonists of histidine kinase polypeptides and/or polynucleotides) found using screens provided by the invention, or known in the art. particularly narrow-spectrum antibiotics, should be useful in the treatment of H. pylori infection. Such treatment should decrease the advent of H. pylori -induced cancers, such as gastrointestinal carcmoma. 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 in this specification are herein incorporated by reference in 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 priority is also incorporated by reference herein m its entirety in 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 individual or from an organism infecting, infesting or inhabiting an individual, including but not limited to. cells, tissues and waste, such as. bone blood, serum, cerebrospinal fluid, semen, saliva, muscle, cartilage, organ tissue, skin, urine stool or autopsv matenals "Dιsease(s)" means any disease caused bv or related to infection by a bactena. including . for example, disease, such as. infections of the upper respiratory tract (e g . otitis media, bactenal tracheitis. acute epiglottitis. thy roiditis) loyyer respiratory (e g . empvema lung abscess) cardiac {s. g infective endocarditis) gastromtestmal (e g . secretory diaπhoea. splemc absces retropentoneal abscess). CNS (e g cerebral abscess), eve (e g . blephantis conjunctivitis, keratitis endophthalrrutis preseptal and orbital celluhtis. darcryocystitis). kidney and urinary tract (e g . epididvm-tis. mtrarenal and pennephπc absces. toxic shock syndrome), skin (e g , impetigo, fol cuhtis. cutaneous abscesses, celluhtis wound infection bactenal mvositis) bone and jomt (e g , septic arthntis. 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 polvpeptide sequences or two or more polynucleotide sequences, as the case may be, as determmed 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 descnbed in (Computational Molecular Biology, Lesk, A M . ed . Oxford University Press. New York. 1988, Biocomputing Informatics and Genome Projects. Smith. D W . ed , Academic Press. New York, 1993. Computer Analysis of Sequence Data. Part I. 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. Gπbskov. 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 Biol 215 403-410 (1990) The BLAST X program is publicly available from NCBI and other sources (BLAST Manual. Altschul. S . et al , NCBI NLM NIH Bethesda, MD 20894. Altschul. S . et al . J Mol Biol 215 403-410 (1990) The w ell known Smith Waterman algorithm may also be used to determine 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 compansons

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

(1) Polynucleotide embodiments further mclude an isolated polynucleotide compnsmg a polynucleotide sequence havmg at least a 95, 97 or 100% identity to the reference sequence of SEQ ID NO 1. wherem said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO 1 or may mclude up to a certain integer number of nucleotide alterations as compared to the reference sequence, herem said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion. or insertion, and wherein said alterations mav 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 in the reference sequence or m 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 in SEQ ID NO 1 bv the mteger defining the percent identity divided by 100 and then subtracting that product from said total number of nucleotides in SEQ ID NO 1. or

ⁿn ≤ ^λn " (^xn • 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 integer prior to subtracting it from x_n Alterations of a poly nucleotide sequence encoding the polvpeptide of SEQ ID NO 2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations

(2) Polypeptide embodiments further include an isolated polypeptide comprising a polypeptide havmg 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, wherein said alterations are selected from the group consisting of at least one ammo acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the ammo- or carboxy-termmal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the ammo acids in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of amino acid alterations is determined bv multiply ing the total number of amino acids in SEQ ID NO 2 bv the integer defining the percent identity dn ided b\ 100 and then subtracting that product from said total number of amino acids in SEQ ID NO 2 or

n_a < x_a - (x_a • y),

wherein n_a is the number of amino acid alterations, x_a is the total number of amino acids in 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 where any non-mteger product of x_a and y is rounded down to the nearest integer prior to subtracting it from x_a

"Indrvιdual(s)" means a multicellular eukaryote, including, but not limited to a metazoan. a mammal, an ovid. a bov d. a simian, a primate, and a human

"Isolated" means altered "by the hand of man" from its natural state. i e . if it occurs m nature, it has been changed or removed from its ongmal environment, or both For example, a polynucleotide or a polypeptide naturally present in a livmg orgamsm is not isolated but die same poK nucltotidL or polypeptide separated from the coexisting matenals of its natural state is "isolated" as die tenn is employed herem \loreov er. a polynucleotide or polypeptide that is introduced mto an orgamsm bv transformation genetic manipulation or by any- other recombinant method is "isolated" even if it is still present in said organism, which organism may be living or non-living.

"Organism(s)" means a (i) prokaryote, including but not limited to. a member of the genus Streptococcus. Staphylococcus. Bordetella. Corynebacterium. Mycobacterium. Neisse a. Haemophilus. Actinomycetes. Streptomycetes. Nocardia. Enterobacter, Yersinia, Fancisella. Pasturella. Moraxella. Acinetobacter, Erysipelothrix. Branhamella, Actinobacillus. Streptobacillus. Listeria. Calymmatobacterium. Brucella, Bacillus. Clostridium, Treponema. Escherichia. Salmonella. Kleibsiella. Vibrio. Proteus: Erwinia. Borrelia. Leptospira. Spirillum. Gampvlohaaer. Shi gel la / eawnel/a Pseudomonas. Aeromonas: Rickettsia. Chlamydia. Borrelia and Mycυplasma. and further including, 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 pneumυniae. Streptococcus pyogenes. Streptococcus agalactiae. Streptococcus faecalis, Streptococcus faecium. Streptococcus clurans: Neisseria gonorrheae. Neisseria meningitidis. Staphylococcus aureus. Staphylococcus epidermidis. Corynebacterium diptheriae. Gardnerella vaginalis, Mycobacterium tuberculosis. Mycobacterium bovis. Mycobacterium ulcerans, Mycobacterium leprae, Actinomyctes israelii, Listeria monocytogenes. Bordetella pertusis, Bordatella parapertusis, Bordetella bronchiseptica, Escherichia coli, Shigella dysenteriae, Haemophilus influenzae, Haemophilus aegyptius, Haemophilus parain luenzae, Haemophilus ducreyi, Bordetella, Salmonella typhi, Citrobacter freundii, Proteus mirabilis, Proteus vulgaris, Yersinia pestis. Kleibsiella pneumoniae, Serratia marcessens, Serratia liquefaciens, Vibrio cholera. Shigella dysenterii, Shigellaflexneri, Pseudomonas aeruginosa, Franscisella tularensis, Brucella abortis, Bacillus anthracis. Bacillus cereus, Clostridium perfringens, Clostridium tetani. Clostridium botulinum. Treponema pallidum. Rickettsia rickettsii and Chlamydia trachomitis, (ii) an archaeon, including but not limited to Archaebacter, and (iii) a unicellular or filamentous eukaryote, including but not limited to. a protozoan, a fungus, a member of the genus Saccharomyces, Kluveromyces. or Candida, and a member of the species Saccharomyces ceriviseae, Kluveromyces lactis, or Candida albicans.

"Polynucleotide(s)" generally refers to any polyribonucleotide or polydeoxyribonucleotide. that may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide(s)" include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and triple-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or. more typically, double-stranded, or triple-stranded regions, or a mixture of single- and double-stranded regions. In addition, "polynucleotide" as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules One of the molecules of a tnple-he cal region often is an ohgonucleotide As used herein, the term "polynucleotιde(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 "polynucleotιde(s)" as that term is intended herem Moreover. DNAs or RNAs compnsmg unusual bases, such as inosine. or modified bases, such as tntylated bases, to name just two examples, are polynucleotides as die term is used herem It will be appreciated that a great vanety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill m die art The term "polynucleotιde(s)" as it is employed herem embraces such chemically, enzymaticallv or metabohcallv 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 polynucleotides often refened to as ohgonucleotide(s)

"Polypeptide(s)" refers to any peptide or protem compnsmg two or more ammo acids jomed to each other by peptide bonds or modified peptide bonds "Polypeptιde(s)" refers to both short chains, commonly refened to as peptides. ohgopeptides and ohgomers and to longer chains generally refened to as proteins Polypeptides may compnse ammo acids other than die 20 gene encoded ammo acids "Polypeptιde(s)" mclude those modified either by natural processes, such as processmg and odier post-translational modifications, but also by chemical modification techmques Such modifications are well descnbed in basic texts and m more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill m the art It will be appreciated that the same type of modification may be present m the same or varymg degree at several sites m a given polypeptide Also, a given polypeptide mav 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-πbosylation. amidation. covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide denvative. covalent attachment of a hpid or hpid denvative. covalent attachment of phosphotidylinositol, cross-linking, cyc zation. disulfide bond formation, demethylation. formation of covalent cross-links, formation of cysteme, formation of pyroglutamate, formvlation gamma-carboxvlation. GPI anchor formation hv droxvlation. lodination. methylation. myπstoylation oxidation, proteolvtic processmg. phosphorv lation prenv lation racemization. glycosylation. hpid attachment, sulfation. gamma-carboxylation of glutamic acid residues hydroxv lation and ADP-nbosylation. selenoylation. sulfation. transfer-RNA mediated addition of ammo acids to proteins, such as argrnylation. 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 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS. B C Johnson. Ed . Academic Press. New York (1983). Seifter et zl . 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 Cv chc. branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well

"Recombinant expression svstem(s)" refers to expression systems or portions thereof or polynucleotides of the mvention mtroduced or transformed into a host cell or host cell lvsate tor the production of the polynucleotides and polypeptides of the mvention

^■'Vaπant(s)^"' as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties A typical v ariant of a polynucleotide differs m nucleotide sequence from another, reference polynucleotide Changes in the nucleotide sequence of the variant mav or may not alter the amino acid sequence of a polvpeptide 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 in ammo acid sequence from another, reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the vanant are closely similar overall and. in many regions, identical A vaπant and reference polypeptide may differ in ammo acid sequence by one or more substitutions, additions, deletions in any combination A substituted or inserted amino acid residue may or mav not be one encoded by the genetic code The present invention also includes mclude variants of each ot tl polypeptides of the mvention. that is polypeptides that vary from the referents by conserv ativ e ammo acid substitutions, wherebv a residue is substituted bv 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 Tvr 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 anv combmation A variant of a polynucleotide or polypeptide mav be a naturally occurring such as an allehc vanant. or it may be a variant that is not known to occur naturally Non- naturally occurπng vanants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans EXAMPLES

The examples below are earned out usmg standard techmques. 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 die mv ention Example 1 Strain selection, Library Production and Sequencing The polynucleotide havmg 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 coh The sequencing data from two or more clones comprising overlapping Staphylococcus aureus DNAs was used to construct the contiguous DNA sequence in SEQ ID NO 1 Libraries mav be prepared bv routine methods toi example

Methods 1 and 2 below

Total cellular DNA is isolated from Staphylococcus aureus WCUH 29 according to standard procedures and size-fractionated bv either of two methods

Method 1 Total cellular DNA is mechanically sheared by passage through a needle in order to size- fractionate accordmg to standard procedures DNA fragments of up to 1 lkbp m size are rendered blunt by treatment with exonuclease and DNA polymerase. and EcoRI linkers added Fragments are ligated into the vector Lambda ZapII that has been cut with EcoRI. the library packaged by standard procedures and E coh infected with the packaged library The library is amplified by standard procedures

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combination of restriction enzvmes 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 ligated to the DNA and the fragments then ligated into the vector Lambda ZapII that have been cut with EcoRI, the hbraiy packaged by standard procedures, and E coh infected with the packaged library The library is amplified by standard procedures

Claims

What is claimed is:

1 An isolated polvpeptide selected from the group consisting of

(1) an isolated polypeptide comprising an ammo 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 comprising nucleotides 222 to 1258 of SEQ ID NO 1

2 An isolated polynucleotide selected from the group consisting of

(I) an isolated polynucleotide compnsmg a polynucleotide sequence encodmg a polypeptide diat has at least 95% identity to the ammo acid sequence of SEQ ID NO 2. over the entire lengdi of SEQ ID NO 2.

(n) an isolated polynucleotide compnsmg a polynucleotide sequence diat lias at least 95% identity over its entire length to a polynucleotide sequence encodmg the polypeptide of SEQ ID NO 2.

(m) an isolated polynucleotide compnsmg a nucleotide sequence that has at least 95 % identity to that of SEQ ID NO 1 over the entire length of that portion of SEQ ID NO 1 which encodes SEQ ID NO 2.

(iv) an isolated polynucleotide compnsmg a nucleotide sequence encodmg the polypeptide of SEQ ID NO 2,

(v) an isolated polynucleotide that is nucleotides 222 to 1258 of SEQ ID NO 1,

(vi) an isolated polynucleotide of at least 30 nucleotides m length obtainable by screemng an appropnate library under strmgent hybndization conditions with a probe having the sequence of SEQ ID NO 1 or a fragment thereof of at least 30 nucleotides m length.

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

(vm) a polynucleotide sequence complementary to said isolated polynucleotide of (I), (11). (in), (iv).

3 A method for the treatment of an individual

(l) in need of enhanced activity or expression of or immunological 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 individual a therapeutically effective amount of an antagonist to said polypeptide. or (b) administering to the individual 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 hgand. substrate, or receptor, or

(d) administering to the individual an amount of a polypeptide that induces an immunological response to said polypeptide m said individual

4 A process for diagnosing or prognosing a disease or a susceptibility to a disease in an individual 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 in an orgamsm in said mdividual, or

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

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

(I) an isolated polypeptide comprising an ammo acid sequence selected from the group having 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 polypeptide comprising the ammo 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 comprising nucleotides 222 to 1258 of SEQ ID NO 1. compnsmg the step of cultunng 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 expressmg a polypeptide selected from the group consisting of

(i) an isolated polypeptide compnsmg 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 polvpeptide comprising the amino acid sequence of SEQ ID NO 2

(in) an isolated polypeptide that is the amino acid sequonc - of SEQ I D \() 2 tnd

(iv) a polypeptide that is encoded by a recombinant polynucleotide comprising nucleotides 222 to 1258 of SEQ ID NO 1. said process comprising the step of transforming or transfectmg a cell ith an expression system compnsmg a polynucleotide capable of producing said polvpeptide of (1). (n), (in) or (iv) when said expression system is present m a compatible host cell such the host cell, under appropnate culture conditions, produces said polypeptide of (1), (11). (in) or (iv)

7 A host cell or a membrane 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 ov er the entire length of SEQ ID

NO 2.

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

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

(iv) a polypeptide that is encoded bv a recombinant polynucleotide comprising nucleotides 222 to 1258 of SEQ ID NO 1

8 An antibody lmmunospecific for the polypeptide of claim 1

9 A method for screenmg to identify compounds that agonize or that inhibit the function of the polypeptide of claim 1 that compnses a method selected from the group consisting of

(a) measuπng the binding of a candidate compound to the polypeptide (or to the cells or membranes bearmg the polypeptide) or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound,

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

(c) testing whether the candidate compound results in a signal generated bv activation or inhibition of the polypeptide. using detection systems appropriate to the cells or cell membranes beaπng the polypeptide.

(d) mixing a candidate compound with a solution comprising a poh peptide 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 agomst or antagonist to the polypeptide of claim 1

; ι -