EP1080181A1 - clpX OF STREPTOCOCCUS PNEUMONIAE - Google Patents

Abstract

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

Description

clpX OF STREPTOCOCCUS PNEUMONIAE

RELATED APPLICATIONS

This application claims benefit of US Provisional Patent Application Number 60/085.803. filed May 18. 1998

FIELD 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 poKnucleotides and polypeptides of the clp proteins (ATP-dependent proteases) farruh . as well as their variants herein referred to as "clpX " "clpX polynucleotιde(s)." and "clpX polypeptιde(s)" as the case may be

BACKGROUND OF THE INVENTION Tire Streptococci make up a medically important genera of microbes known to cause several types of disease in humans, including, for example, otitis media, conjunctivitis, pneumonia, bacteremia meningitis, sinusitis pleural empyema and endocarditis and most particularly meningitis, such as for example infection of cerebrospinal fluid Since its isolation more man 100 vears ago. Streptococcus pneumomae has been one of the more intensively studied microbes For example, much of our earh understanding that DNA is. in fact, the genetic mateπal was predicated on the work of Gnfflth and of Aveiy. Macleod and McCarty using this microbe Despite the vast amount of research with S pneumomae manv questions concerning the virulence of this microbe remain It is particularh preferred to emplos Strcptococcal genes and gene products as targets for the development of antibiotics The frequency of Streptococcus pneumomae infections has risen dramatically in the past few decades This has been attributed to the emergence of multiply antibiotic resistant strains and an increasing population of people with weakened immune systems It is no longer uncommon to isolate Streptococcus pneumomae strains that are resistant to some or all of the standard antibiotics This phenomenon has created an unmet medical need and demand for new anti-microbial agents vaccines drug screening methods and diagnostic tests for this organism

Moreover, the drug discovery process is currently undergoing a fundamental revolution as it embraces "functional genormcs," that is, high throughput genome- or gene-based biolog) This approach is rapidly superseding earlier approaches based on "positional cloning" and other methods Functional genomics relies heavily on the various tools of biornformatics to identify gene sequences of potential interest from the many molecular biology databases now available as well as from other sources There is a continuing and significant need to identify and characterize further genes and other polynucleotides sequences and their related polypeptides, as targets for drug discovery

Clearly, there exists a need for polynucleotides and polypeptides such as the clpX embodiments of the invention, that have a present benefit of, among other things, being useful to screen compounds for antimicrobial activity Such factors are also useful to determine their role in pathogenesis of infection, dysfunction and disease There is also a need for identification and characterization of such factors and their antagonists and agonists to find ways to prevent, ameliorate or correct such infection, dysfunction and disease

SUMMARY OF THE INVENTION

The present invention relates to clpX in particular clpX polypeptides and clpX polynucleotides, recombmant mateπals and methods for their production In another aspect, the invention relates to methods for using such polypeptides and polynucleotides. rncluding treatment of microbial diseases, amongst others In a further aspect, the invention relates to methods for identifying agonists and antagonists using the materials provided by the invention, and for treating microbial infections and conditions associated with such infections with the identified agonist or antagonist compounds In a still further aspect, the invention relates to diagnostic assays for detecting diseases associated with microbial infections and conditions associated with such infections, such as assays for detecting clpX expression or activity

Various changes and modifications within the spirit 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 other parts of the present disclosure

DESCRIPTION OF THE INVENTION

The invention relates to clpX polypeptides and polynucleotides as described in greater detail below In particular, the invention relates to polypeptides and polynucleotides of a clpX of Streptococcus pneumomae, that is related by amino acid sequence homology to Bos taurus clpX polypeptide The invention relates especially to clpX having a nucleotide and amino acid sequences set out rn Table 1 as SEQ ID NO 1 and SEQ ID NO 2 respectively Note that sequences recited in the Sequence Listing below as "DNA^" represent an exemplification of the invention, since those of ordinary skill will recognize that such sequences can be usefully employed in polynucleotides in general, including πbopolynucleotides TABLE 1 clpX Polynucleotide and Polypeptide Sequences

(A) Streptococcus pneumomae clpX polynucleotide sequence [SEQ ID NO: l]. 5 ' - atgtctacaaatagaaaaaatgatatgatggtttattgctcattttgtggcaaaaaccaagaagaagtac aaaaaataat tgctggcaacaatgcttttatttgtaatgaatgcgtggagttagctcaggaaatcattcgagaagaattg gttgaggaag tcttggcagacttgtctgaggtgccaaaaccaattgaactcctccatatcttgaaccactatgtaattgg tcaagatcgt gccaagcgtgccttggcagtggcggtttataaccactacaaacgcatcaatttccacgatacacgcgaag agtcagaaga tgtggatttgcagaagtcaaacattttgatgattggcccaactggttcagggaaaactttccttgcccag accttggcta agagcttgaatgtaccttttgctattgcggatgcgacagctctgacggaggctggttatgtgggtgagga tgtggaaaat atactcctcaaactcttgcaggttgctgactttaacatcgaacgtgcagagcgtggcattatctatgtgg atgaaattga caagattgccaagaagagtgagaatgtgtctatcacacgtgatgtttctggtgaaggggtgcaacaagcc cttctcaaga ttattgagggaactgttgctagcgtaccgcctcaaggtggacgcaaacatccacaacaagagatgattca agtggataca aaaaatatcctcttcatcgtgggtggtgcttttgatggtattgaagaaattgtcaaacaacgtctgggtg aaaaagtcat cggatttggtcaaaacaataaggcgattgacgaaaacagctcatacatgcaagaaatcatcgctgaagac attcaaaaat ttggtattatccctgagttgattggacgcttgcctgtttttgcggctcttgagcaattgaccgttgatga cttggttcgc atcttgaaagagccaagaaatgccttggtgaaacaataccaaaccttgctttcttatgatgatgttgagt tggaatttga cgacgaagcccttcaagagattgctaataaagcaatcgaacggaagacaggggcgcgtggacttcgctcc atcatcgaag aaaccatgctagatgttatgtttgaggtgccgagtcaggaaaatgtgaaattggttcgcatcactaaaga aactgtcgat ggaacggataaaccgatcctagaaacagcctag-3 ' (B) Streptococcus pneumomae clpX polypeptide sequence deduced from a polynucleotide sequence m this table [SEQ ID NO 2]

NH₂-

MSTNRKNDMMVYCSFCGKNQEEVQKIIAGNNAFICNECVELAQEIIREELVEEVLADLSEVPKPIELLHI LNHYVIGQDR

AKRALAVAVYNHYKRINFHDTREESEDVD QKSNILMIGPTGSGKTFLAQTLAKSLNVPFAIADATALTE

AGYVGEDVEN

ILLKLLQVADFNIERAERGIIYVDEIDKIAKKSENVSITRDVSGEGVQQALLKIIEGTVASVPPQGGRKH

PQQEMIQVDT KNILFIVGGAFDGIEEIVKQRLGEKVIGFGQNNKAIDENSSYMQEIIAEDIQKFGIIPELIGRLPVFAAL

EQLTVDDLVR

ILKEPRNALVKQYQTLLSYDDVELEFDDEALQEIA KAIERKTGARGLRSIIEETMLDVMFEVPSQENVK

LVRITKETVD

GTDKPILETA-COOH

Deposited materials

A deposit comprising a Streptococcus pneumomae 0100993 strain has been deposited with the National Collections of Industrial and Marine Bacteπa Ltd (herem "NCIMB"). 23 St Machar Drive. Aberdeen AB2 1RY, Scotland on 11 Apπl 1996 and assigned deposit number 40794 The deposit was descπbed as Streptococcus pneumomae 0100993 on deposit

On 17 Apπl 1996 a Streptococcus pneumomae 0100993 DNA library in E coli was srmilarh deposited with the NCIMB and assigned deposit number 40800 The Streptococcus pneumomae stram deposit is referred to herein as "the deposited stram" or as "the DNA of the deposited stram "

The deposited stram compπses a full length clpX gene The sequence of the polynucleotides compnsed in tire deposited stram. as well as the ammo acid sequence of any polypeptide encoded thereby, are controlling m the event of any conflict with any descnption of sequences herem

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

In one aspect of the invention there is provided an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Streptococcus pneumomae 0100993 stram. which polypeptide is compnsed m the deposited stram Further provided by the mvention are clpX polynucleotide sequences m the deposited stram, such as DNA and RNA, and am o acid sequences encoded thereby Also provided by the mvention are clpX polypeptide and polynucleotide sequences isolated from the deposited stram

Polypeptides ClpX polypeptide of the mvention is substantially phylogenetically related to other protems of the clp proteins (ATP-dependent proteases) family

In one aspect of the mvention there are provided polypeptides of Streptococcus pneumomae referred to herem as "clpX" and "clpX polypeptides" as well as biologically, diagnostically. prophylactically, clinically or therapeutically useful vaπants thereof, and compositions compnsmg the same

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

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

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

The mvention also mcludes a polypeptide consisting of or compnsmg a polypeptide of the formula

X-(R₁)_m-(R₂)-(R₃)_n-Y wherem, at the ammo termmus, X is hydrogen, a metal or any other moiety descnbed herem for modified polypeptides, and at the carboxyl terminus. Y is hydrogen, a metal or any other moiety descnbed herem for modified polypeptides, Ri and R3 are any ammo acid residue or modified ammo acid residue, m is an integer between 1 and 1000 or zero, n is an mteger between 1 and 1000 or zero, and R2 is an am o acid sequence of the mvention, particularly an ammo acid sequence selected from Table 1 or modified forms thereof In the formula above, R is onented so that its ammo terminal ammo acid residue is at the left, covalently bound to Ri and its carboxy terminal ammo acid residue is at the nght, covalently bound to R3 Any stretch of ammo acid residues denoted by either Ri or R3, where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer Other preferred embodiments of the mvention are provided where m is an mteger between 1 and 50. 100 or 500, and n is an mteger between 1 and 50, 100, or 500

It is most preferred that a polypeptide of the mvention is denved from Streptococcus pneumomae. however, it may preferably be obtained from other organisms of the same taxonomic genus A polypeptide of the mvention may also be obtained, for example, from organisms of the same taxonomic family or order

A fragment is a vaπant polypeptide having an ammo acid sequence that is entirely the same as part but not all of any ammo acid sequence of any polypeptide of the mvention As with clpX polypeptides. fragments may be "free-standing." or compnsed within a larger polypeptide of which they form a part or region, most preferably as a smgle contmuous region m a smgle larger polypeptide Preferred fragments mclude. for example, truncation polypeptides having a portion of an ammo acid sequence of Table 1 [SEQ ID NO 2], or of vanants thereof, such as a contmuous senes of residues that mcludes an ammo- and/or carboxyl-termmal ammo acid sequence Degradation forms of the polypeptides of the mvention produced by or in a host cell, particularly a Streptococcus pneumomae. are also preferred Further preferred are fragments characterized by structural or functional attnbutes such as fragments that compnse alpha-helix and alpha-helix formmg regions, beta-sheet and beta-sheet-formmg regions, turn and tum-formmg regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions Further preferred fragments 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 of SEQ ID NO:2, or an isolated polypeptide comprising an ammo acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated or deleted from the amino acid sequence of SEQ ID NO.2 Fragments of the polypeptides of the mvention may be employed for producmg the corresponding full-length polypeptide by peptide synthesis, therefore, these vanants may be employed as intermediates for producmg the full-length polypeptides of the mvention Polynucleotides It is an object of the mvention to provide polynucleotides that encode clpX polypeptides, particularly polynucleotides that encode a polypeptide herem designated clpX

In a particularly preferred embodiment of the mvention the polynucleotide compnses a region encoding clpX polypeptides compnsmg a sequence set out m Table 1 [SEQ ID NO 1] that mcludes a full length gene, or a variant thereof The Applicants believe that this full length gene is essential to the growth and/or survival of an organism that possesses it, such as Streptococcus pneumomae

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

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

In another particularly preferred embodiment of the invention there is a clpX polypeptide from Streptococcus pneumomae comprising or consisting of an ammo acid sequence of Table 1 [SEQ ID NO 2], or a variant thereof Usmg the information provided herem. such as a polynucleotide sequence set out in Table 1

[SEQ ID NO 1]. a polynucleotide of the mvention encoding clpX polypeptide may be obtained usmg standard cloning and screening methods, such as those for cloning and sequencmg chromosomal DNA fragments from bactena usmg Streptococcus pneumomae 0100993 cells as starting matenal. followed by obtaining a full length clone For example, to obtain a polynucleotide sequence of the invention. such as a polynucleotide sequence given in Table 1 [SEQ ID NO 1], typically a library of clones of chromosomal DNA of Streptococcus pneumomae 0100993 m E coli or some other suitable host is probed with a radiolabeled ohgonucleotide, preferably a 17-mer or longer, derived from a partial sequence Clones carrying DNA identical to that of the probe can then be distinguished using stringent hybridization conditions By sequencing the individual clones thus identified by hybridization with sequencing primers designed from the original polypeptide or polynucleotide sequence it is then possible to extend the polynucleotide sequence m both directions to determine a full length gene sequence Conveniently , such sequencing is performed, for example, usmg 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 m particular Screening By Hybndization 1 90 and Sequencmg Denatured Double-Stranded DNA Templates 13 70) Direct genomic DNA sequencing may also be performed to obtain a full length gene sequence Illustrative of the mvention, each polynucleotide set out m Table 1 [SEQ ID NO 1] was discovered m a DNA library denved from Streptococcus pneumomae 0100993

Moreover, each DNA sequence set out m Table 1 [SEQ ID NO 1] contains an open readmg frame encoding a protem havmg about the number of amino acid residues set forth in Table 1 [SEQ ID NO 2] with a deduced molecular weight that can be calculated usmg ammo acid residue molecular weight values well known to those skilled m the art The polynucleotide of SEQ ID NO 1, between nucleotide number 1 and the stop codon that begms at nucleotide number 1231 of SEQ ID NO 1. encodes the polypeptide of SEQ ID NO 2

In a further aspect, the present mvention provides for an isolated polynucleotide comprising or consisting of (a) a polynucleotide sequence that has at least 95% identity, even more preferably at least 97%. still more preferably at least 99%. yet still more preferably at least 99 5% 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 that has at least 95% identity, even more preferably at least 97. still more preferably at least 99%, yet still more preferably at least 99 5% or 100% exact, to the ammo acid sequence of SEQ ID NO 2, over the entire length of SEQ ID NO 2

A polynucleotide encoding a polypeptide of the present mvention. mcludmg homologs and orthologs from species other than Streptococcus pneumomae. may be obtained by a process that compnses the steps of screening an appropnate library under stringent hybndization conditions with a labeled or detectable probe consisting of or compnsmg the sequence of SEQ ID NO 1 or a fragment thereof, and isolatmg a full-length gene and/or genomic clones compnsmg said polynucleotide sequence

The mvention provides a polynucleotide sequence identical over its entire length to a coding sequence (open readmg frame) m Table 1 [SEQ ID NO 1] Also provided by the mvention is a codmg sequence for a mature polypeptide or a fragment thereof, by itself as well as a codmg sequence for a mature polypeptide or a fragment in readmg frame with another codmg sequence, such as a sequence encoding a leader or secretory sequence, a pre-, or pro- or prepro-protern sequence The polynucleotide of the mvention may also compnse at least one non-coding sequence, mcludmg for example, but not limited to at least one non-coding 5' and 3' sequence, such as the transcribed but non- translated sequences, termmation signals (such as rho-dependent and rho-mdependent ter mation signals), nbosome binding sites, Kozak sequences, sequences that stabilize mRNA. mtrons, and polyadenylation signals The polynucleotide sequence may also compnse additional codmg sequence encoding additional ammo acids For example, a marker sequence that facilitates punfication of a fused polypeptide can be encoded In certain embodiments of the mvention. the marker sequence is a hexa-histidme peptide, as provided m the pQE vector (Qiagen, Inc ) and descnbed in Gentz et al , Proc Nail Acad Set , USA 86 821-824 (1989). or an HA peptide tag (Wilson et a . Cell 37 767 ( 1984). both of that may be useful m puπfying polypeptide sequence fused to them Polynucleotides of the mvention also mclude, but are not limited to. polynucleotides compnsmg a structural gene and its naturally associated sequences that control gene expression A preferred embodiment of the mvention is a polynucleotide of consisting of or compnsmg nucleotide 1 to the nucleotide immediately upstream of or mcludmg nucleotide 1231 set forth m SEQ ID NO 1 of Table 1. both of that encode a clpX polypeptide

The mvention also mcludes a polynucleotide consisting of or compnsmg a polvnucleotide of the formula X-(Rι )_m-(R₂)-(R₃)_n-Y wherem, at the 5¹ end of the molecule. X is hydrogen, a metal or a modified nucleotide residue, or together with Y defines a covalent bond, and at the 3' end of the molecule. Y is hydrogen, a metal, or a modified nucleotide residue, or together with X defines the covalent bond, each occurrence of Ri and R3 is independently any nucleic acid residue or modified nucleic acid residue, m is an integer between 1 and 3000 or zero , n is an integer between 1 and 3000 or zero, and R₂ is a nucleic acid sequence or modified nucleic acid sequence of the invention, particularly a nucleic acid sequence selected from Table 1 or a modified nucleic acid sequence thereof In the polynucleotide formula above. R₂ is oriented so that its 5' end nucleic acid residue is at the left, bound to R\ and its 3' end nucleic acid residue is at the right, bound to R3 Any stretch of nucleic acid residues denoted by either R j and/or R₂. where m and/or n is greater than 1. may be either a heteropolymer or a homopolymer, preferably a heteropolymer Where, in a preferred embodiment. X and Y together define a covalent bond, the polynucleotide of the above formula is a closed, circular polynucleotide, that can be a double-stranded polynucleotide wherem the formula shows a first strand to which the second strand is complementary In another preferred embodiment m and/or n is an integer between 1 and 1000. Other 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 l and 50. 100. or 500

It is most preferred that a polynucleotide of the mvention is denved from Streptococcus pneumomae. however, it may preferably be obtained from other organisms of the same taxonomic genus A polynucleotide of the mvention may also be obtained, for example, from organisms of the same taxonomic family or order

The term "polynucleotide encoding a polypeptide" as used herem encompasses polynucleotides that mclude a sequence encoding a polypeptide of the mvention, particularly a bactenal polypeptide and more particularly a polypeptide of the Streptococcus pneumomae clpX havmg an ammo acid sequence set out m Table 1 [SEQ ID NO 2] The term also encompasses polynucleotides that mclude a smgle contmuous region or discontinuous regions encoding the polypeptide (for example, polynucleotides mterrupted by mtegrated phage. an mtegrated msertion sequence, an mtegrated vector sequence, an mtegrated transposon sequence, or due to RNA editing or genomic DNA reorganization) together with additional regions, that also may compnse codmg and/or non-coding sequences

The mvention further relates to vanants of the polynucleotides descnbed herem that encode vanants of a polypeptide having a deduced ammo acid sequence of Table 1 [SEQ ID NO 2] Fragments of polynucleotides of the mvention may be used, for example, to synthesize full-length polynucleotides of the mvention Further particularly preferred embodiments are polynucleotides encoding clpX vanants. that have the ammo acid sequence of clpX polypeptide of Table 1 [SEQ ID NO 2] m which several, a few. 5 to 10. 1 to 5, 1 to 3, 2. 1 or no ammo acid residues are substituted, modified, deleted and/or added, in any combination Especially preferred among these are silent substitutions, additions and deletions, that do not alter the properties and activities of clpX polypeptide Preferred isolated polynucleotide embodiments also mclude polynucleotide fragments, such as a polynucleotide comprising a nuclic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids from the polynucleotide sequence of SEQ ID NO: l, or an polynucleotide comprising a nucleic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids truncated or deleted from the 5 ' and/or 3 ' end of the polynucleotide sequence of SEQ ID NO- 1

Further preferred embodiments of the mvention are polynucleotides that are at least 95%, 97% or 99 5% identical over their entire length to a polynucleotide encoding clpX polypeptide havmg an ammo acid sequence set out m Table 1 [SEQ ID NO 2], and polynucleotides that are complementary to such polynucleotides Most highly preferred are polynucleotides that compnse a region that is at least 95% are especially preferred Furthermore, those 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 preferred, with at least 99 5% bemg the more preferred Preferred embodiments are polynucleotides encoding polypeptides that retain substantially the same biological function or activity as a mature polypeptide encoded by a DNA of Table 1 [SEQ ID NO l]

In accordance with certain preferred embodiments of this mvention there are provided polynucleotides that hybndize, particularly under stπngent conditions, to clpX polynucleotide sequences, such as those polynucleotides in Table 1

The mvention further relates to polynucleotides that hybndize to the polynucleotide sequences provided herem In this regard, the mvention especially relates to polynucleotides that hybndize under stπngent conditions to the polynucleotides descnbed herem As herem used, the terms "stringent conditions" and "stringent hybndization conditions" mean hybndization occumng only if there is at least 95% and preferably at least 97% identity between the sequences A specific example of stringent hybridization conditions is overnight incubation at 42°C m a solution comprising 50% formamide, 5x SSC (150mM NaCl 15mM tπsodmm citrate). 50 mM sodium phosphate (pH7 6), 5x Denhardt's solution. 10% dextran sulfate. and 20 micrograms/ml of denatured. sheared salmon sperm DNA, followed by washmg the hybridization support m 0 lx SSC at about 65 °C Hybridization and wash conditions are well known and exemplified in Sambrook. et al . Molecular Cloning A Laboratory Manual. Second Edition, Cold Spring Harbor, N Y . (1989). particularly Chapter 11 therein 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 screemng an appropriate library comprising a complete gene for a polynucleotide sequence set forth m SEQ ID NO 1 under stringent hybridization conditions with a probe havmg the sequence of said polynucleotide sequence set forth in SEQ ID NO 1 or a fragment thereof, and isolating said polynucleotide sequence Fragments useful for obtaining such a polynucleotide include, for example, probes and primers fully described elsewhere herein

As discussed elsewhere herem regarding polynucleotide assays of the mvention, for instance. the polynucleotides of the mvention, may be used as a hybndization probe for RNA. cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding clpX and to isolate cDNA and genomic clones of other genes that have a high identity, particularly high sequence ldentits . to a clpX gene Such probes generally will compnse at least 15 nucleotide residues or base pairs Preferably, such probes will have at least 30 nucleotide residues or base pairs and may have at least 50 nucleotide residues or base pairs Particularly preferred probes will have at least 20 nucleotide residues or base pairs and will have lee than 30 nucleotide residues or base pairs A codmg region of a clpX gene may be isolated by screening usmg a DNA sequence provided m Table 1 [SEQ ID NO 1] to synthesize an o gonucleotide probe A labeled o gonucleotide havmg a sequence complementary to that of a gene of the mvention is then used to screen a library of cDNA, genomic DNA or mRNA to determine which members of the library the probe hybndizes to There are several methods available and well known to those skilled m the art to obtain full-length DNAs, or extend short DNAs. for example those based on the method of Rapid Amplification of cDNA ends (RACE) (see. for example, Frohman, et al , PNAS USA 85 8998- 9002, 1988) Recent modifications of the 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 withm the amplified product (typically an adaptor specific primer that anneals further 3' m the adaptor sequence and a gene specific primer that anneals further 5' m the selected gene sequence) The products of this reaction can then be analyzed by DNA sequencing and a full-length DNA constructed either by joining the product directly to the existing DNA to give a complete sequence, or carrying out a separate full-length PCR using the new sequence information for the design of the 5' primer The polynucleotides and polypeptides of the mvention may be employed, for example, as research reagents and matenals for discovery of treatments of and diagnostics for diseases, particularly human diseases, as further discussed herem relatmg to polynucleotide assays

The polynucleotides of the invention that are ohgonucleotides derived from a sequence of Table 1 [SEQ ID NOS 1 or 2] may be used in the processes herein as described, but preferably for PCR, to determine whether or not the polynucleotides identified herein m whole or in part are transcribed in bacteria in infected tissue It is recognized that such sequences will also have utility in diagnosis of the stage of mfection and type of infection the pathogen has attained

The mvention also provides polynucleotides that encode a polypeptide that is a mature protem plus additional ammo or carboxyl-termmal ammo acids, or ammo acids mteπor to a mature polypeptide (when a mature form has more than one polypeptide cham, for instance) Such sequences may play a role processmg of a protem from precursor to a mature form, may allow protem transport, may lengthen or shorten protem half-life or may facilitate manipulation of a protem for assay or production, among other things As generally is the case in vivo, the additional ammo acids may be processed away from a mature protem by cellular enzymes For each and every polynucleotide of the mvention there is provided a polynucleotide complementary to it It is preferred that these complementary polynucleotides are fully complementary to each polynucleotide with which they are complementary

A precursor protem, havmg a mature form of the polypeptide fused to one or more prosequences may be an mactive form of the polypeptide When prosequences are removed such mactive precursors generally are activated Some or all of the prosequences may be removed before activation Generally, such precursors are called proproteins

As will be recognized, the entire polypeptide encoded by an open readmg frame is often not required for activity Accordingly, it has become routine m molecular biology to map the boundanes of the pπmary structure required for activity with N-termmal and C-terminal deletion experiments These experiments utilize exonuclease digestion or convement restnction sites to cleave codmg nucleic acid sequence For example. Promega (Madison. WI) sell an Erase-a-base™ system that uses Exonuclease III designed to facilitate analysis of the deletion products (protocol available at www promega com) The digested endpoints can be repaired (e g . by hgation to synthetic linkers) to the extent necessary to preserve an open reading frame In this way. the nucleic acid of SEQ ID NO 1 readily provides contiguous fragments of SEQ ID NO 2 sufficient to provide an activity, such as an enzymatic, binding or antibody-inducing activity Nucleic acid sequences encoding such fragments of SEQ ID NO 2 and vanants thereof as descnbed herem are within the mvention. as are polypeptides so encoded In sum, a polynucleotide of the mvention may encode a mature protem. a mature protem plus a leader sequence (which may be referred to as a preprotem). a precursor of a mature protem havmg one or more prosequences that are not the leader sequences of a preprotem, or a preproprotem. that is a precursor to a proprotem. havmg a leader sequence and one or more prosequences. that generally are removed during processmg steps that produce active and mature foπns of tire polypeptide Vectors, Host Cells, Expression Systems

The mvention also relates to vectors that compnse a polynucleotide or polynucleotides of the mvention, host cells that are genetically engineered with vectors of the mvention and the production of polypeptides of the mvention by recombinant techniques Cell-free translation systems can also be employed to produce such proteins usmg RNAs denved from the DNA constructs of the mvention Recombinant polypeptides of the present mvention may be prepared by processes well known m those skilled m the art from genetically engineered host cells compnsmg expression systems Accordingly, a further aspect, the present mvention relates to expression systems that compnse a polynucleotide or polynucleotides of the present mvention, to host cells that are genetically engineered with such expression systems, and to the production of polypeptides of the mvention by recombinant techniques

For recombinant production of the polypeptides of the mvention, host cells can be genetically engmeered to incorporate expression systems or portions thereof or polynucleotides of the mvention Introduction of a polynucleotide mto the host cell can be effected by methods descnbed m many standard laboratory manuals, such as Davis, et al . BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, et al , MOLECULAR CLONING A LABORATORY MANUAL. 2nd Ed . Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N Y (1989), such as. calcium phosphate transfection. DEAE-dextran mediated transfection. transvection, micro jection. catiomc lipid-mediated transfection. electroporation, transduction, scrape loading, ballistic mtroduction and infection

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

A great vanety of expression systems can be used to produce the polypeptides of the mvention Such vectors mclude. among others, chromosomal-, episomal- and virus-deπved vectors, for example, vectors denved from bacteπal plasmids. from bacteπophage, from transposons. from yeast episomes. from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40. vaccinia viruses, adenoviruses. fowl pox viruses, pseudorabies viruses, picornaviruses and retroviruses. and vectors denved from combinations thereof, such as those denved from plasmid and bactenophage genetic elements, such as cosmids and phagemids The expression system constructs may compnse control regions that regulate as well as engender expression Generally, any system or vector suitable to mamtam, propagate or express polynucleotides and/or to express a polypeptide in a host may be used for expression in this regard The appropπate DNA sequence may be inserted mto the expression system by any of a vanety of well- known and routme techniques, such as, foi example, those set forth m Sambrook et al . MOLECULAR CLONING, A LABORATORY MANUAL, (supra) In recombinant expression systems in eukaryotes, for secretion of a translated protem mto the lumen of the endoplasmic reticulum. mto the penplasmic space or mto the extracellular environment, appropnate secretion signals may be incorporated mto the expressed polypeptide These signals may be endogenous to the polypeptide or they may be heterologous signals Polypeptides of the mvention can be recovered and puπfied from recombinant cell cultures by well-known methods mcludmg ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography. phosphocellulose chromatography, hydrophobic mteraction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectm chromatography Most preferably, high performance liquid chromatography is employed for puπfication Well known techniques for refolding protem may be employed to regenerate active conformation when the polypeptide is denatured duπng isolation and or puπfication

Diagnostic, Prognostic, Serotyping and Mutation Assays

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

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

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

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

This mvention also relates to the use of polynucleotides of the present mvention as diagnostic reagents Detection of a mutated form of a polynucleotide of the mvention, preferable, SEQ ID NO 1. that is associated with a disease or pathogenicity will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, a prognosis of a course of disease, a determination of a stage of disease, or a susceptibility to a disease, that results from under-expression, over-expression or altered expression of the polynucleotide Organisms, particularly infectious oiganisms. carrying mutations in such polynucleotide may be detected at the polynucleotide level by a vanety of techniques, such as those descnbed elsewhere herem The differences in a polynucleotide and/or polypeptide sequence between organisms possessing a first phenotype and organisms possessing a different, second different phenotype can also be determined If a mutation is observed m some or all organisms possessing the first phenotype but not in any orgamsms possessing the second phenotype, then the mutation is likely to be the causative agent of the first phenotype Cells from an organism carrying mutations or polymorphisms (allehc vaπations) m a polynucleotide and/or polypeptide of the mvention may also be detected at the polynucleotide or polypeptide level by a vanety of techniques, to allow for serotyping. for example For example. RT- PCR can be used to detect mutations m the RNA It is particularly preferred to use RT-PCR conjunction with automated detection systems, such as. for example, GeneScan RNA. cDNA or genomic DNA may also be used for the same purpose, PCR As an example, PCR primers complementary to a polynucleotide encoding clpX polypeptide can be used to identify and analyze mutations The mvention further provides these primers with 1, 2. 3 or 4 nucleotides removed from the 5' and/or the 3' end These primers may be used for. among other things, amplifying clpX DNA and/or RNA isolated from a sample denved from an mdividual. such as a bodily matenal The primers may be used to amplify a polynucleotide isolated from an infected mdividual. such that the polynucleotide may then be subject to vaπous techmques for elucidation of the polynucleotide sequence In this way. mutations m the polynucleotide sequence may be detected and used to diagnose and/or prognose the infection or its stage or course, or to serotype and/or classify the infectious agent The mvention further provides a process for diagnosing, disease, preferably bacterial infections, more preferably infections caused by Streptococcus pneumomae . 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 1] Increased or decreased expression of a clpX polynucleotide can be measured using any on of the methods well known m 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 in accordance with the mvention for detecting over-expression of clpX polypeptide compared to normal control tissue samples may be used to detect the presence of an infection, for example Assay techmques that can be used to deteπrune levels of a clpX polypeptide. m a sample denved from a host, such as a bodily matenal. are well-known to those of skill m the art Such assay methods mclude radioimmunoassays. competitive-bmdmg assay s. Western Blot analysis, antibody sandwich assays, antibody detection and ELISA assays Antagonists and Agonists - Assays and Molecules Polypeptides and polynucleotides of the mvention may also be used to assess the binding of small molecule substrates and gands m. for example, cells, cell-free preparations, chemical bianes. and natural product mixtures These substrates and hgands may be natural substrates and hgands or may be structural or functional mrmetics See. e g . Co gan et al . Current Protocols in Immunology 1(2) Chapter 5 (1991) Polypeptides and polynucleotides of the present mvention are responsible for many biological functions, mcludmg many disease states, m particular the Diseases herem mentioned It is therefore desirable to devise screening methods to identify compounds that agonize (e g . stimulate) or that antagonize (e g ,ιnhιbιt) the function of the polypeptide or polynucleotide Accordingly , m a further aspect, the present mvention provides for a method of screening compounds to identify those that agonize or that antagonize the function of a polypeptide or polynucleotide of the mvention. as well as related polypeptides and polynucleotides In general, agomsts or antagomsts (e g . inhibitors) may be employed for therapeutic and prophylactic purposes for such Diseases as herem mentioned Compounds may be identified from a vanety of sources, for example, cells, cell-free preparations. chemical libraπes. and natural product mixtures Such agomsts and antagomsts so-identified may be natural or modified substrates, hgands. receptors, enzymes, etc . as the case may be, of clpX polypeptides and polynucleotides, or may be structural or functional mimetics thereof (see Coligan et al . Current Protocols in Immunology 1(2) Chapter 5 (1991))

The screening methods may simply measure the binding of a candidate compound to the polypeptide or polynucleotide, or to cells or membranes bearing the polypeptide or polynucleotide, or a fusion protem of the polypeptide by means of a label directly or indirectly associated with the candidate compound Alternatively the screening method may involve competition with a labeled competitor Further, these screening methods may test whether the candidate compound results m a signal generated by activation or inhibition of the polypeptide or polynucleotide. usmg detection systems appropπate to the cells comprising the polypeptide or polynucleotide Inhibitors of activation are generally assayed in 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 m screening methods for inverse agonists, in the absence of an agonist or antagonist, by testing whether the candidate compound results m inhibition of activation of the polypeptide or polynucleotide. as the case may be Further, the screening methods may simply comprise the steps of mixing a candidate compound with a solution comprising a polypeptide or polynucleotide of the present invention, to form a mixture, measuring clpX polypeptide and/or polynucleotide activity in the mixture, and comparing the clpX polypeptide and/or polynucleotide activity of the mixture to a standard Fusion proteins, such as those made from Fc portion and clpX polypeptide. as herein described, can also be used for high-throughput screening assays to identify antagonists of the polypeptide of the present mvention. as well as of phylogenetically and and/or functionally related polypeptides (see D Bennett et al , J Mol Recognition. 8 52-58 (1995), and K Johanson et al . J Biol Chem. 270(16) 9459-9471 (1995))

The polynucleotides, polypeptides and antibodies that bind to and/or interact with a polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and/or polypeptide m cells For example, an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide usmg monoclonal and polyclonal antibodies by standard methods known 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 screening compounds to identify those that enhance (agomst) or block (antagomst) the action of clpX polypeptides or polynucleotides. particularly those compounds that are bacteπstatic and/or bactencidal The method of screening may mvolve high- throughput techmques For example, to screen for agomsts or antagomsts. a synthetic reaction mix. a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, compnsmg clpX polypeptide and a labeled substrate or gand of such polypeptide is incubated in the absence or the presence of a candidate molecule that may be a clpX agomst or antagomst The ability of the candidate molecule to agonize or antagonize the clpX polypeptide is reflected m decreased binding of the labeled gand or decreased production of product from such substrate Molecules that bmd gratuitously, i e . without mducmg the effects of clpX polypeptide are most likely to be good antagomsts 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 agonists Detection of the rate or level of, as the case may be, production of product from substrate, signal transduction, or chemical channel activity may be enhanced by usmg a reporter system Reporter systems that may be useful m this regard mclude but are not limited to coloπmetπc. labeled substrate converted into product, a reporter gene that is responsive to changes in clpX polynucleotide or polypeptide activity, and binding assays known m the art

Polypeptides of the invention may be used to identify membrane bound or soluble receptors, if any, for such polypeptide. through standard receptor binding techniques known in the art These techniques include, but are not limited to, hgand binding and crosslinking assays m which the polypeptide is labeled with a radioactive isotope (for instance. ^⁵I). chemically modified (for instance, biotmylated). or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (e g , cells, cell membranes, cell supernatants, tissue extracts, bodily matenals) Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy These screening methods may also be used to identify agonists and antagonists of the polypeptide that compete with the binding of the polypeptide to its receptor(s). if any Standard methods for conducting such assays are well understood in the art

The fluorescence polarization value for a fluorescently-tagged molecule depends on the rotational correlation time or tumblmg rate Protem complexes, such as formed by clpX polypeptide associating with another clpX polypeptide or other polypeptide. labeled to comprise a fluorescently-labeled molecule will have higher polarization values than a fluorescently labeled monomenc protem It is preferred that this method be used to characterize small molecules that disrupt polypeptide complexes

Fluorescence energy transfer may also be used characterize small molecules that interfere with the formation of clpX polypeptide dimers, tπmers. tetramers or higher order structures, or structures formed by clpX polypeptide bound to another polypeptide ClpX 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 dimenzation will inhibit fluorescence energy transfer

Surface plasmon resonance can be used to monitor the effect of small molecules on clpX polypeptide self-association as well as an association of clpX polypeptide and another polypeptide or small molecule ClpX polypeptide can be coupled to a sensor chip at low site density such that covalently bound molecules will be monomenc Solution protem can then passed over the clpX polypeptide -coated surface and specific binding can be detected m realtime by monitoring the change m resonance angle caused by a change m local refractive index This technique can be used to characterize the effect of small molecules on kinetic rates and equilibrium binding constants for clpX polypeptide self-association as well as an association of clpX polypeptide and another polypeptide or small molecule A scintillation proximity assay may be used to characterize the interaction between an association of clpX polypeptide with another clpX polypeptide or a different polypeptide ClpX polypeptide can be coupled to a scintillation-filled bead Addition of radio-labeled clpX polypeptide results in binding where the radioactive source molecule is in close proximity to the scintillation fluid Thus, signal is emitted upon clpX polypeptide binding and compounds that prevent clpX polypeptide self-association or an association of clpX polypeptide and another polypeptide or small molecule will dim ish signal

In other embodiments of the mvention there are provided methods for identifying compounds that bmd to or otherwise mteract with and inhibit or activate an activity or expression of a polypeptide and/or polynucleotide of the mvention compnsmg contacting a polypeptide and/or polynucleotide of the mvention with a compound to be screened under conditions to permit bmdmg to or other interaction between the compound and the polypeptide and/or polynucleotide to assess the bmdmg to or other interaction with the compound, such bmdmg or interaction preferably bemg associated with a second component capable of providing a detectable signal m response to the bmdmg or mteraction of the polypeptide and/or polynucleotide with the compound, and determining whether the compound bmds to or otherwise interacts with and activates or inhibits an activity or expression of the polypeptide and/or polynucleotide by detectmg the presence or absence of a signal generated from the bmdmg or mteraction of the compound with the polypeptide and/or polynucleotide Another example of an assay for clpX agomsts is a competitive assay that combmes clpX and a potential agomst with clpX-bmdmg molecules, recombinant clpX bmdmg molecules, natural substrates or hgands. or substrate or gand m metics. under appropnate conditions for a competitive inhibition assay ClpX can be labeled, such as by radioactivity or a coloπmetnc compound, such that the number of clpX molecules bound to a bmdmg molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagomst

It will be readily appreciated by the skilled artisan that a polypeptide and/or polynucleotide of the present invention may also be used in a method for the structure-based design of an agonist or antagonist of the polypeptide and/or polynucleotide, by (a) determining in the first instance the three-dimensional structure of the polypeptide and/or polynucleotide, or complexes thereof, (b) deducmg 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 motifts). 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 may be performed usmg automated and computer-controlled steps

In a further aspect, the present mvention provides methods of treatmg abnormal conditions such as. for instance, a Disease, related to either an excess of. an under-expression of, an elevated activity of. or a decreased activity of clpX polypeptide and/or polynucleotide

If the expression and/or activity of the polypeptide and/or polynucleotide is in excess, several approaches are available One approach compπses admmistermg to an mdividual in need thereof an inhibitor compound (antagomst) as herem descnbed. optionally in combination with a pharmaceutically acceptable earner, m an amount effective to inhibit the function and/or expression of the polypeptide and/or polynucleotide, such as. for example, by blocking the bmdmg of hgands. substrates, receptors, enzymes, etc , or by rnhibitmg a second signal, and thereby alleviating the abnormal condition In another approach, soluble forms of the polypeptides still capable of bmdmg the hgand. substrate, enzymes, receptors, etc m competition with endogenous polypeptide and/or polynucleotide may be administered Typical examples of such competitors include fragments of the clpX polypeptide and/or polypeptide In still another approach, expression of the gene encoding endogenous clpX polypeptide can be inhibited using expression blocking techniques This blocking may be targeted against any step in gene expression, but is preferably targeted against transcription and/or translation An examples of a known technique of this sort mvolve the use of antisense sequences, either internally generated or separately administered (see, for example, O'Connor. J Neurochem

(1991) 56 560 in Ohgodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)) Alternatively, ohgonucleotides that form triple helices with the gene can be supplied (see, for example, Lee et al , Nucleic Acids Res (1979) 6 3073. Cooney et al Science (1988) 241 456, Dervan et al Science (1991) 251 1360) These ohgomers can be administered er se or the relevant ohgomers can be expressed in vivo

Each of the polynucleotide sequences provided herein may be used m the discovery and development of antibacterial compounds The encoded protein, upon expression, can be used as a target for the screemng of antibacterial drugs Additionally , the polynucleotide sequences encoding the ammo terminal regions of the encoded protem or Shme-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the coding sequence of interest

The invention also provides the use of the polypeptide, polynucleotide, agomst 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 m the prevention of adhesion of bacteria, in particular gram positive and/or gram negative bacteria, to eukaryotic, preferably mammalian, extracellular matrix proteins on m-dwellmg devices or to extracellular matrix proteins m wounds, to block bacterial adhesion between eukaryotic. preferably mammalian, extracellular matrix proteins and bacterial clpX proteins that mediate tissue damage and/or. to block the normal progression of pathogenesis in infections initiated other than by the implantation of m-dwellmg devices or by other surgical techniques

In accordance with yet another aspect of the mvention. there are provided clpX agomsts and antagomsts, preferably bacteπstatic or bactencidal agomsts and antagomsts

The antagomsts and agomsts of the mvention may be employed, for instance, to prevent. inhibit and/or treat diseases

Hehcobacter pylori (herem "H pylori") bacteria mfect the stomachs of over one-third of the world's population causmg stomach cancer, ulcers, and gastritis (International Agency for Research on Cancer (1994) Schistosomes, Liver Flukes and Hehcobacter Pylori (International Agency for Research on Cancer, Lyon. France, http //www uicc ch/ecp/ecp2904 htm) Moreover, the International Agency for Research on Cancer recently recognized a cause-and- effect relationship between H pylori and gastric adenocarcinoma, classifying the bacterium as a Group I (definite) carcinogen Preferred antimicrobial compounds of the invention (agonists and antagomsts of clpX polypeptides and or polynucleotides) found using screens provided by the invention, or known m the art, particularly narrow-spectrum antibiotics, should be useful in the treatment of H pylori infection Such treatment should decrease the advent of H /jy/σrz-induced cancers, such as gastrointestinal carcinoma Such treatment should also prevent, inhibit and/or cure gastric ulcers and gastritis

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

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

"Bodily mateπal(s) means any matenal denved from an mdividual or from an organism infecting, infesting or inhabiting an mdividual. mcludmg but not limited to. cells, tissues and waste. such as. bone, blood, serum, cerebrospmal fluid, semen, saliva, muscle, cartilage, organ tissue, skin, urine, stool or autopsy mateπals

"Dιsease(s)" means any disease caused by or related to infection by a bacteπa. mcludmg . for example, otitis media, conjunctivitis, pneumonia, bacteremia. menmgitis. sinusitis, pleural empyema and endocarditis, and most particularly menmgitis, such as for example infection of cerebrospmal 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

"Identity," as known m the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determmed by comparmg the sequences In the art. "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be. as determined by the match between strings of such sequences "Identity" can be readily calculated by known methods, mcludmg but not limited to those descnbed m (Computational Molecular Biology, Lesk. A M , ed , Oxford University Press, New York, 1988, Biocomputing Informatics and Genome Projects. Smith. D W . ed .

Academic Press, New York. 1993. Computer Analysis of Sequence Data, Part I, Griffin. A M . and Griffin. H G , eds , Humana Press, New Jersey, 1994, Sequence Analysis in Molecular

Biology, von Hemje, G , Academic Press. 1987, and Sequence Analysis Primer, Gπbsko\, M and Devereux, J . eds . M Stockton Press. New York. 1991, and Caπllo. H , and Lipman, D ,

SIAM J Applied Math , 48 1073 (1988) Methods to determine identity are designed to give the largest match between the sequences tested Moreover, methods to determme identity are codified in publicly available computer programs Computer program methods to determme identity between two sequences include, but are not limited to, the GCG program package (Devereux. J et al . Nucleic Acids Research 12(1) 387 (1984)). BLASTP. BLASTN and FASTA (Altschul

S F et al , J Molec Biol 215 403-410 (1990) The BLAST X program is publicly available from NCBI and other sources (BLAST Manual. Altschul. S , et al . NCBI NLM NIH Bethesda

MD 20894. Altschul. S et al . J Mol Biol 215 403-410 (1990) The well known Smith

Waterman algorithm may also be used to deteπnme identity Parameters for polypeptide sequence comparison include the following Algorithm

Needleman and Wunsch. J Mol Biol 48 443-453 (1970)

Comparison matrix BLOSSUM62 from Hentikoff and Hentikoff Proc Natl Acad Sci USA

89 10915-10919 (1992)

Gap Penalty 12 Gap Length Penalty 4

A program useful with these parameters is publicly available as the "gap" program from Genetics

Computer Group, Madison WI The aforementioned parameters are the default parameters for peptide comparisons (along with no penalty for end gaps)

Parameters for polynucleotide comparison include the following Algorithm Needleman and Wunsch. J Mol Biol 48 443-453 (1970)

Comparison matrix matches = +10. mismatch = 0

Gap Penalty 50

Gap Length Penalty 3

Available as The "gap" program from Genetics Computer Group. Madison WI These are the default parameters for nucleic acid comparisons

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

(1) Polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide sequence having at least a 95, 97. 99 5 or 100% identity to the reference sequence of SEQ ID NO 1, wherem said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO 1 or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherem 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 θι in one or more contiguous groups within the reference sequence, and wherem said number of nucleotide alterations is determined by multiplying the total number of nucleotides m SEQ ID NO 1 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleotides in SEQ ID NO 1. or

n_n < x_n - (x_n • y),

wherem n_n is the number of nucleotide alterations x_n is the total number of nucleotides in SEQ ID NO 1, y is 0 95 for 95%, 0 97 for 97%. 0 995 for 99 5% or 1 00 for 100%. and • is the symbol for the multiplication operator, and wherein any non-integer product of x_n and y is rounded down to the neaiest integer prior to subtracting it from x_n Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO 2 may create nonsense. missense or frameshift mutations m this codmg sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations

(2) Polypeptide embodiments further include an isolated polypeptide comprising a polypeptide having at least a 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO 2. wherem said polypeptide sequence may be identical to the reference sequence of SEQ ID NO 2 or may include up to a certain integer number of amino acid alterations as compared to the reference sequence, wherem said alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution or insertion, and wherein said alterations may occur at the ammo- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids m the reference sequence or m one or more contiguous groups withm the reference sequence, and wherem said number of ammo acid alterations is determined by multiplying the total number of ammo acids in SEQ ID NO 2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in SEQ ID NO 2. or ⁿa ≤ ^xa ^" (^xa * y),

wherem n_a is the number of ammo acid alterations. x_a is the total number of amino acids m SEQ ID NO 2, y is 0 95 for 95%. 0 97 for 97% or 1 00 for 100%. and • is the symbol for the multiplication operator, and wherein any non-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 primate, and a human "Isolated" means altered "by the hand of man" from its natural state. / e . if it occurs in nature, it has been changed or removed from its oπgmal environment, or both For example, a polynucleotide or a polypeptide naturally present m a living organism is not "isolated.^" but the same polynucleotide or polypeptide separated from the coexisting mateπals of its natural state is "isolated", as the term is employed herem Moreover, a polynucleotide or polypeptide that is introduced mto an organism by transformation, genetic mampulation or by any other recombmant method is "isolated" even if it is still present m said organism, which organism may be living or non-living

"Organιsm(s)" means a (l) prokaryote. mcludmg but not limited to. a member of the genus Streptococcus, Staphylococcus, Bordetella, Corynebacterium, Mycobactenum, Neissena, Haemoph us, Actinomycetes, Streptomycetes, Nocardia, Enterobacter. Yersinia, Fancisella, Pasturella, Moraxella, Acmetobacter, Erysipelothnx, Branhamella, Actinobacillus, Streptobacillus. Listena, Calymmatobactenum, Brucella, Bacillus, Clostndium, Treponema, Eschenchia, Salmonella, Kleibsiella, Vibrio, Proteus, Erwin a, Borreha, Leptospira Spirillum, Campylobacter. Shigella, Legwnella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia. Borreha and Mycoplasma. and further mcludmg. but not limited to, a member of the species or group. Group A Streptococcus Group B Streptococcus, Group C Streptococcus, Group D Streptococcus, Group G Streptococcus, Streptococcus pneumomae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus faecahs, Streptococcus faecium, Streptococcus durans, Neissena gonorrheae, Neissena memngitidis, Staphylococcus aureus, Staphylococcus epidermidis, Coiynebactenum dψthenae. Gardnerella vaginalis, Mycobactenum tuberculosis. Mycobactenum bovis, Mycobactenum ulcerans, Mycobactenum leprae, Actinomyctes israeln, Listena monocytogenes. Bordetella pertusis, Bordatella parapertusis, Bordetella bronchiseptica, Eschenchia coli Shigella dysentenae, Haemophilus influenzae, Haemophύus aegyptius, Haemophύus parainfluenzae, Haemophύus ducreyi, Bordetella, Salmonella typhi, C trobacter freundπ, Proteus mirabihs, Proteus vulgans, Yersinia pestis, Kleibsiella pneumomae, Serratia marcessens, Serratia hquefaciens, Vibrio cholera. Shigella dysentern, Shigella flexnen, Pseudomonas aeruginosa, Franscisella tularensis, Brucella abortis, Bacillus anthracis, Bacillus cereus, Clostndium perfnngens, Clostndium tetani, Clostndium botulinum, Treponema palhdum, Rickettsia nckettsn and Chlamydia trachomitis, (n) an archaeon, mcludmg but not limited to Archaebacter . and (m) a unicellular or filamentous eukaryote. mcludmg but not limited to. a protozoan, a fungus, a member of the genus Saccharomyces, Kluveromyces, or Candida, and a member of the species Saccharomyces cenviseae, Kluveromyces lactis, or Candida albicans

"Polynucleotιde(s)" generally refers to any polynbonucleotide or polydeoxyπbonucleotide. that may be unmodified RNA or DNA or modified RNA or DNA "Polynucleotιde(s)" mclude. without limitation, smgle- and double-stranded DNA. DNA that is a mixture of single- and double-stranded regions or smgle-. double- and tnple-stranded regions, smgle- and double-stranded RNA. and RNA that is mixture of smgle- and double-stranded regions, hybnd molecules compnsmg DNA and RNA that may be single-stranded or. more typically, double-stranded, or tnple-stranded regions, or a mixture of smgle- and double-stranded regions In addition, "polynucleotide" as used herem refers to tπple- stranded regions compnsmg RNA or DNA or both RNA and DNA The strands m such regions may be from the same molecule or from different molecules The regions may mclude all of one or more of the molecules, but more typically mvolve only a region of some of the molecules One of the molecules of a tπple-he cal region often is an ohgonucleotide As used herem. the term "polynucleotιde(s)" also mcludes DNAs or RNAs as descnbed above that compnse one or more modified bases Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotιde(s)" as that teπn is intended herem Moreover. DNAs or RNAs compnsmg unusual bases, such as mosme. or modified bases, such as tntylated bases, to name just two examples, are polynucleotides as the term is used herem It will be appreciated that a great vanety of modifications have been made to DNA and RNA that serve many useful puφoses known to those of skill m the art The term "polynucleotιde(s)" as it is employed herem embraces such chemically, enzymatically or metabolically modified forms of polynucleotides. as well as the chemical forms of DNA and RNA characteπstic of viruses and cells, mcludmg. for example, simple and complex cells "Polynucleotιde(s)" also embraces short polynucleotides often referred to as ohgonucleotιde(s)

"Polypeptιde(s)" refers to any peptide or protem compnsmg two or more ammo acids jomed to each other by peptide bonds or modified peptide bonds "Polypeptιde(s)" refers to both short chains, commonly referred to as peptides. ohgopeptides and ohgomers and to longer chains generally referred to as proteins Polypeptides may compnse ammo acids other than the 20 gene encoded ammo acids "Polypeptιde(s)" mclude those modified either by natural processes, such as processing and other post- translational modifications, but also by chemical modification techniques Such modifications are well descnbed m basic texts and m more detailed monographs, as well as m a voluminous research literature, and they are well known to those of skill m the art It will be appreciated that the same type of modification may be present m the same or varying degree at several sites m a given polypeptide Also, a given polypeptide may compnse many types of modifications Modifications can occur anywhere m a polypeptide. mcludmg the peptide backbone, the ammo acid side-chams. and the ammo or carboxyl termini Modifications mclude. for example, acetylation. acylation. ADP-πbosylation. amidation. covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide denvative, covalent attachment of a lipid or lipid deπvative, covalent attachment of phosphotidylinositol, cross-linking, cyclization. disulfide bond formation, demethylation. formation of covalent cross-links, formation of cysteme, formation of pyroglutamate. formylation. gamma-carboxylation. GPI anchor formation, hydroxylation. lodmation. methylation. mynstoylation. oxidation, proteolytic processmg, phosphorylation. prenylation. racerπization. glycosylation. lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP- πbosylation. selenoylation, sulfation. transfer-RNA mediated addition of amino acids to protems. such as argmvlation, 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 m POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B C Johnson. Ed , Academic Press, New York (1983), Seifter et al . Meth Enzymol 182 626-646 (1990) and Rattan et al . Protein Synthesis Posttranslational Modifications and Aging, Ann N Y Acad Sci 663 48-62 (1992) Polypeptides may be branched or cyclic, with or without branching Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well

"Recombmant expression system(s)" refers to expression systems or portions thereof or polynucleotides of the mvention mtroduced or transformed mto a host cell or host cell lysate for the production of the polynucleotides and polypeptides of the mvention

"Vaπant(s)" as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retams essential properties A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide Changes m the nucleotide sequence of the variant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result in ammo acid substitutions, additions, deletions, fusion proteins and truncations in the polypeptide encoded by the reference sequence, as discussed below A typical variant of a polypeptide differs m amino acid sequence from another, reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and. in many regions, identical A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions m any combination A substituted or inserted amino acid residue may or may not be one encoded by the genetic code The present mvention also mcludes mclude vanants of each of the polypeptides of the mvention. that is polypeptides that vary from the referents by conservative a mo acid substitutions, whereby a residue is substituted by another with like characteπstics Typical such substitutions are among Ala. Val, Leu and He. among Ser and Thr. among the acidic residues Asp and Glu, among Asn and Gin, and among the basic residues Lys and Arg. or aromatic residues Phe and Tyr Particularly preferred are vanants m winch several, 5-10. 1-5. 1-3, 1-2 or 1 ammo acids are substituted, deleted, or added m any combmation A variant of a polynucleotide or polypeptide may be a naturally occurring such as an alle c 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 recombinant methods known to skilled artisans

EXAMPLES

The examples beloyy are earned out usmg standard techniques, that are well known and routme to those of skill in the art, except where otherwise descnbed m detail The examples are illustrative, but do not limit the mvention

Example 1 Strain selection, Library Production and Sequencing

The polvnucleotide having a DNA sequence given in Table 1 [SEQ ID NO 1] was obtained from a library of clones of chromosomal DNA of Streptococcus pneumomae in E coli The sequencing data from two or more clones comprising overlapping Streptococcus pneumomae DNAs was used to construct the contiguous DNA sequence in SEQ ID NO 1 Libraries may be prepared by routine methods, for example Methods 1 and 2 below

Total cellular DNA is isolated from Streptococcus pneumomae 0100993 according to standard procedures and size-fractionated by either of two methods Method 1

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

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combination of restriction enzymes appropπate to generate a series of fragments for clonmg 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 clpX Characterization

The S. pneumoniae clpX 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 m the mouse is efficiently disrupted and processed m 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 give 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 protem free preparations of RNA obtained are suitable for Reverse 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 Soy Agar. BBL) plates containing 5% horse blood and allowed to grow overnight at 37°C m a C02 incubator Bacterial growth is scraped mto 5 ml of phosphate-buffered salme (PBS) and adjusted to an A600 ~ 0 6 (4 x 106/ml) Mice (male CBA/J-1 mice, approximately 20g) were anaesthetized with isoflurane and 50 microhters of the prepared bacterial inoculum is delivered by mtranasal instillation Animals are allowed to recover and observed twice daily for signs of moπbundancy Forty-eight hours after infection the animals are euthanized by carbon dioxide overdose 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 m a cryovial and immediately frozen in liquid nitrogen, the other half is used for bacterial enumeration after homogemzation 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 mto a dry ice ethanol bath In a microbiological safety cabinet the samples are disrupted up to eight at a time while the remaining samples are kept frozen m the dry ice ethanol bath To disrupt the bacteria withm the tissue sample. 50-100 mg of the tissue is transfered to a FastRNA tube containing a silica ceramic matrix (BIO 101) Immediately . 1 ml of extraction reagents

(FastRNA reagents, BIO 101) are added to give a sample to reagent volume ratio of approximately 1 to 20 The tubes are shaken m a reciprocating shaker (FastPrep FP120.

BIO 101) at 6000 φm for 20-120 sec The crude RNA preparation is extracted with chloroform isoamyl alcohol, and precipitated with DEPC-treated/Isopropanol Precipitation

Solution (BIO 101) 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-dned 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 Rnasm

(Promega) is added

RNA preparations are stored at -80 oC for up to one month For longer term 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 ethidium 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 run 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 pneumomae The size of the hybridising band is compared to that of control RNA isolated from m vitro grown Streptococcus pneumoniae 0100993 m the Northern blot Correct sized bacterial 16S rRNA bands can be detected total RNA samples which show degradation of the mammalian RNA when visualised on TBE gels c) The removal of DNA from Streptococcus pneumomae-deπved RNA

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

DNAase treated RNA was resuspended in 100 microlitres of DEPC treated water with the addition of Rnasm as described 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 Superscript Preamphfication System 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 transcriptase are also run Both +/-RT samples are treated with RNaseH before proceeding to the PCR reaction e) The use of PCR to deteπnme the presence of a bacterial cDNA species

PCR reactions are set up on ice m 0 2ml tubes by adding the following components 43 microlitres PCR Master Mix (Advanced Biotechnologies Ltd ), 1 microhtre PCR primers (optimally 18-25 basepairs in length and designed to possess similar annealing temperatures), each primer at lOmM initial concentration, and 5 microlitres cDNA

PCR reactions are run on a Perkm Elmer GeneAmp PCR System 9600 as follows 2 minutes at 94 oC, then 50 cycles of 30 seconds each at 94 oC, 50 oC and 72 oC followed by 7 minutes at 72 oC and then a hold temperature of 20 oC (the number of cycles is optimally 30-50 to determme the appearance or lack of a PCR product and optimally 8-30 cycles if an estimation of the starting quantity of cDNA from the RT reaction is to be made). 10 microhtre ahquots are then run out on 1 % 1 x TBE gels stained with ethidium bromide, with PCR product, if present, sizes estimated by comparison to a 100 bp DNA Ladder (Gibco BRL. Life Technologies) Alternatively if the PCR products are conveniently labelled by the use of a labelled PCR primer (e g labelled at the 5'end with a dye) a suitable aliquot of the PCR product is run out on a polyacrylamide sequencing gel and its presence and quantity detected using a suitable gel scanning system (e g ABI PπsmTM 377 Sequencer using GeneScanTM software as supplied by Perkm Elmer)

RT/PCR controls may mclude +/- reverse transcriptase reactions, 16S rRNA primers or DNA specific primer pairs designed to produce PCR products from non-transcribed Streptococcus pneumoniae 0100993 genomic sequences

To test the efficiency of the primer pairs they are used in DNA PCR with Streptococcus 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 unmformative Of those which give the correct size product with DNA PCR two classes are distinguished in RT/PCR 1 Genes which are not transcribed in vivo reproducibly fail to give a product in RT/PCR. and 2 Genes which are transcribed 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) m -RT controls Example 3

The clpX 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 folloyvmg the DNA sequence encoding the clpX gene contained m Seq ID NO 1

The allehc replacement cassette was introduced into S pneumomae R6 by transformation Competent cells yvere prepared accordmg 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 erythromycm resistance gene Cells were plated in agar containing lug erythromycm per ml Following incubation at 37°C for 36 hours, colonies are picked and grown overnight in Todd-Hewitt broth supplemented with 0 5% yeast extract Typically 1000 transformants containing the appropriate allehc replacement are obtained If no transformants are obtained m three separate transformation experiments as was the case for this gene clpX. then the gene is considered as being essential in vitro

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 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 amino acid sequence of SEQ ID NO 2. and

(iv) a polypeptide that is encoded by a recombinant polynucleotide comprising the polyncleotide sequence of SEQ ID NO 1

2 An isolated polynucleotide selected from the group consist g of

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

(n) an isolated polynucleotide compnsmg a polynucleotide sequence that has at least 95% identify over its entire lengtii to a polynucleotide sequence encoding 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 lover the entire length that portion of SEQ ID NO 1 which encodes SEQ ID NO 2.

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

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

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

(vn) an isolated polynucleotide encoding a mature polypeptide expressed by the clpX gene compnsed m the Streptococcus pneumoniae, and

(vm) a polynucleotide sequence complementary to said isolated polynucleotide of (1), (n). (m).

3 A method for the treatment of an individual

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

(a) administering to the individual a therapeutically effective amount of an antagonist to said polypeptide. or

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

(c) admimstenng 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 prognosmg 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

(I) an isolated polypeptide comprising an ammo acid sequence selected from the group having at least 95% identity to the ammo acid sequence of SEQ ID NO 2 over the entire length of

SEQ ID NO 2,

(u) an isolated polypeptide comprising the ammo acid sequence of SEQ ID NO 2 (in) an isolated polypeptide that is the ammo acid sequence of SEQ ID NO 2. and (iv) a polypeptide that is encoded by a recombinant polynucleotide comprising the polynucleotide sequence of SEQ ID NO 1 , comprising the step of cultuπng a host cell under conditions sufficient for the production of the polypeptide

6 A process for producing a host cell comprising an expression system or a membrane thereof 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 ammo acid sequence of SEQ ID NO 2 over the entire length of SEQ ID NO 2, (n) an isolated polypeptide comprising the ammo acid sequence of SEQ ID NO 2, (in) an isolated polypeptide that is the 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 transfecting a cell with an expression system compnsmg a polynucleotide capable of producing said polypeptide of (l), (n), (in) or (iv) when said expression system is present in a compatible host cell such the host cell, under appropriate culture conditions, produces said polypeptide of (I), (n). (in) or (iv)

7 A host cell or a membrane expressmg a polypeptide selected from the group consisting of

(l) 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

8 An antibody lmmunospecific for the polypeptide of claim 1

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

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

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

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

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

10 An agonist or antagonist to the polypeptide of claim 1