WO2001023599A1 - Streptococcus pneumoniae ykqc - Google Patents

Abstract

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

Description

Streptococcus pneumoniae ykqC

HELD OF THE INVENTION

This invention relates to newly identified polynucleotides and polypeptides. and their production and uses, as well as their variants, agonists and antagonists, and their uses In particular, the invention relates to polynucleotides and polypepUdes of the ykqC (mRNA 3 '-end processing factor) family, as well as their variants, herein referred to as "ykqC," "ykqC polynucleotιde(s)." and "ykqC polypeptιde(s)" as the case ma be

BACKGROUND OF THE INVENTION

The Streptococci make up a medically important genera of microbes known to cause several types of disease in humans, including, for example, otitis media, conjunctivitis, pneumonia, bacteremia. meningitis, sinusitis, pleural emp\ ema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospmal fluid Since its isolation more than 100 years ago. Streptococcus pneumoniae has been one of the more intensively studied microbes For example, much of our early understanding that D A is. in fact the genetic matenal was predicated on the work of Griffith and of Avery, Macleod and McCarty usuig tins microbe Despite the vast amount of research with S pneumoniae. many questions concerning the virulence of tins microbe remam It is particularly preferred to employ Streptococcal genes and gene products as targets for tine development of antibiotics The frequency of Streptococcus pneumoniae infections has πsen dramaticallv in the past few decades Tins has been attributed to the emergence of multiph antibiotic resistant strains and an increasing population ot people with weakened immune s stems It is no longer uncommon to isolate Streptococcus pneumoniae strains that arc resistant to some or all of the standard antibiotics Tins phenomenon has created an unmet medical need and demand for new anti-nncrobial agents, vaccines, drug screening mediods. and diagnostic tests for tins 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 bιolog\ Tins approach is rapidly supersedmg earlier approaches based on "positional cloning" and other methods Functional genomics relies heaviK on the \anous tools of bioinformatics to identify gene sequences of potential mterest from the many molecular biology databases now available as well as from otlier sources There is a continuing and significant need to identify and characteπze further genes and other polynucleotides sequences and their related poKpeptides. as targets for drug discover,

Clearh . there exists a need for polvnucleoUdes and poh eptides. such as the ykqC embodiments ot the invention, that

a present benefit of. among otlier things, being useful to screen compounds for antimicrobial activity Such factors are also useful to determine their role in pathogenesis of infection. dysfuncUon and disease There is also a need for ldentificaUon 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 ykqC. in particular ykqC polypeptides and ykqC polynucleotides. recombmant materials and methods for their production hi another aspect, the invention relates to methods for using such pohpeptides and polvnucleoUdes. including treatment of rmcrobial 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 rmcrobial infections and conditions associated with such infections with the identified agomst or antagonist compounds In a still further aspect, the invention relates to diagnostic assays for detecting diseases associated with rmcrobial infections and conditions associated with such infections, such as assays for detecting ykqC expression or activity

Various changes and modifications within the spiπt and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following descriptions and from reading the odicr parts of the present disclosure

DESCRIPTION OF THE INVENTION

The mvention relates to >kqC polypeptides and polynucleotides as described ui greater detail below hi particular, die mvention relates to pohpeptidcs and pohiiucleotides of a ykqC of Streptococcus pneumoniae. diat is related b\ amino acid sequence homologv to B subti s ykqC polypeptide The invention relates especialh to \kqC having a nucleotide and ammo acid sequences set out in Table 1 as SEQ ID NO 1 and SEQ ID NO 2 respectiveh 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 usefullv employed in polynucleotides in general, including πbopolynucleotides

TABLE 1 ykqC Polynucleotide and Polypeptide Sequences

(A) Streptococcus pneumoniae ykqC polvnucleotide sequence [SEQ ID NO 1J

5 ' -

ATGGCCTACACTCTTAAACCTGAAGAAGTCGGCGTTTTTGCCATCGGTGGTCTAGGAGAAATCGGGAAAAACACT TACGGAATTGAATACCAAGACGAGATTATCATCGTCGATGCTGGGATTAAATTCCCAGAAGATGACTTGCTTGGT ATCGACTATGTCATTCCTGACTACTCTTACATCGTAGACAATATCGACCGCGTCAAGGCTGTTTTAATCACACAC GGACACGAGGACCAC

- ? - ATTGGTGGGATTCCGTTCCTACTCAAGCAAGCAAATGTCCCTATTTATGCTGGACCGCTTGCCTTGGCTTTGATC CGTGGGAAACTCGAAGAACACGGCCTCTTGCGCAACGCCAAACTTTACGAAATCAACCACAACACCGAGTTGACC TTTAAAAATCTCAAGGCAACTTTCTTTAGAACGACTCACTCTATTCCAGAACCTTTGGGAATTGTCATCCATACT CCTCAAGGGAAAATC ATCTGTACAGGTGACTTTAAGTTCGACTTTACTCCAGTTGGAGAACCTGCGGACTTGCACCGTATGGCAGCCCTA GGTGAAGATGGAGTGCTTTGCTTGCTCTCTGACTCGACCAATGCTGAGGTTCCAACCTTTACAAACTCTGAAAAG GTTGTCGGCCAGTCCATCATGAAGATTATCCAAGGTATCGAGGGACGTATCATCTTTGCATCCTTCGCCTCAAAT ATCTTCCGTCTCCAA CAAGCAACGGAAGCTGCTGTTAAGACTGGGCGCAAGATTGCGGTCTTTGGTCGCTCTATGGAAAAGGCCATTGTC AACGGAATCGATCTTGGCTACATCAAAGCTCCTAAGGGAACCTTTATTGAGCCAAATGAAATCAAAGACTATCCT GCAGGTGAGATTCTGATCCTCTGTACAGGTAGTCAAGGTGAACCTATGGCAGCCCTCTCTCGTATCGCCAACGGA ACCCACCGTCAGGTA

CAACTCCAACCAGGCGATACCGTTATCTTCTCTTCTAGTCCCATCCCTGGAAACACTACTAGCGTCAACAAGCTG ATTAACATCATTTCTGAACCTGGTGTCGAAGTTATCCACGGTAAAGTGAACAATATCCATACATCTGGACACGGT GGTCAGCAAGAGCAAAAACTCATGCTCCGCTTGATTAAGCCAAAATACTTCATGCCTGTCCACGGTGAATACCGC ATGCAAAAAGTCCAC

GCTGGACTAGCAGTGGATACTGGTGTTGAGAAGGACAATATCTTTATCATGAGCAATGGCGATGTGCTTGCCCTT ACTGCTGACTCAGCTCGTATCGCAGGTCATTTCAACGCCCAAGATATCTATGTCGATGGAAATCGTATCGGTGAA ATTGGCGCAGCTGTCCTCAAAGATCGTCGCGATCTATCTGAAGACGGTGTCGTTCTGGCAGTCGCAACTGTTGAC TTCAAATCGCAGATG

ATTCTATCTGGTCCAGACATCCTCAGCCGAGGCTTTGTCTACATGAGAGAGTCTGGCGACTTGATTCGCCAAAGC CAGCGTATCCTCTTCAATGCCATTCGTATCGCACTGAAAAATAAGGATGCTAGCGTGCAATCTGTCAATGGTGCC ATTGTCAACGCTATTCGCCCCTTCCTCTATGAAAATACCGAACGTGAACCGATCATCATCCCGATGATCCTCACA CCAGATGAAGAATAA-3 '

(B) Streptococcus pneumoniae ykqC polypeptide sequence deduced from a polynucleotide sequence in this table |SEQ ID N0.2|

NH~-

MAYTLKPEEVGVFAIGGLGEIGKNTYGIEYQDEIIIVDAGIKFPEDDLLGIDYVIPDYSYIVDNIDRVKAV ITH GHEDHIGGIPF LKQANVPIYAGPLALALIRGKLEEHGLLRNAKLYEINHNTELTFKNLKATFFRTTHSIPEPLG

IVIHTPQGKIICTGDFKFDFTPVGEPADLHRMAALGEDGVLCLLSDSTNAEVPTFTNSEKWGQSIMKIIQGIEG

RIIFASFASNIFR Q

QATEAAVKTGRKIAVFGRSMEKAIVNGIDLGYIKAPKGTFIEPNEIKDYPAGEILILCTGSQGEPMAALSRIAIxIG

THRQVQLQPGDTVIFSSSPIPGNTTSVNKLINIISEAGVEVIHGKV NIHTSGHGGQQEQKLMLRLIKPKYFMPV HGEYR QKVHAGLAVDTGVEKDNIFIMSHGDVLALTADSARIAGHFNAQDIYVDGNRIGEIGAAVLKDRRDLSED

GW AVATVDFKΞQM

I SGPDILSRGFVYMRESGDLIRQSQRI FNAIRIALKNKDASVQSYNGAIVNAIRPFLYENTEREPIIIPMILT

PDEE

-COOH

Deposited materials A deposit comprising a Streptococcus pneumomae 0100993 strain has been deposited with the National Collections of Industrial and Marine Bactena Ltd (herein "NCIMB"), 23 St Machar Dπve, Aberdeen AB2 1RY. Scotland on 11 Apπl 1996 and assigned deposit number 40794 The deposit was descπbed as Streptococcus pneumoniae 0100993 on deposit On 17 Apπl 1996 a Streptococcus pneumoniae 0100993 DNA library in E coli was similarly deposited widi the NCIMB and assigned deposit number 40800 The Streptococcus pneumomae stram deposit is refeπed to herein as "the deposited stram" or as "the DNA of the deposited stram "

The deposited stram compnses a full length ykqC gene The sequence of die polynucleotides compπsed in the deposited stram. as well as the ammo acid sequence of any polypeptide encoded thereby, are controlling in the event of am conflict with any descπption of sequences herein

The deposit of the deposited stram has been made under the terms of die Budapest Treaty on d e International Recognition of the Deposit of Micro-organisms for Purposes of Patent Procedure The deposited strain will be irrevocably and without restnction or condition released to the public upon die issuance of a patent The deposited stram is provided merelv as convenience to diose 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 deπved dierefrom. and no such license is hereby granted

In one aspect of the mvention there is provided an isolated nucleic acid molecule encodmg a mature polypeptide expressible by die Streptococcus pneumomae 0100993 strain, which pohpeptide is compπsed in die deposited stram Furtiier provided by die mvention are ykqC polynucleotide sequences in d e deposited strain such as DNA and RNA. and amino acid sequences encoded

Also provided bv die invention are \ kqC pohpeptide and polvnucleotide sequences isolated from die deposited stram

Polypeptides

YkqC pohpeptide of die mvention is substantially phylogcneticalh related to odicr proteins of die ykqC (mRNA 3'-end processing factor) famih

In one aspect of the mvention there are provided polypeptides oϊ Streptococcus pneumoniae reteπed to here as "vkqC" and "\kqC polypeptides" as well as biologicalh diagnosticalh proplw lacticalh clinicallv or d erapeuticallv useful \ anants thereof, and compositions compπsing the same

Among d e particularlv preferred embodiments of die mvention are vanants of vkqC pohpeptidi. encoded b\ naturalh occurring alleles of a vkqC gene

The present mvention furtiier provides for an isolated polypeptide dtat (a) comprises or consists of an amino acid sequence that has at least 95% identity, most preferablv at least 97-99% or exact ldenti to that of SEQ ID NO 2 over the entire length of SEQ ID NO 2, (b) a polypeptide encoded bv an isolated polvnucleotide comprising or consisting of a polvnucleotide 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: l; (c) a polypeptide encoded by an isolated polvnucleotide comprising or consisting of a polvnucleotide sequence encoding a polypeptide that has at least 95% identity, even more preferably at least 97-99%o or exact identity, to the ammo acid sequence of SEQ ID NO.2. over the entire lengtii of SEQ ID NO:2.

The polypeptides of the invention include a pohpeptide of Table 1 [SEQ ID NO:2] (in particular a mature polypeptide) as well as pohpeptides and fragments, particularly those that has a biological activity of ykqC. and also those that have at least 95% identity to a polypeptide of Table 1 [SEQ ID NO:2J 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 d e formula

X-(R₁)_m.(R₂)-(R₃)_n-Y wherein, at die ammo terminus. X is hydrogen, a metal or any other moiety descπbed herein for modified pohpeptides. and at the carboxyl terminus. Y is hydrogen, a metal or any other moiety described herein for modified polypeptides. R_j and R are any amino acid residue or modified amino acid residue, m is an integer between 1 and 1000 or zero, n is an mteger between 1 and 1000 or zero, and R₂ is an ammo acid sequence of die invention, particularly an ammo acid sequence selected from Table 1 or modified foπns diereof In die formula above. R₂ is onented so that its amino terminal amino acid residue is at d e left, covalently bound to R_j and its carboxy terminal ammo acid residue is at die right, covalently bound to R3. Any stretch of ammo acid residues denoted by eidier R_] or R3. where m and or n is greater than 1, may be eidier a hetcropolymcr or a homopolymer. preferably a heteropolymcr Odicr preferred embodiments of die invention arc provided where m is an integer between 1 and 50. 100 or 500. and 11 is an integer between 1 and 50. 100. or 500

It is most preferred diat a pohpeptide of die invention is deπved from Streptococcus pneumomai however, it may preferably be obtamed from odicr orgamsms of die same taxononnc genus A polypeptide of die invention may also be obtamed. for example, from orgamsms of the same taxononnc family or order

A fragment is a vaπant pohpeptide having an ammo acid sequence that is entirely die same as part but not all of any amino acid sequence of any polypeptide of die mvention As widi ykqC pohpeptides. fragments may be "free-standing." or compnsed within a larger pohpeptide of which diey form a part or region, most preferably as a s gle continuous region in a single larger pohpeptide Preferred fragments mclude. for example, truncation pohpeptides having a portion of an ammo acid sequence of Table 1 [SEQ ID N0.2J. or of vaπants diereof. such as a continuous series of residues diat includes an amino- and or carboxy 1 -terminal amino acid sequence Degradation forms of die pohpeptides of die mvention produced by or m a host cell, particularly a Streptococcus pneumoniae. are also prefeπed Furtiier preferred are fragments characteπzed by structural or functional attributes such as fragments diat compπse alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-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 mdex 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 ol 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 ot SEQ ID NO:2. Fragments of the polypeptides of die mvention may be employed for produc g the corresponding full-length polypeptide by peptide syndiesis. therefore, these vaπants may be employed as intemiediates for producmg die full-length polypeptides of die mvention Polynucleotides It is an object of the mvention to provide polynucleotides that encode ykqC polypeptides. particularh polvnucleotidcs that encode a pohpeptide herein designated ykqC hi a particularly preferred embodiment of die mvention die polynucleotide compnses a region encoding ykqC polypeptides compnsmg a sequence set out in Table 1 [SEQ ID NO 1 1 diat includes a full lcngdi gene, or a variant thereof This mvention provides that dns full length gene is essential to die growth and/or survival of an organism that possesses it. such as Streptococcus pneumomae As a furtiier aspect of die invention diere are provided isolated nucleic acid molecules encoding and/or expressing

polypeptides and polynucleotides. particularly Streptococcus pneumoniae ykqC polypeptides and pohnucleotides. including, for example, unprocessed RNAs.

RNAs inRNAs cDNAs. genomic DNAs. B- and Z-DNAs Furtiier embodiments of die invention include biologicalh diagnostically . proplw lactically . clmicalh or therapeutical h useful pohnucleotides and polypeptides. and vaπants diereof. and compositions compnsmg die same

Anodier aspect of die mvention relates to isolated polynucleotides, including at least one full length gene, d at encodes a kqC pohpeptide hav g a deduced amino acid sequence of Table 1 [SEQ ID NO 2| and pohnucleotides closelv related d ereto and vaπants thereof

In another particularly preferred embodiment of the invention there is a vkqC poly peptide from Streptococcus pneumoniae comprising or consisting of an amino acid sequence of Table 1 | SEQ ID NO 2|. or a variant thereof

Using die information provided herem. such as a polynucleotide sequence set out in Table 1 | SEQ ID NO 1]. a polynucleotide of d e mvention encoding ykqC polypeptide mav be obtamed using standard cloning and screemng mediods. such as diose for clonmg and sequencing cliromosomal DNA fragments from bactena using Streptococcus pneumoniae 0100993 cells as starting material, followed by obtaining a full length clone For example, to obtain a polynucleotide sequence of the invention, such as a polynucleotide sequence given in Table 1 [SEQ ID NO: l]. typically a library of clones of chromosomal DNA oϊ Streptococcus pneumoniae 0100993 in E.coli or some other suitable host is probed with a radiolabeled oligonucleotide. 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 hybridization conditions. By sequencing the individual clones thus identified by hybridization with sequencing primers designed from the original polypeptide or polynucleotide 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, using denatured double stranded DNA prepared from a plasmid clone Suitable techniques are described 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 obtain a full length gene sequence Illustrative of the mvention. each polynucleotide set out in Table 1 [SEQ ID NO- 11 was discovered in a DNA library derived from Streptococcus pneumomae 0100993

Moreover, each DNA sequence set out in Table 1 [SEQ ID NO: IJ contains an open reading frame encoding a protein having about the number of ammo acid residues set forth in Table 1 [SEQ ID N0.2J widi a deduced molecular weight that can be calculated usmg amino acid residue molecular weight values well known to diose skilled in die art The polynucleotide of SEQ ID NO.1. between nuclcotide number 1 and die stop codon diat begins at nuclcotide number 1678 of SEQ ID NO 1. encodes the polypeptide of SEQ ID NO.2

In a further aspect, die present mvention provides for an isolated polynucleotide comprising or consisting of: (a) a polynucleotide sequence that has at least 95% identity, even more prcferabiy 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 encoding a polypeptide diat 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 lengdi of SEQ ID NO.2

A polynucleotide encoding a polypeptide of the present mvention. including homologs and orthologs from species odier than Streptococcus pneumoniae. may be obtamed by a process that compnses die steps of screemng an appropnate library under stnngent hybridization conditions widi a labeled or detectable probe consistmg of or compnsmg die sequence of SEQ ID NO. l or a fragment diereof. and isolating a full-lengdi gene and/or genonnc clones compnsmg 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.l] 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 in reading frame with another codmg sequence, such as a sequence encodmg a leader or secretory sequence, a pre-, or pro- or prepro-protem sequence The polynucleotide of the mvention may also compπse at least one non-coding sequence, including for example, but not limited to at least one non-coding 5' and 3' sequence, such as die transcπbed but non-translated sequences, termination signals (such as rho-dependent and rho-independent termination signals), nbosome binding sites. Kozak sequences, sequences that stabilize mRNA. introns. and polyadenylation signals The polynucleotide sequence may also compπse additional codmg sequence encodmg additional ammo acids For example, a marker sequence that facilitates puπfication of a fused polypeptide can be encoded In certain embodiments of die mvention. the marker sequence is a hexa-histidine peptide. as provided m the pQE vector (Qiagen. hie ) and descπbed in Gentz et al . Proc Nail Acacl Sci . USA 86 821-824 (1989). or an HA peptide tag (Wilson et al . Cell 37 767 ( 1 84). both of that may be useful m puπfying polypeptide sequence fused to them Polynucleotides of die mvention also mclude. but are not limited to. polynucleotides compnsmg a structural gene and its naturally associated sequences that control gene expression

A prefeπed embodiment of the mvention is a polynucleotide of consisting of or compnsmg nucleotide 1 to the nucleotide immediately upstream of or including nucleotide 1678 set forth in SEQ ID NO 1 of Table 1. bodi of that encode a ykqC pohpeptide The invention also includes a polvnucleotide consisting of or compnsmg a polynucleotide of die formula

X-(R₁)_m-(R₂)-(R₃)_n-Y wherein, at the 5' end of the molecule. X is h\ drogen. 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 hvdrogen, 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 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 polvnucleotide formula above. R₂ is oriented so that its 5' end nucleic acid residue is at the left, bound to R_j and its 3' end nucleic acid residue is at the right, bound to R3 Any stretch of nucleic acid residues denoted by either R_] and/or R . where m and/or n is greater than 1.

be either a heteropolvmer or a ho opolvmer. preferably a heteropolymcr Where, in a preferred embodiment. X and Y together define a covalent bond, the polynucleotide of the above formula is a closed, circular polvnucleotide. 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. 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 polynucleotide of the mvention is deπved from Streptococcus pneumoniae. however, it may preferably be obtamed from other orgamsms of the same taxononnc genus A polynucleotide of die mvention may also be obtamed. for example, from orgamsms of the same taxononnc family or order

The term "polynucleotide encodmg a polypeptide" as used herein encompasses polynucleotides tiiat mclude a sequence encodmg a polypeptide of the mvention, particularly a bacteπal polypeptide and more particularly a pohpeptide of the Streptococcus pneumoniae ykqC havmg an ammo acid sequence set out in Table 1 [SEQ ID NO.2] The term also encompasses polynucleotides that mclude a smgle continuous region or discontinuous regions encodmg the polypeptide (for example, polynucleotides interrupted bv integrated phage. an mtegrated insertion sequence, an mtegrated vector sequence, an mtegrated transposon sequence, or due to RNA editing or genormc DNA reorganization) together wndi additional regions, diat also may compnse codmg and/or non-coding sequences

The mvention further relates to vanants of die polynucleotides descnbed herein diat encode vaπants of a polypeptide havmg a deduced ammo acid sequence of Table 1 [SEQ ID NO 2J Fragments of polynucleotides of the mvention may be used, for example, to synthesize full-lcngdi pohnucleotides of die invention Furtiier particularly preferred embodiments are polynucleotides encoding ykqC vaπants. that

die annno acid sequence of vkqC polypeptide of Table 1 [SEQ ID NO 2] which several, a few. 5 to 10 1 to 5. 1 to 3. 2. 1 or no annno acid residues are substituted, modified, deleted and or added, in an\ combination Especially preferred among d ese are silent substitutions, additions and deletions, diat do not alter die properties and activities of vkqC polypeptide Prefeπcd isolated polvnucleotide 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 ot SEQ ID NO 1 , 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 ot the polynucleotide sequence ot SEQ ID NO 1

Furtiier preferred embodiments of die invention are pohnucleotides diat are at least 95% or 97% identical o\er dieir entire lengdi to a polvnucleotide encodmg \kqC polypeptide having an ammo acid sequence set out in Table 1 [SEQ ID NO 2]. and pohnucleotides diat are complementars to such pohnucleotides Most highly preferred are polynucleotides that compnse a region that is at least 95% are especially preferred Furthermore, tirøse with at least 97% are highly preferred among those with at least 95%, and among these those with at least 98% and at least 99% are particularly highly prefeπed, widi at least 99% being the more preferred.

Preferred embodiments are polynucleotides encoding polypeptides that retain substantially die same biological function or activity as a mature polypeptide encoded by a DNA of Table 1 [SEQ ID NO: 1],

In accordance wid certain prefeπed embodiments of this mvention there are provided polynucleotides that hybridize, particularly under stringent conditions, to ykqC polynucleotide sequences, such as those polynucleotides in Table 1.

The invention further relates to polynucleotides that hybridize to the polynucleotide sequences provided herein. In this regard, the invention especially relates to polynucleotides that hybridize under stringent conditions to the pohnucleotides described herein. A specific example of stringent hybridization conditions is overnight incubation at 42°C m a solution comprising: 50% formamide. 5x SSC (150mM NaCI. 15mM tπsodium citrate). 50 mM sodium phosphate (pH7.6). 5x Denhardt's solution. 10% dextran sulfate. and 20 micrograms/ml of denatured, sheared salmon speπn DNA. followed by washing the hybridization support in O. lx SSC at about 65°C Hybridization and wash conditions are well known and exemplified in Sambrook. et al.. Molecular Cloning: A Laboratory Manual. Second Edition. Cold Spring Harbor. N.Y.. (1989), particularly Chapter 1 1 therein. Solution hybridization may also be used with the polynucleotide sequences provided by the invention

The invention also provides a polynucleotide consisting of or comprising a polynucleotide sequence obtained by screening an appropriate library comprising a complete gene for a polynucleotide sequence set forth in SEQ ID NO 1 under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence set forth in SEQ ID NO l or a fragment thereof, and isolating said polynucleotide sequence Fragments useful for obtaining such a polynucleotide include, for example, probes and primers fully described elsewhere herein As discussed elsewhere herem regarding polynucleotide assays of die invention, for instance, the polynucleotides of die mvention. may be used as a hybridization probe for RNA. cDNA and genomic DNA to isolate full-lengdi cDNAs and genomic clones encodmg ykqC and to isolate cDNA and genomic clones of odier genes diat

e a high identity, particularly high sequence identity, to a ykqC gene Such probes generally will compπse at least 1 nucleotide residues or base pairs Preferably, such probes will have at least 30 nucleotide residues or base pairs and may have at least 50 nuclcotide residues or base pairs Particular ly preferred probes will have at least 20 nucleotide residues or base pairs and will have lee dian 30 nucleotide residues or base pairs

A codmg region of a ykqC gene may be isolated by screemng usmg a DNA sequence provided Table 1 [SEQ ID NO 1J to synthesize an ohgonucleotide 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 library the probe hybπdizes 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 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 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 die selected gene sequence) The products of this reaction can then be analyzed by DNA sequencing and a full-length DNA constructed either by |Oining the product directly to the existing DNA to give a complete sequence, or carrying out a separate full- length PCR using the new sequence information for the design of the 5' primer

The polynucleotides and pohpeptides of d e invention may be employed, for example, as research reagents and matenals for discovery of treatments of and diagnostics for diseases, particularly human diseases, as furtiier discussed herein relat g to pohnucleotidc assays The pohnucleotides of the invention that are ohgonucleotides derived from a sequence of Table

1 [SEQ ID NOS 1 or 2] mav be used in the processes herein as described, but preferably for PCR. to detcπnine whether or not the pohnucleotides identified herein in whole or in part are transcribed in bacteria in infected tissue It is recognized that such sequences will also have utιlιt\ in diagnosis of the stage of infection and type of infection the pathogen has attained The invention also provides pohnucleotides diat encode a polypeptide diat is a mature protein plus additional ammo or carboxy 1-tcrmιnal ammo acids, or annno acids intenor to a mature pohpeptide (when a mature form has more than one pohpeptide cham. for mstance) Such sequences may pla\ a role in processing of a protein from precursor to a mature foπn.

allow protein transport. ma\ lengthen or shorten protein half-life or ma\ facilitate manipulation of a protein for assa\ or production, among other dnngs As generally is die case in vivo, die additional ammo acids mav be processed awa\ from a matuii protem b\ cellular enzymes

For each and every polynucleotide of die invention diere is provided a polynucleotide complementary to it It is preferred diat diese complementary pohnucleotides are fullv complementary to each polynucleotide w ιdι winch dιe\ are complementary A precursor protein, havmg a mature form of the polypeptide fused to one or more prosequences may be an inactive form of the pohpeptide. When prosequences are removed such inactive precursors generally are activated. Some or all of the prosequences may be removed before activation. Generally, such precursors are called proproteins As will be recognized, die entire polypeptide encoded by an open reading frame is often not required for activity. Accordingly, it has become routine in molecular biology to map die boundanes of die primary structure required for activity widi N-terminal and C-terminal deletion experiments. These experiments utilize exonuclease digestion or convenient restriction sites to cleave coding nucleic acid sequence. For example. Promega (Madison. Wl) 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 endpomts can be repaired (e.g.. by hgation to synthetic linkers) to die extent necessary to preserve an open readmg frame In this way. die nucleic acid of SEQ ID NO.l readily provides contiguous fragments of SEQ ID N0:2 sufficient to provide an activity, such as an enzymatic, binding or antibody-inducing activity Nucleic acid sequences encodmg such fragments of SEQ ID NO.2 and vanants thereof as described herem are within die mvention. as are pohpeptides so encoded

As is known in the art. portions of the N-terminal and/or C-tcrminal sequence of a protein can generally be removed without serious consequence to the function of the protein 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, any contiguous fragment of SEQ ID NO:2 which retains at least 20%, preferably at least 50%. of an activity of the pohpeptide 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 comprises at least 70% of the amino acid residues of SEQ ID NO 2. preferably at least 80%. 90% or 95% of the residues In sum. a polynucleotide of the invention may encode a mature protem. a mature protein plus a leader sequence (diat may be refeπed to as a preprotem). a precursor of a mature protein having one or more prosequences diat are not die leader sequences of a preprotem. or a preproprotem. diat is a precursor to a proprotem. havmg a leader sequence and one or more prosequences. tiiat generally are removed dunng processmg steps diat produce active and mature forms of die pohpeptide Vectors, Host Cells, Expression Systems

The invention also relates to vectors that compπse a polynucleotide or polynucleotides of the invention, host cells that are genetically engmeered widi vectors of die mvention and die production of pohpeptides of d e invention by recombmant techniques Cell-free translation systems can also be employed to produce such protems usmg RNAs deπved from die DNA constructs of die invention Recombmant polypeptides of the present mvention may be prepared by processes well known m those skdled 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 die production of pohpeptides of the mvention by recombmant techmques

For recombmant 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 descπbed 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 Spπng Harbor. N Y (1989). such as. calcium phosphate transfection. DEAE-dextran mediated transfection. transvection. micromjection. cationic lipid-mediated transfection, electroporation. transduction. scrape loading, ballistic introduction and infection

Representative examples of appropnate hosts mclude bactenal cells, such as cells of streptococci staphvlococci. enterococci E cob, streptomyces. cyanobactena. Bacillus subti s. and Streptococcus pneumomae. fungal cells, such as cells of a yeast. Kluveromyce . Saccharomyces. a basidiomvccte. Candida albicans and Aspergillus. insect cells such as cells of Drosophtla S2 and Spυdoptera 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 gvmnosperm or angiosperm A great vanety of expression systems can be used to produce die polypeptides of die invention Such vectors mclude. among others, chromosomal-, episomal- and virus-deπvcd vectors, for example vectors deπved from bactenal plasnnds. from bacteπophage. from transposons. from yeast episomcs. from insertion elements, from yeast cliromosomal elements, from viruses such as baculoviruscs. papova uniscs such s SV40. vaccinia viruses, adenoλ'iruses. fowl po\ viruses, pseudorabies viruses, picomavi ses and retroviruses. and vectors denved from combinations thereof, such as those deπved from plasnnd and bacteπophage genetic elements, such as cosnnds and phagemids The expression system constructs ma\ compπse control regions that regulate as well as engender expression Generally, any system or vector suitable to mamtam. propagate or express pohnucleotides and or to express a polypeptide m a host ma\ be used for expression m dns regard The appropnate DNA sequence

be inserted into die expression

stem bv am of a vanetv of well-known and routine techmques. such as. for example, diose set forth in Sambrook a al . MOLECULAR CLONING A LABORATORY MANUAL, (supra) hi recombmant expression systems in eukaryotes. for secretion of a translated protein to die lumen of die endoplasmic reticulum. mto die penplasnuc space or mto die extracellular environment, appropnate secretion signals may be incorporated mto the expressed pohpeptide These signals may be endogenous to the polypeptide or they may be heterologous signals

Polypeptides of the mvention can be recovered and puπfied from recombmant cell cultures by well- known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography. and lectin chromatography Most preferably, high performance liquid chromatography is employed for puπfication Well known techmques 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 ykqC polynucleotides and polypeptides of the mvention for use as diagnostic reagents Detection of ykqC polynucleotides 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 Eukaryotes, particularly mammals, and especial lv humans, particularly those infected or suspected to be infected with an organism compnsmg die ykqC gene or protem, may be detected at the nucleic acid or ammo acid level by a vanety of well known tcclnnques as well as by mediods provided herein

Pohpeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtained from a putativcly infected and/or infected individual's bodily matenals Polynucleotides from any of diese sources. particularly DNA or RNA. may be used directly for detection or may be amplified enzymatically bv using PCR or am odicr amplification technique pπor to analysis RNA. particularly mRNA. cDNA and genomic DNA mav also be used m die same ways Using amplification, charactenzation of die species and strain of infectious or resident organism present m an individual, may be made by an analysis of die genotype ot a selected polvnucleotide of the organism Deletions and insertions can be detected by a change in size of die 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 die same species Point mutations can be identified bv hvbndizing amplified DNA to labeled ykqC polynucleotide sequences Perfectlv or significanth matched sequences can be distinguished from imperfectly or more significantly mismatched duplexes bv DNase or RNase digestion, for DNA or RNA respectively, or bv detecting differences in melting temperatures or renaturation kmetics Polvnucleotide sequence differences mav also be detected bv alterations m the electrophoretic mobihtv of polynucleotide fragments m gels as compared to a reference sequence Tins may be earned out idi or without denaturing agents Polynucleotide differences may also be detected bv direct DNA or RNA sequencmg See. for example. Mvers et al . Science, 230 1242 (1985) Sequence changes at specific locations also mav be revealed bv nuclease protection assays, such as RNase. V 1 and S 1 protection assay or a chemical cleavage method. See. for example, Cotton et al., Proc. Natl. Acad. Sci., USA, 85: 4397-4401 (1985).

In another embodiment, an array of o gonucleotides probes comprising ykqC 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 variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability (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: l . 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 antibody 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. Tins mvention also relates to the use of polynucleotides of the present mvention as diagnostic reagents. Detection of a mutated form of a polvnucleotide of die invention, preferable. SEQ ID NO.1. diat is associated widi a disease or patiiogenicity 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, diat results from under-expression. over-expression or altered expression of die polynucleotide Orgamsms. particularly infectious organisms, carrying mutations in such polynucleotide may be detected at die polynucleotide level by a vancty of techmques. such as diose descnbcd elsewhere herein

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 deteπnincd. If a mutation is observed in 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 (allelic variations) in a polynucleotide and/or polypeptide of die mvention may also be detected at die polynucleotide or polypeptide level by a v aπctv of techmques. to allow for serotypmg. for example For example. RT-PCR can be used to detect mutations die RNA It is particularly preferred to use RT-PCR in conjunction widi automated detection systems, such as. for example. GeneScan RNA. cDNA or genonnc DNA may also be used for die same purpose. PCR As an example. PCR pπmers complementary to a polynucleotide encodmg ykqC polypeptide can be used to identity' and analyze mutations The mvention furtiier provides tiiese pπmers widi 1. 2. 3 or 4 nucleotidcs removed from die 5' and/or die 3' end These pnmers may be used for. among odier dnngs. amplifying ykqC DNA and/or RNA isolated from a sample denved from an individual, such as a bodily material. The primers may be used to amplify a polynucleotide isolated from an infected individual, such that the polynucleotide may then be subject to various techniques for elucidation of die polynucleotide sequence, hi diis way. mutations in the polynucleotide sequence may be detected and used to diagnose and or prognose die infection or its stage or course, or to serotype and/or classify the infectious agent.

The invention further provides a process for diagnosing, disease, preferably bacterial infections, more preferably infections caused by Streptococcus pneumoniae. comprising determining from a sample derived from an individual, such as a bodily material, an increased level of expression of polynucleotide having a sequence of Table 1 [SEQ ID NO: l] Increased or decreased expression of a ykqC 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 ykqC polypeptide compared to normal control tissue samples may be used to detect die presence of an infection, for example. Assay techmques that can be used to determine levels of a ykqC polypeptide. in a sample deπved from a host, such as a bodily material, are well-known to those of skill m the art. Such assay mediods include radioimmunoassays. competitive-binding assays. Western Blot analysis, antibody sandwich assays, antibody detection and ELISA assays

Antagonists and Agonists - Assays and Molecules

Pohpeptidcs and polynucleotides of die invention may also be used to assess die b dmg of small molecule substrates and hgands m. for example, cells, cell-free preparations, chemical braπcs. and natural product mixtures These substrates and hgands may be natural substrates and gands or may be structural or functional nnmetics See, e.g.. Cohgan et al.. Current Protocols in Immunology 1 (2) Chapter 5 ( 1 91 ) Polypeptides and polynucleotides of die present mvention are responsible for many biological functions, including many disease states, m particular die Diseases herem mentioned. It is therefore desirable to devise screemng methods to identify compounds that agonize (e.g.. stimulate) or diat antagonize (e.g .inhibit) die function of the polypeptide or polynucleotide Accordingly, m a furtiier aspect, die present mvention provides for a method of screemng compounds to identify those diat agomze or that antagonize die function of a polypeptide or polynucleotide of die mvention. as well as related polypeptides and polynucleotides In general, agomsts or antagonists (e.g . inhibitors) may be employed for dicrapeutic and prophylactic purposes for such Diseases as herem mentioned Compounds may be identified from a variety of sources, for example, cells, cell-free preparations, chemical hbraπes. and natural product mixtures Such agomsts and antagonists so-identified may be natural or modified substrates, hgands. receptors, enzymes, etc . as the case may be. of ykqC polypeptides and polynucleotides; or may be structural or functional mimetics thereof (see Co gan et al, Current Protocols m Immunology l(2).Chapter 5 (1991))

The screemng methods may simply measure the binding of a candidate compound to the polypeptide or polvnucleotide. 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 screenmg method may involve competition with a labeled competitor Further, these screemng methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide or polynucleotide. using detection systems appropriate to the cells comprising the polypeptide or polynucleotide Inhibitors of activation are generally assayed m the presence of a known agonist and the effect on activation by the agonist 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 in 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 ykqC polypeptide and/or polynucleotide activity in the mixture, and comparing the ykqC polypeptide and or polynucleotide activity of the mixture to a standard Fusion proteins, such as those made from Fc portion and ykqC 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 phylogenctically and and/or functionally related polypeptides (see D Bennett et al . 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 poly peptide 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 using monoclonal and polvclonal antibodies by 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 screemng compounds to identify those diat enhance (agonist) or block (antagonist) the action of ykqC pohpeptides or polynucleotides. particularly diose compounds diat are bacteπstatic and/or bacteπcidal The method of screemng may involve high-throughput techniques 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 any thereof, compnsmg ykqC 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 ykqC agonist or antagonist The ability of die candidate molecule to agomze or antagonize the ykqC polypeptide is reflected m decreased binding of the labeled gand or decreased production of product from such substrate Molecules that bmd gratuitously, ; e , witiiout ducmg die effects of ykqC 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 us g a reporter system Reporter systems that may be useful in this regard mclude but are not limited to colonmetnc. labeled substrate converted mto product, a reporter gene that is responsive to changes in ykqC pol nucleotide or polypeptide activity, and binding assays known m the art

Pohpeptides of the mvention 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. hgand binding and crosslinking assays m which the polypeptide is labeled with a radioactive isotope (for instance. ^^I). chemically modified (for instance, biotinyiated). 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 supcπiatants. tissue extracts, bodily materials) Other methods include biophysical techniques such as surface plasmon resonance and spcctroscopy 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 fluorescenth -tagged molecule depends on the rotational correlation time or tumbling rate Protein complexes, such as foπned by ykqC poly peptid associating with another ykqC polypeptide or other polypeptide. labeled to comprise a fluorescenth - labeled molecule ill have higher polarization values than a fluorescently labeled monomeπc prote It is preferred that this method be used to characterize small molecules that disnipt polypeptide complexes

Fluorescence energv transfer may also be used characterize small molecules that interfere with the foπnation of vkqC polypeptide dimers. tπmers. tetramers or higher order stmctures. or stmctures foπned bv v kqC polypeptide bound to another poly peptide YkqC 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 energv transfer Surface plasmon resonance can be used to monitor the effect of small molecules on ykqC polypeptide self-association as well as an association of ykqC polypeptide and another polypeptide or small molecule YkqC polypeptide can be coupled to a sensor chip at low site density such that covalently bound molecules will be monomenc Solution protein can then passed over the ykqC polypeptide -coated surface and specific binding 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 ykqC polypeptide self-association as vv ell as an association of ykqC polypeptide and another polypeptide or small molecule A scintillation proximity assay may be used to characterize the interaction between an association of ykqC polypeptide with another ykqC polypeptide or a different polypeptide YkqC polypeptide can be coupled to a scintillation-filled bead Addition of radio-labeled ykqC polypeptide results m binding where the radioactive source molecule is in close proximity to the scintillation fluid Thus, signal is emitted upon ykqC polypeptide binding and compounds that prevent ykqC poh eptide self-association or an association of ykqC polypeptide and another polypeptide or small molecule will diminish signal

In odier embodiments of the mvention diere are provided methods for identifying compounds that bmd to or odierwise mteract with and inhibit or activate an activity or expression of a polypeptide and/or pol nucleotide of die invention compnsmg contactmg a polypeptide and/or polvnucleotide of die invention widi a compound to be screened under conditions to peπint binding to or odicr interaction between die compound and die polypeptide and/or polvnucleotide to assess die bmdmg to or other interaction widi die compound, such bmdmg or interaction preferably be g associated widi a second component capable of providing a detectable signal in response to die binding or mteraction of die pohpeptide and/or polvnucleotide witii die compound, and determining whedier the compound bmds to or otiierwisc interacts idi and activates or inhibits an activitv or expression of die polypeptide and or polvnucleotide bv detecting die presence or absence of a signal generated from die bmdmg or mteraction of die compound widi die pohpeptide and/or polvnucleotide

Another example of an assav for vkqC agomsts is a competitive assav that combines vkqC and a potential agomst with ykqC-binding molecules recombmant vkqC bmdmg molecules, natural substrates or hgands or substrate or gand mimetics. under appropnate conditions for a competitive inhibition assav YkqC can be labeled, such as bv radioactivitv or a coloπmetnc compound, such diat die number of v kqC molecules bound to a bmdmg molecule or converted to product can be determmed accurateh to assess die effectiveness of die potential antagonist It will be readily appreciated by the skilled artisan that a polypeptide and/or polvnucleotide 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 polvnucleotide, 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 motifts) of an agonist or antagonist, (c) synthesizing candidate compounds that are predicted to bind to or react with the deduced binding sιte(s) reactive sιte(s). and/or motιf(s). and (d) testing whether the candidate compounds are indeed agonists or antagonists It will be further appreciated that this will normally be an iterative process and this iterative process mav be performed using automated and computer-controlled steps hi a further aspect the present mvention provides mediods of treating abnormal conditions such as for instance a Disease related to either an excess of an under-cxpression of an elevated activitv of or a decreased activitv of vkqC polypeptide and/or polvnucleotide If the expression and/or activity of die polypeptide and/or polynucleotide is in excess, several approaches are available One approach compnses administering to an individual m need thereof an inhibitor compound (antagonist) as herem descnbed optionally in combination widi a pharmaceutically acceptable earner in an amount effective to inhibit the function and or expression of die polypeptide and or polvnucleotide such as for example by blocking die bmdmg of hgands. substrates receptors enzvmes etc or bv inhibiting a second signal, and therebv alleviatmg die abnomial condition In another approach soluble f onus of the pohpeptides still capable of binding the hgand substrate enzvmes receptors etc in competition with endogenous pohpeptide and or polvnucleotide mav be administered Tvpical examples of such competitors include fragments of the vkqC po peptide and or poh peptide

In still another approach expression of the gene encoding endogenous vkqC polypeptide can be inhibited using expression blocking techniques Tins blocking mav be targeted against anv step in gene expression but is preferabh targeted against transcription and/or translation An examples of a kno technique of this sort invol e the use of antisense sequences either lntemallv generated or scparatch administered (see for example O Connor J Neurochem ( 1991 ) 56 560 in Ohgodeoxvnucleotides as Antisense Inhibitors of Gene Expression CRC Press Boca Raton FL ( I 988)) Alternativeh ohgonucleotides that fonn triple helices with the gene can be supplied (see for example Lee et al Nucleic Acids Res ( 1979) 6 3073 Coonev et al Science ( 1988) 241 456 Dervan et al Science ( 1991 ) 2 1 1360) These ohgomers can be administered pet 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 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 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, m particular gram positive and or gram negative bacteria, to eukaryotic, preferably mammalian, extracellular matrix proteins on m-dwelhng devices or to extracellular matrix proteins m wounds, to block bacterial adhesion between eukaryotic. preferably mammalian, extracellular matrix proteins and bacterial ykqC 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 widi yet another aspect of die mvention, there are provided ykqC agomsts and antagomsts, preferably bactenstatic or bacteπcidal agomsts and antagomsts

The antagomsts and agomsts of the mvention may be employed, for instance, to prevent, inhibit and/or treat diseases Antagomsts of die mvention mclude. among others, small organic molecules, peptides, polypeptides and antibodies diat bmd to a polynucleotide and/or polypeptide of die invention and diercby 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 diat bmds die same sites on a bmdmg molecule, such as a bmd g molecule, without mducmg ykqC-mduced activities, thereby preventing die action or expression of ykqC polypeptides and/or polynucleotides by excludmg ykqC polypeptides and/or polynucleotides from bmdmg

Antagomsts of die invention also mclude a small molecule diat binds to and occupies die binding site of die pohpeptide diereb preventing bmdmg to cellular bmdmg molecules, such diat normal biological activitv is prevented Examples of small molecules mclude but are not lmnted to small organic molecules peptides or peptide-like molecules Odier antagomsts mclude antisense molecules (see Okano, J Neurochein 56 560 (1991 ). OUGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSlOh. CRC Press. Boca Raton. FL (1988). for a descπption of these molecules) Prefeπed antagomsts mclude compounds related to and vanants of ykqC Other examples of pohpeptide antagomsts mclude antibodies or. in some cases, o gonucleotides or proteins that are closely related to the hgands. substrates, receptors, enzymes, etc.. as die case may be. of die polypeptide, e g , a fragment of the hgands. substrates, receptors, enzymes, etc . or small molecules diat bmd to the polypeptide of the present mvention but do not elicit a response, so that die activity of die polypeptide is prevented

Small molecules of the mvention preferably have a molecular weight belo 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

Helicobacter pylori (herein "H pylori") bacteria infect the stomachs of over one-third of the world's population causing stomach cancer, ulcers, and gastritis (International Agency for Research on Cancer (1994) Schistosomes, Liver Flukes and Helicobacter Pylori (International Agency for Research on Cancer. Lyon. France, http //www.uιcc.ch/ecp/ecp2904.htm) Moreover, the International Agency for Research on Cancer recently recognized a cause-and-effect relationship between H pylori and gastric adenocarcinoma. classifying the bacterium as a Group I (definite) carcinogen Preferred antimicrobial compounds of the invention (agonists and antagonists of ykqC 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. pylon-mάuced 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 in this specification are herein incorporated by reference in their cntirctv as if each indiv ldual publication or reference were specificalh and individually indicated to be incorporated bv reference herein as being fully set forth Any patent application to which this application claims priority is also incorporated by reference herein in its entirety in the manner described above for publications and references

GLOSSARY

The follow g definitions are provided to facilitate understandmg of certain teπns used frequcntlv herem

"Bodily mateπal(s) means any matenal deπved 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, skm. urine, stool or autopsv mateπals

- 9? - "Dιsease(s)" means any disease caused by or related to infection by a bacteπa, including . for example, otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid

"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

"Identit ." 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 die 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 "Identit " can be readily calculated by known methods, including but not limited to those described in

(Computational Molecular Biology. Lesk. A M . cd . Oxford University Press. New York. 1 88. 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 Jerse . 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 Lφman, 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 . Molec Bwl 215 403-410 (1990) The BLAST X program is pubhclv available from NCB1 and other sources (BLAST Manual . Altschul. S et al . NCBI NLM NIH Bethesda. MD 20894. Altschul. S . et al J Mυl Bwl 215 403-410 ( 1990) The well known Smith Waterman algorithm may also be used to determine identitv 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 Penaltv 4

A program useful ith 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 pohpeptides. 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 mav mclude 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, including transition and transversion. or insertion, and wherein 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 in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is deteπnined by multiplying the total number of nucleotides in SEQ ID NO 1 by the integer defining the percent identity divided by 1 0 and then subtracting that product from said total number of nucleotides in SEQ ID NO 1. or

n_n < λ_n - (x_n • ),

wherein n_n is the number of nuclcotide alterations. λ_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 anv non-integer product of x_n and y is rounded down to the nearest integer prior to subtracting it from λ_ι: Alterations of a polvnucleotide sequence encoding the poly peptide of SEQ ID NO 2 mav create nonsense, missense or frameshift mutations in this coding sequence and therebv altei the polypeptide encoded bv the pol nucleotide following such alterations

(2) Poly peptide 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. wherein said polypeptide sequence may be identical to the reference sequence of SEQ ID NO 2 or mav 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 consistmg of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherem said alterations may occur at the ammo- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, mterspersed 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 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 by 100 and then subtracting that product from said total number of annno acids in SEQ ID NO 2. or

n_a < x_a - (x_a • y),

wherein n_a is the number of ammo 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 wherem any non-integer 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 pπmate, and a human

"Isolated" means altered "by die hand of man" from its natural state, i e , if it occurs in nature it has been changed or removed from its oπgmal environment or bod For example, a polvnucleotide or a pohpeptide naturalh present a living organism is not ' isolated " but die same polvnucleotide or pohpeptide separated from die coexisting materials of its natural state is "isolated", as die tenn is emploved herem Moreover a polvnucleotide or polypeptide diat is introduced mto an organism bv transformation genetic manipulation or bv anv other recombmant mediod is "isolated" even if it is still present in said organism which organism mav be living or non-living "Organιsm(s)" means a (I) prokarvote, including but not limited to a member of die genus

Streptococcus Staphvlococcus Bordetella Corynebactenum Mvcobactenum Neissena Haemophilus Actinomvcetcs Streptomycetes Nocardia Enterobactei \ersιnιa Fancisella Paslurella Moiaxella Ac netobactei Erysψelothπx Branhamella Actinobacillus Streptobacillus Listena

Calvmmatobactenum Brucella Bacillus Clostndium Ireponema Eschenchia Salmonella Kleibsiella Vibrio Proteus Erwinia Borreha Leptospiia Spirillum Campvlobaclci Shigella Leφonella Pseudomonas Aeromonas Rickettsia Chlamvdia Bon eba and Mycoplasma and furtiier mcludmg but not lmnted to a member of die species or group Group A Streptococcus Group B Streptococcus Group C Streptococcus Group D Streptococcus Group G Streptococcus Streptococcus pneumomae Streptococcus pyogenes Streptococcus agalact ae Streptococcus faecahs Streptococcus faecium Streptococcus durans Neisseria gonorrheae. Neissena menmgit dis, Staphylococcus aureus. Staphylococcus epidermidis, Corynebacterium dψthenae, Gardnerella vaginahs, Mycobacterium tuberculosis, Mycobactenum bovis. Mycobactenum ulcerans, Mycobacte ium leprae. Actinomyctes israelu, Listena monocytogenes, Bordetella pertusis, Bordatella parapertusis. Bordetella bronchiseptica, Esche ichia coli, Shigella dysentenae, Haemophύus influenzae. Haemoph us aegyptius, Haemophύus paramfluenzae, Haemophύus ducreyi, Bordetella, Salmonella typh , Citrobacter freundn, Proteus mirabihs, Proteus vulgans, Yersinia pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratia hquefaciens, Vibrio cholera, Shigella dysenteru, Shigella flexnen, Pseudomonas aeniginosa, Franscisella tularensis, Brucella abortis. Bacillus anthracis. Bacillus cereus, Clostndium perfnngens, Clostndium tetani, Clostridium bυtulmum, Treponema palbdum Rickettsia rickettsu and Chlamvdia trachomitis, (n) an archaeon. mcludmg but not limited to Archaebacler. and (iii) a unicellular or filamentous eukaryote. including but not limited to, a protozoan, a fungus, a member of die genus Saccharomyces, Kluveromyces, or Candida, and a member of die species Saccharomyces cenviseae, Kluveromyces Iactis. or Candida albwans

"Polynucleotιde(s)" generally refers to any polyπbonucleotide or polydeoxyπbonucleotide. diat may be umnodified RNA or DNA or modified RNA or DNA "Polynucleotιde(s)" mclude. widiout limitation, smgle- and double-stranded DNA. DNA that is a mixture of single- and double-stranded regions or single-. double- and tπple-stranded regions, smgle- and double-stranded RNA. and RNA diat is mixture of smgle- and double-stranded regions, hybnd molecules compnsmg DNA and RNA diat 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 herein refers to tnple-stranded regions compnsmg RNA or DNA or both RNA and DNA The strands in such regions may be from die same molecule or from different molecules The regions mav mclude all of one or more of die molecules, but more typically involv e onlv a region of some of die molecules One of die molecules of a tπple-hclical region often is an ohgonucleotide As used herein die teπu "pohnucleotιde(s)" also includes DNAs or RNAs as described above diat compnse one or more modified bases Thus. DNAs or RNAs widi backbones modified for stability or for odier reasons are "polynucleotιde(s)" as diat teπn is mtended herein Moreover. DNAs or RNAs comprising unusual bases such as mosine. or modified bases, such as tntylated bases, to name just two examples, are polynucleotides as die tcnn is used herein It will be appreciated diat a great variety of modifications have been made to DNA and RNA diat serve many useful purposes known to diose of skill in die art The tenn "polynucleotιde(s)" as it is employed herein embraces such chenncally. enzymatically or metabolically modified fonns 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 ohgonucleotιde(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 "Polypeptide(s)" refers to both short chams. commonly refeπed to as peptides. o gopeptides and ohgomers and to longer chams generally refeπed to as proteins Polypeptides may compπse ammo acids odier than die 20 gene encoded a mo acids "Polypeptιde(s)" mclude those modified either by natural processes, such as processing and other 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 m a voluminous research literature, and they are well known to those of skill in the art It will be appreciated that the same type of modification mav be present in the same or varying degree at several sites m a given polypeptide Also, a given polypeptide may compπse many types of modifications Modifications can occur anywhere m a polypeptide. including die peptide backbone, die ammo acid side-chains, and die ammo or carboxyl termini Modifications include, for example acetyiation. acylation. ADP-πbosy lation. amidation, covalent attachment of flav . covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide deπvative. covalent attachment of a pid or pid deπvative. covalent attachment of phosphotidylinositol. cross-linkmg. cvclization. disulfide bond formation, demethviation. formation of covalent cross-links, formation of cysteme. formation of pyroglutamatc. foπnylation. gamma-carboxyiation. GPI anchor formation, hydroxylation. lodination mcdiyiation, myπstoy lation. oxidation, proteolytic processing, phosphorylation, prenylation. racennzation glycosy lation. hpid attachment, sulfation. gamma-carboxy lation of glutamic acid residues, hydroxylation and ADP-πbosylation. selenoylation. sulfation. transfer-RNA mediated addition of ammo acids to proteins, such as argmylation. and ubiquitmation See. for mstance. 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 POSIT RANSIATIONAI COVALENT MODIFICATION OF PROTEINS. B C Johnson. Ed . Academic Press New York ( 1983) Seifter et al . Meth Enzymol 182 626-646 ( 1990) and Rattan et al . Protem Svnthesis Posttranslational Modifications and Aging. Ann N Y Acad Sci 663 48-62 (1992) Polypeptides may be branched or cyclic widi or without branching Cvc c. branched and branched circular polypeptides may result from posttranslational natural processes and may be made by entirely syndietic methods, as well

"Recombmant expression svstem(s)" refers to expression sv stems or portions diereof or pohnucleotides of die mvention mtroduced or transfoπned into a host cell or host cell lvsate for die production of die pohnucleotides and polypeptides of die lnv ention

"Vanant(s)^* as the teπn is used herein, is a polvnucleotide or poly peptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties A typical variant of a polynucleotide differs m nucleotide sequence from another, reference polvnucleotide Changes m the nucleotide sequence of the variant mav or mav 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 in the polypeptide encoded by the reference sequence, as discussed below A typical variant of a polypeptide differs in amino 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 variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination A substituted or inserted ammo acid residue may or may not be one encoded by the genetic code The present mvention also includes mclude vanants of each of die polypeptides of die mvention. that is polypeptides that vary from the referents by conservative ammo acid substitutions, whereby a residue is substimted 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 die basic residues Lys and Arg. or aromatic residues Phe and Tyr Particularly preferred are vanants in which several. 5-10. 1-5. 1-3. 1-2 or 1 ammo acids are substituted, deleted, or added in anv combmation A variant of a polynucleotide or polypeptide may be a naturally occurring such as an allehc variant, or it may be a variant that is not known to occur naturally Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombmant methods known to skilled artisans EXAMPLES

The examples below are earned out using standard techniques, that are well known and routine to diose of skill in die art. except where odierwise descnbed detail The examples are illustrative, but do not limit die mvention Example 1 Strain selection, Library Production and Sequencing

The polv nucleotide having a DNA sequence given in Table 1 |SEQ ID NO 1 ) was obtained from a library of clones of chromosomal DNA oϊ Streptococcus pneumoniae in L coli The sequencing data from two or more clones comprising overlapping Streptococcus pneumoniae DNAs was used to construct the contiguous DNA sequence in SEQ ID NO 1 Libraries may be prepared bv routine methods, for example Methods 1 and 2 below

Total cellular DNA is isolated from Streptococcus pneumoniae 0100993 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 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 into the vector Lambda ZapII that has been cut with EcoRI, the library packaged by standard procedures and E coli infected with the packaged library The library is amplified by standard procedures

Method 2 Total cellular DNA is partially hydrolyzed with a one or a combination of restriction 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 hgated to the DNA and the fragments then hgated into the vector Lambda ZapII that have been cut with EcoRI. the library packaged by standard procedures, and E coli infected with the packaged library The library is amplified by standard procedures

Example 2 vkqC Characterization

The S. pneumoniae vkqC gene is expressed during infection in a respiratory tract infection model

The determination of expression during infection of a gene from Streptococcus pneumoniae Excised lungs from a 48 hour respiratory tract infection of Streptococcus pneumoniae 0100993 in the mouse is efficiently disrupted and processed in the presence of chaotropic agents and RNAase inhibitor to provide a mixture of animal and bacterial RNA The optimal conditions for disruption and processing to giv e stable preparations and high yields of bacterial RNA are followed by the use of hybridisation to a radiolabelled ohgonucleotide specific to Streptococcus pneumoniae 16S RNA on Northern blots The RNAase free. DNAase free. DNA and protein free preparations of RNA obtained are suitable for Rev erse Transcription PCR (RT-PCR) using unique primer pairs designed from the sequence of each gene of Streptococcus pneumoniae 0100993

a) Isolation of tissue infected with Streptococcus pneumoniae 0100993 from a mouse animal model of infection (lungs)

Streptococcus pneumoniae 0100993 is seeded onto TSA (Tryptic Sov Agar. BBL) plates containing 5% horse blood and allowed to grow overnight at 37°C in a C02 incubator Bacterial growth is scraped into 5 ml of phosphate-buffered saline (PBS) and ad)usted to an A600 ~ 0 6 (4 x 106/ml) Mice (male CBA/J-1 mice, approximately 20g) were anaesthetized with lsoflurane and 50 microhters of the prepared bacterial inoculum is delivered by intranasal instillation Animals are allowed to recov er and observed twice dailv for signs of moπbundancy Forty-eight hours after infection the animals are euthanized by carbon dioxide ov erdose and their torsos swabbed with ethanol and then RNAZap The torso is then opened, and the lungs are aseptically removed Half of each pair of lungs is placed in a cryovial and immediately frozen in liquid nitrogen: the other half is used for bacterial enumeration after homogenization of the tissue m 1 ml of PBS

b) Isolation of Streptococcus pneumoniae 0100993 RNA from infected tissue samples Infected tissue samples, in 2-ml cryo-strorage tubes, are removed from -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 in 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 silica/ceramic matrix (BIO 101) Immediately. 1 ml of extraction reagents (FastRNA reagents. BIO101 ) 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. BIO 101) at 6000 rpm for 20-120 sec The crude RNA preparation is extracted with chloroform/isoamy 1 alcohol, and precipitated with DEPC-treated lsopropanol Precipitation Solution (BIO101 ) RNA preparations are stored m 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, followed by 5-10 minutes at 55 oC Finally, after at least 1 minute on ice, 200 units of Rnasin (Promega) is added

RNA preparations are stored at -80 oC for up to one month For longer teπn storage the RNA precipitate can be stored at the wash stage of the protocol in 75% ethanol for at least one year at -20 oC

Quality of the RNA isolated is assessed by running samples on 1 % agarose gels 1 x TBE gels stained with etlndium bromide are used to visualise total RNA yields To demonstrate the isolation of bacterial RNA from the infected tissue 1 x MOPS. 2 2M formaldehyde gels are nin and vacuum blotted to Hybond-N (Amersham) The blot is then hybridised with a 32P-labelled ohgonucletide probe, of sequence 5 ' AACTGAGACTGGCTTTAAGAGATTA 3 ' [SEQ ID NO 3 |. specific to 16S rRNA of Streptococcus pneumoniae The size of the hvbπdising band is compared to that of control RN Λ isolated from in vitro grown Streptococcus pneumoniae 0100993 in the Northern blot Correct sized bacterial 16S rRNA bands can be detected in total RNA samples which show degradation of the mammalian RNA when visualised on TBE gels

c) The removal of DNA from Streptococcus pneumoniae-deπved RNA

DNA was removed from 50 microgram samples of RNA by a 30 minute treatment at 37°C with 20 units of RN Aase-free DNAasel (GenHunter) in the buffer supplied in a final volume of 57 microhters The DNAase was mactivated and removed by treatment with TRIzol LS Reagent (Gibco BRL. Life Technologies) according to the manufacturers protocol

DNAase treated RNA was resuspended in 100 microhtres of DEPC treated water with the addition of Rnasin as descnbed before

d) The preparation of cDNA from RNA samples derived from infected tissue

3 microgram samples of DNAase treated RNA are reverse transcribed using a SuperScπpt Preamphfication Sy stem for First Strand cDNA Synthesis kit (Gibco BRL. Life Technologies) according to the manufacturers instructions 150 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 determine the presence of a bacterial cDNA species PCR reactions are set up on ice in 0 2ml tubes by adding the following components 43 microhtres PCR Master Mix (Advanced Biotechnologies Ltd ), 1 microhtre PCR pπmers (optimally 18-25 basepairs in 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 Perkin Elmer GeneAmp PCR System 9600 as follows 2 minutes at 94 oC. then 50 cvcles of 30 seconds each at 94 oC 50 oC and 72 oC followed bv 7 minutes at 72 oC and then a hold temperature of 20 oC (the number of cycles is optimalh 30-50 to determine the appearance or lack of a PCR product and optimalh 8-30 cvcles if an estimation of the starting quantitv 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 by comparison to a 100 bp DNA Ladder (Gibco BRL. Life Technologies) Alternativeh if the PCR products arc convenientlv labelled bv the use of a labelled PCR primer (e g labelled at the 5'end with a dve) a suitable aliquot of the PCR product is run out on a polvacrv lamide sequencing gel and its presence and quantitv detected using a suitable gel scanning system (e g ABI PπsmTM 377 Sequencer using GeneScanTM software as supplied bv Perkin Elmer)

RT/PCR controls may include +/- reverse transcπptase reactions. 16S rRNA pπmers or DNA specific primer pairs designed to produce PCR products from non-transcribed Streptococcus pneumoniae 0100993 genomic sequences To test the efficiency of the pnmer pairs they are used in 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 uninfor ative 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 reproduciblv 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 m the +RT samples than the signal (if at all present) in -RT controls

Example 3

The ykqC gene is essential for S. pneumoniae in vitro growth.

Demonstration of gene essentiality to bacterial viability

An allehc replacement cassette was generated using PCR technology The cassette consisted of a pair of 500bp chromosomal DNA fragments flanking an erythromycm resistance gene The chromosomal DNA sequences are the 500bp preceding and following the DNA sequence encoding the ykqC gene contained in Seq ID NO 1

The allehc replacement cassette was introduced mto S pneumoniae R6 bv transfoπuation Competent cells were prepared according to published protocols DNA was introduced into the cells by incubation of ng quantities of allehc replacement cassette with 10" cells at 30°C for 30 minutes The cells were transferred to 37°C for 90 minutes to allow expression of the crvthromycin resistance gene Cells were plated in agar containing lug ervthromvcin per ml Follo ing incubation at 37°C for 36 hours, colonies are picked and grown overnight in Todd-Hevvitt broth supplemented with 0 5% veast extract Typically 1000 transformants containing the appropriate allehc replacement are obtained If no transformants are obtained in three separate transfoπnation experiments as was the case for this gene ykqC. then the gene is considered as being essential in vitro

\2 -

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 ammo acid sequence of SEQ ID NO 2 over the entire lengdi of SEQ ID NO.2, (n) an isolated polypeptide compnsmg the ammo acid sequence of SEQ ID NO.2, (in) an isolated polypeptide that is the amino acid sequence of SEQ ID NO 2, and (IV) a polypeptide that is encoded by a recombmant polynucleotide comprising the polyncleotide sequence of SEQ ID NO 1

2 An isolated polynucleotide selected from the group consisting of

(l) 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.

(π) an isolated polynucleotide compnsmg a polvnucleotide sequence that has at least 95% identity over its entire length to a polynucleotide sequence encodmg the polypeptide of SEQ ID

NO.2,

(in) 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 SEQ ID NO 1 ,

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

NO 2.

(v) an isolated pol nucleotide that is the polvnucleotide of SEQ ID NO 1 ,

(vi) an isolated polynucleotide of at least 30 nucleotides in lengdi obtainable by screening an appropnate library under stringent hybndization conditions widi a probe havmg die sequence of SEQ

ID NO 1 or a fragment thereof of of at least 30 nucleotides in lengdi,

(vn) an isolated polvnucleotide encodmg a mature polypeptide expressed by the ykqC gene compnsed m die Streptococcus pneumoniae. and

(vm) a polvnucleotide sequence complementary to said isolated polvnucleotide of (I). (π). (in), (i ) (v). (vi) or (vn)

3 A method for the treatment of an individual

(I) in 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

- JJ - (n) havmg need to inhibit activity or expression of the polypeptide of claim 1 compnsmg

(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 in 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 individual comprising the step of

(a) determining the presence or absence of a mutation in the nucleotide sequence encoding said polypeptide in 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

(0 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 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 recombmant polynucleotide comprising the polvnucleotide sequence of SEQ ID NO 1, comprising the step of culturing a host cell under conditions sufficient for the production of the polypeptide

6 A process for producing 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 compnsmg the amino acid sequence of SEQ ID NO 2, (in) an isolated polypeptide that is the amino 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 transfectmg a cell with an expression system compnsmg a polynucleotide capable of producing said polypeptide of (I), (n), (in) or (iv) when said expression system is present m a compatible host cell such the host cell, under appropriate culture conditions, produces said polypeptide of (i). (n), (in) or (iv)

7 A host cell or a membrane expressing a polypeptide selected from the group consisting of (I) 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 amino 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

8 An antibodv lmmunospecific for the polypeptide of claim 1

9 A method for screening to ldentifv compounds diat agonize or diat inhibit die function of iL polypeptide of claim 1 diat compnses a mediod selected from die group consisting of

(a) measuring the binding of a candidate compound to the polypeptide (or to the cells or membranes bearing the polypeptide) or a fusion protein thereof bv means of a label directly or indirecth 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 m the presence of a labeled competitor

(c) testing whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide. using detection sv stems appropriate to the cells or cell membranes bearing the polypeptide. (d) mixing a candidate compound with a solution comprising a polypeptide of claim 1. to form a mixture, measunng 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 m cells, using for instance, an ELISA assay

10 An agomst or antagonist to the polypeptide of claim 1