WO2000049033A1 - yybQ - Google Patents

Abstract

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

Description

yybQ

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 polynucleotides and polypeptides of the inorganic phosphatase family, as well as their vanants, herein referred to as "yybQ," "yybQ polynucleoude(s)," and "yybQ polypeptιde(s)" as the case may be

BACKGROUND OF THE INVENTION

The Streptococci make up a medically important genera of microbes known to cause several types of disease in humans, including, for example, otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid Since its isolation more than 100 years ago, Streptococcus pneumomae has been one of the more intensively studied microbes For example, much of our early understanding that DNA is, in fact, the genetic matenal was predicated on the work of Griffith and of Avery, Macleod and McCarty using this microbe Despite the vast amount of research with S pneumomae, many questions concerning the virulence of this microbe remain It is particularly preferred to employ

Streptococcal genes and gene products as targets for the development of antibiotics

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- icrobial agents, vaccines, drug screening methods, and diagnostic tests for this organism

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

Clearly, there exists a need for polynucleotides and polypeptides, such as the yybQ 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 yybQ, m particular yybQ polypeptides and yybQ polynucleotides, recombinant matenals and methods for their production In another aspect, the invention relates to methods for using such polypeptides and polynucleotides, including treatment of microbial diseases, amongst others In a further aspect, the mvention relates to methods for identifying agonists and antagonists using the mateπals provided by the mvention, and for treatmg 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 yybQ 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 yybQ polypeptides and polynucleotides as descπbed in greater detail below In particular, the invention relates to polypeptides and polynucleotides of a yybQ of Streptococcus pneumomae, that is related by amrno acid sequence homology to coaggregation-relevant adhesin [Streptococcus gordomi] polypeptide The invention relates especially to yybQ having a nucleotide and amino acid sequences set out in Table 1 as SEQ ED NO 1 and SEQ ID NO 2 respectively Note that sequences recited in the Sequence Listmg below as "DNA" represent an exemplification of the mvention, since those of ordinary skill will recognize that such sequences can be usefully employed m polynucleotides in general, including πbopolynucleotides

TABLE 1 yybQ Polynucleotide and Polypeptide Sequences

(A) Streptococcus pneumomae yybQ polynucleotide sequence [SEQ ID NO 1] 5 ' -ATGTCCAAGATTCTAGTATTTGGTCACCAAAATCCAGACTCAGATGCCATCGGATCATCTGTAGCTTTTG CCTACCTTGCAAAAGAAGCTTACGGTTTGGATACGGAAGCTGTTGCCCTTGGAACTCCAAATGAAGAAAC AGCCTTTGTCTTGAACTATTTTGGTGTGGAAGCACCACGTGTTATCACTTCTGCCAAAGCAGAGGGGGCA GAGCAAGTTATCTTGACTGACCACAATGAATTCCAACAATCTGTATCAGATATCGCTGAAGTAGAAGTTT ACGGTGTTGTAGACCACCACCGTGTGGCTAACTTTGAAACTGCAAGCCCACTTTACATGCGTTTGGAGCC AGTTGGATCAGCGTCTTCAATCGTTTACCGTATGTTCAAAGAACATGGTGTAGCTGTGCCTAAAGAGATT GCAGGTTTGATGCTTTCAGGTTTGATTTCAGATACCCTTCTTTTGAAATCACCAACAACACACCCAACAG ATAAAATCATTGCTCCTGAATTGGCTGAATTGGCTGGTGTGAACTTGGAAGAATATGGTTTGGCAATGTT GAAAGCTGGTACCAACTTGGCTAACAAATCTGCTGAAGAATTGATTGACATCGATGCTAAGACTTTTGAA CTCAACGGAAATAATGTCCGTGTTGCCCAAGTGAACACAGTTGACATCGCTGAAGTTTTGGAACGCCAAG CAGAAATTGAAGCTGCAATGCAAGCTGCCAACGAATCAAACGGCTACTCTGACTTTGTCTTGATGATTAC AGATATCGTCAACTCAAACTCAGAAATCTTGGCTCTTGGTGCCAATATGGACAAGGTCGAAGCGGCTTTC AACTTCAAACTTGAAAACAATCATGCCTTCCTTGCTGGTGCCGTTTCACGTAAGAAACAAGTGGTACCTC AATTGACTGAAAGCTTTAATGCGTAA-3 '

(B) Streptococcus pneumomae yybQ polypeptide sequence deduced from a polynucleotide sequence m this table [SEQ ID NO 2]

NH₂-MSKILVFGHQNPDSDAI GSSVAFAYLAKEAYGLDTEAVALGTPNEETAFVLNYFGVEAPRVITSAKAEGA EQVILTDHNEFQQSVSDIAEVEVYGWDHHRVANFETASPLYMRLEPVGSAS SIVYRMFKEHGVAVPKEI AGLMLSGLI SDTLLLKSPTTHPTDKI IAPE1-AEI-AGVNLEEYG AMLKAGTNLANKSAEELIDI DAKTFE LNGNNVRVAQVNTVDIAEVLERQAEIEAAMQAANESNGYSDFVLMITDIVNSNSEI LALGANMDKVEAAF NFKLENNHAFLAGAVSRKKQWPQLTESFNA-COOH

Deposited materials

A deposit compπsrng a Streptococcus pneumomae 0100993 strain has been deposited with the National Collections of Industπal and Maπne Bacteπa Ltd (herein "NCIMB"), 23 St Machar Dπve, Aberdeen AB2 1RY, Scotland on 11 Apπl 1996 and assigned deposit number 40794 The deposit was descnbed as Streptococcus pneumomae 0100993 on deposit

On 17 Apπl 1996 a Streptococcus pneumomae 0100993 DNA library in E coll was similarly deposited with the NCIMB and assigned deposit number 40800 The Streptococcus pneumomae strain deposit is refeπed to herein as "the deposited strain" or as "the DNA of the deposited strain "

The deposited strain compπses a full length yybQ gene The sequence of the polynucleotides compπsed in the deposited strain, as well as the ammo acid sequence of any polypeptide encoded thereby, are controlling in the event of any conflict with any descπption of sequences herem

The deposit of the deposited strain has been made under the terms of the Budapest Treaty on the International Recogmtion of the Deposit of Micro-organisms for Purposes of Patent Procedure The deposited strain will be irrevocably and without restriction or condition released to the public upon the issuance of a patent The deposited strain is provided merely as convenience to those of skill in the art and is not an admission that a deposit is required for enablement, such as that required under 35 U S C §112 A license may be required to make, use or sell the deposited strain, and compounds deπved 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 strain, which polypeptide is compπsed in the deposited strain Further provided by the invention are yybQ polynucleotide sequences in the deposited strain, such as DNA and RNN and ammo acid sequences encoded thereby Also provided by the mvention are yybQ polypeptide and polynucleotide sequences isolated from the deposited strain

Polypeptides

YybQ polypeptide of the invention is substantially phylogeneucally related to other proteins of the inorganic phosphatase family

In one aspect of the invention there are provided polypeptides of Streptococcus pneumomae refeπed to herein as "yybQ" and "yybQ polypeptides" as well as biologically, diagnostically prophylactically, clinically or therapeutically useful vaπants thereof, and compositions compπsing the same Among the particularly prefeπed embodiments of the mvention are vaπants of yybQ polypeptide encoded by naturally occurring alleles of a yybQ gene

The present mvention further provides for an isolated polypeptide that (a) composes or consists of an amino acid sequence that has at least 95% identity, most preferably at least 97-99% or exact identity, to that of SEQ ID NO 2 over the entire length of SEQ ID NO 2, (b) a polypeptide encoded by an isolated polynucleotide compπsmg or consisting of a polynucleotide sequence that has at least 95%o 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 compπsmg or consisting of a polynucleotide sequence encoding a polypeptide that has at least 95 % identity, even more preferably at least 91-99% or exact identity, to the amino acid sequence of SEQ ID NO 2, over the entire length of SEQ ID NO 2

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

The mvention also includes a polypeptide consisting of or compπsmg a polypeptide of the formula

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

It is most preferred that a polypeptide of the 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 yybQ polypeptides, fragments may be "free-standing," or compπsed within a larger polypeptide of which they form a part or region, most preferably as a single contmuous region a smgle larger polypeptide

Prefeπed fragments include, for example, truncation polypeptides having a portion of an ammo acid sequence of Table 1 [SEQ ID NO 2], or of vaπants thereof, such as a contmuous seπes of residues that includes an ammo- and/or carboxyl-terminal ammo acid sequence Degradation forms of the polypeptides of the mvention produced by or in a host cell, particularly a Streptococcus pneumomae, are also prefeπed Further prefeπed are fragments characterized by structural or functional attπbutes such as fragments that compπse alpha-helrx and alpha-he x forming regions, beta-sheet and beta-sheet-forrning regions, turn and turn-fonmng regions, coil and coil-formmg regions, hydrophihc regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-formmg regions, substrate binding region, and high antigemc dex regions

Further prefeπed fragments include an isolated polypeptide comprising an amino acid sequence havmg 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 comprismg an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous ammo 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 coπespondmg full-length polypeptide by peptide synthesis, therefore, these vaπants 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 yybQ polypeptides, particularly polynucleotides that encode a polypeptide herem designated yybQ In a particularly prefeπed embodiment of the invention the polynucleotide comprises a region encoding yybQ polypeptides comprising a sequence set out in Table 1 [SEQ ID NO:l] that includes 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 pneumoniae. As a further aspect of the invention there are provided isolated nucleic acid molecules encoding and/or expressing yybQ polypeptides and polynucleotides, particularly Streptococcus pneumoniae yybQ polypeptides and polynucleotides, including, for example, unprocessed RNAs, ribozyme RNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs. Further embodiments of the invention include biologically, diagnostically, prophylacticaUy, clinically or therapeutically useful polynucleotides and polypeptides, and variants thereof, and compositions comprising the same.

Another aspect of the invention relates to isolated polynucleotides, including at least one full length gene, that encodes a yybQ polypeptide having a deduced amino acid sequence of Table 1 [SEQ ID NO:2] and polynucleotides closely related thereto and variants thereof.

In another particularly prefeπed embodiment of the invention there is a yybQ polypeptide from Streptococcus pneumoniae comprising or consisting of an amino acid sequence of Table 1 [SEQ ID NO: 2], or a variant thereof.

Using the information provided herein, such as a polynucleotide sequence set out in Table 1 [SEQ ID NO:l], a polynucleotide of the invention encoding yybQ polypeptide may be obtained using standard cloning and screening methods, such as those for cloning and sequencing chromosomal DNA fragments from bacteria using Streptococcus pneumoniae 0100993 cells as starting material, followed by obtaining a full length clone. For example, to obtain a polynucleotide sequence of the invention, such as a polynucleotide sequence given in Table 1 [SEQ ID NO:l], typically a library of clones of chromosomal DNA of Streptococcus pneumoniae 0100993 in E.coli or some other suitable host is probed with a radiolabeled oligonucleotide, 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 in both directions to determine a full length gene sequence. Conveniently, such sequencing is performed, for example, using denatured double stranded DNA prepared from a plasmid clone. Suitable techniques are described by Maniatis, T., Fritsch, E.F. and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989). (see in particular Screening By Hybridization 1.90 and Sequencing Denatured Double-Stranded DNA Templates 13.70). Direct genomic DNA sequencing may also be performed to obtain a full length gene sequence. Illustrative of the mvention, each polynucleotide set out m Table 1 [SEQ ID NO 1] was discovered a DNA library deπved from Streptococcus pneumoniae 0100993

Moreover, each DNA sequence set out in Table 1 [SEQ ID NO 1] contains an open reading frame encoding a protein having about the number of ammo acid residues set forth m Table 1 [SEQ ID NO 2] with a deduced molecular weight that can be calculated using ammo acid residue molecular weight values well known to those skilled in the art The polynucleotide of SEQ ID NO 1, between nucleotide number 1 and the stop codon that begins at nucleotide number 934 of SEQ ID NO 1, encodes the polypeptide of SEQ ED NO 2

In a further aspect, the present mvention provides for an isolated polynucleotide compπsmg or consisting of (a) a polynucleotide sequence that has at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID NO 1 over the entire length of SEQ ID NO 1. or the entire length of that portion of SEQ ID NO 1 which encodes SEQ ID NO 2, (b) a polynucleotide sequence encoding a polypeptide that has at least 95% identity, even more preferably at least 97-99% or 100% exact, to the ammo acid sequence of SEQ ID NO 2, over the entire length of SEQ ID NO 2 A polynucleotide encoding a polypeptide of the present mvention, mcludmg homologs and orthologs from species other than Streptococcus pneumomae, may be obtained by a process that compπses the steps of screening an appropπate library under stringent hybndization conditions with a labeled or detectable probe consisting of or compπsmg the sequence of SEQ ID NO 1 or a fragment thereof, and isolating a full- length gene and/or genomic clones compπsmg said polynucleotide sequence The mvention provides a polynucleotide sequence identical over its entire length to a coding sequence (open reading frame) Table 1 [SEQ ED NO 1] Also provided by the mvention is a coding sequence for a mature polypeptide or a fragment thereof, by itself as well as a coding sequence for a mature polypeptide or a fragment in reading frame with another coding sequence, such as a sequence encoding a leader or secretory sequence, a pre-, or pro- or prepro-protein sequence The polynucleotide of the mvention may also compπse at least one non-codmg sequence, mcludmg for example, but not limited to at least one non-codmg 5' and 3' sequence, such as the transcnbed but non-translated sequences, terrrunation signals (such as rho-dependent and rho-mdependent termination signals), πbosome binding sites, Kozak sequences, sequences that stabilize mRNN mtrons, and polyadenylation signals The polynucleotide sequence may also compπse additional coding sequence encoding additional ammo acids For example, a marker sequence that facihtates purification of a fused polypeptide can be encoded In certam embodiments of the mvention the marker sequence is a hexa-histidine peptide, as provided m the pQE vector (Qiagen, Inc ) and descπbed m Gentz et al , Proc Natl Acad Set , USA 86 821-824 (1989), or an HA peptide tag (Wilson et al , Cell 37 767 (1984), both of that may be useful in puπfymg polypeptide sequence fused to them Polynucleotides of the invention also include, but are not limited to, polynucleotides comprising a structural gene and its naturally associated sequences that control gene expression.

A prefeπed embodiment of the invention is a polynucleotide of consisting of or comprising nucleotide 1 to the nucleotide immediately upstream of or including nucleotide 934 set forth in SEQ ID NO : 1 of Table 1 , both of that encode a yybQ polypeptide.

The invention also includes a polynucleotide consisting of or comprising a polynucleotide of the formula:

X-(R₁)_m-(R₂)-(R₃)_n-Y wherein, at the 5' end of the molecule, X is hydrogen, a metal or a modified nucleotide residue, or together with Y defines a covalent bond, and at the 3' end of the molecule, Y is hydrogen, a metal, or a modified nucleotide residue, or together with X defines the covalent bond, each occuπence of Rj 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 R 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 Rj and its 3' end nucleic acid residue is at the right, bound to R3. Any stretch of nucleic acid residues denoted by either Rj and/or R₂, where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer. Where, in a prefeπed embodiment, X and Y together define a covalent bond, the polynucleotide of the above formula is a closed, circular polynucleotide, that can be a double-stranded polynucleotide wherein the formula shows a first strand to which the second strand is complementary. In another preferred embodiment m and/or n is an integer between 1 and 1000. Other prefeπed embodiments of the invention are provided where m is an integer between 1 and 50, 100 or 500, and n is an integer between 1 and 50, 100, or 500. It is most prefeπed that a polynucleotide of the invention is derived from Streptococcus pneumoniae, however, it may preferably be obtained from other organisms of the same taxonomic genus. A polynucleotide of the invention may also be obtained, for example, from organisms of the same taxonomic family or order.

The term "polynucleotide encoding a polypeptide" as used herein encompasses polynucleotides that include a sequence encoding a polypeptide of the invention, particularly a bacterial polypeptide and more particularly a polypeptide of the Streptococcus pneumoniae yybQ having an amino acid sequence set out in Table 1 [SEQ ED NO:2]. The term also encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, polynucleotides interrupted by integrated phage, an integrated insertion sequence, an integrated vector sequence, an integrated transposon sequence, or due to RNA editing or genomic DNA reorganization) together with additional regions, that also may comprise coding and/or non-coding sequences.

The invention further relates to variants of the polynucleotides described herein that encode variants of a polypeptide having a deduced amino acid sequence of Table 1 [SEQ ID NO:2]. Fragments of polynucleotides of the invention may be used, for example, to synthesize full-length polynucleotides of the invention.

Further particularly prefeπed embodiments are polynucleotides encoding yybQ variants, that have the amino acid sequence of yybQ polypeptide of Table 1 [SEQ ID NO:2] in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, modified, deleted and/or added, in any combination. Especially prefeπed among these are silent substitutions, additions and deletions, that do not alter the properties and activities of yybQ polypeptide.

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

NO: l.

Further prefeπed embodiments of the invention are polynucleotides that are at least 95 % or 97% identical over their entire length to a polynucleotide encoding yybQ polypeptide having an amino acid sequence set out in Table 1 [SEQ ED NO:2], and polynucleotides that are complementary to such polynucleotides. Most highly prefeπed are polynucleotides that comprise a region that is at least 95% are especially prefeπed. Furthermore, those with at least 97% are highly prefeπed among those with at least

95%, and among these those with at least 98% and at least 99% are particularly highly prefeπed, with at least 99% being the more prefeπed. Prefeπed embodiments are polynucleotides encoding polypeptides that retain substantially the same biological function or activity as a mature polypeptide encoded by a DNA of Table 1 [SEQ ID NO: 1].

In accordance with certain prefeπed embodiments of this invention there are provided polynucleotides that hybridize, particularly under stringent conditions, to yybQ polynucleotide sequences, such as those polynucleotides in Table 1. The invention further relates to polynucleotides that hybridize to the polynucleotide sequences provided herein. In this regard, the invention especially relates to polynucleotides that hybridize under stringent conditions to the polynucleotides described herein. As herein used, the terms "stringent conditions" and "stringent hybridization conditions" mean hybridization occurring 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 in a solution comprising: 50% formamide, 5x SSC (150mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml of denatured, sheared salmon sperm DNA, followed by washing the hybridization support in 0. lx SSC at about 65°C. Hybridization and wash conditions are well known and exemplified in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold 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 screening an appropriate library comprising a complete gene for a polynucleotide sequence set forth in SEQ ID NO:l under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence set forth in SEQ ID NO:l or a fragment thereof; and isolating said polynucleotide sequence. Fragments useful for obtaining such a polynucleotide include, for example, probes and primers fully described elsewhere herein.

As discussed elsewhere herein regarding polynucleotide assays of the invention, for instance, the polynucleotides of the invention, may be used as a hybridization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding yybQ and to isolate cDNA and genomic clones of other genes that have a high identity, particularly high sequence identity, to a yybQ gene. Such probes generally will comprise 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 prefeπed probes will have at least 20 nucleotide residues or base pairs and will have lee than 30 nucleotide residues or base pairs.

A coding region of a yybQ gene may be isolated by screening using a DNA sequence provided in Table 1 [SEQ ED NO:l] to synthesize an oligonucleotide probe. A labeled oligonucleotide having a sequence complementary to that of a gene of the invention is then used to screen a library of cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.

There are several methods available and well known to those skilled in the art to obtain full- length DNAs, or extend short DNAs, for example those based on the method of Rapid Amplification of cDNA ends (RACE) (see, for example, Frohman, et al., PNAS USA 85: 8998-9002, 1988). Recent modifications of the technique, exemplified by the Marathon™ technology (Clontech Laboratories Inc.) for example, have significantly simplified the search for longer cDNAs. In the Marathon™ technology, cDNAs have been prepared from mRNA extracted from a chosen tissue and an 'adaptor' sequence ligated 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 oligonucleotide primers. The PCR reaction is then repeated using "nested" primers, that is, primers designed to anneal within the amplified product (typically an adaptor specific primer that anneals further 3' in the adaptor sequence and a gene specific primer that anneals further 5' in the selected gene sequence). The products of this reaction can then be analyzed by DNA sequencing and a full-length DNA constructed either by joining the product directly to the 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 invention may be employed, for example, as research reagents and materials for discovery of treatments of and diagnostics for diseases, particularly human diseases, as further discussed herein relating to polynucleotide assays.

The polynucleotides of the invention that are oligonucleotides derived from a sequence of Table 1 [SEQ ID NOS:l or 2] may be used in the processes herein as described, but preferably for

PCR, to determine whether or not the polynucleotides identified herein in whole or in part are transcribed in bacteria in infected tissue. It is recognized that such sequences will also have utility in diagnosis of the stage of infection and type of infection the pathogen has attained.

The invention also provides polynucleotides that encode a polypeptide that is a mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to a mature polypeptide (when a mature form has more than one polypeptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, may allow protein transport, may lengthen or shorten protein lialf-life or may facilitate manipulation of a protein for assay or production, among other things. As generally is the case in vivo, the additional amino acids may be processed away from a mature protein by cellular enzymes .

For each and every polynucleotide of the invention there is provided a polynucleotide complementary to it. It is prefeπed that these complementary polynucleotides are fully complementary to each polynucleotide with which they are complementary.

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

As will be recognized, the entire polypeptide encoded by an open reading frame is often not required for activity. Accordingly, it has become routine in molecular biology to map the boundaries of the primary structure required for activity with N-teιτninal and C-terminal deletion experiments. These experiments utilize exonuclease digestion or convenient restriction sites to cleave coding nucleic acid sequence. For example, Promega (Madison, WI) sell an Erase-a-base™ system that uses Exonuclease III designed to facilitate analysis of the deletion products (protocol available at www.promega.com). The digested endpoints can be repaired (e.g., by ligation to synthetic linkers) to the extent necessary to preserve an open reading frame In this way, the nucleic acid of SEQ ID NO 1 readily provides contiguous fragments of SEQ ID NO 2 sufficient to provide an activity, such as an enzymatic, binding or antibody-inducing activity Nucleic acid sequences encoding such fragments of SEQ ED NO 2 and vaπants thereof as descπbed herem are within the mvention, as are polypeptides so encoded In sum, a polynucleotide of the mvention may encode a mature protein, a mature protein plus a leader sequence (which may be refeπed to as a preprotem), a precursor of a mature protein having one or more prosequences that are not the leader sequences of a preprotem, or a preproprotein, that is a precursor to a proprotein, having a leader sequence and one or more prosequences, that generally are removed duπng processing steps that produce active and mature forms of the polypeptide Vectors, Host Cells, Expression Systems

The mvention also relates to vectors that compπse a polynucleotide or polynucleotides of the mvention, host cells that are genetically engmeered with vectors of the mvention and the production of polypeptides of the mvention by recombinant techniques Cell-free translation systems can also be employed to produce such proteins using RNAs denved from the DNA constructs of the mvention Recombinant polypeptides of the present mvention may be prepared by processes well known in those skilled in the art from genetically engmeered host cells compπsmg expression systems Accordingly, in a further aspect, the present mvention relates to expression systems that compπse a polynucleotide or polynucleotides of the present mvention, to host cells that are genetically engmeered with such expression systems, and to the production of polypeptides of the mvention 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 descπbed in many standard laboratory manuals, such as Davis, et al , BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, et al , MOLECULAR CLONING A LABORATORY MANUAL, 2nd Ed , Cold Sprmg Harbor Laboratory Press, Cold Sprmg Harbor, NY (1989), such as, calcium phosphate transfection, DEAE- dextran mediated transfection, transvection, micromjection, cationic hpid-mediated transfection, electroporation, transduction scrape loading, ballistic introduction and infection

Representative examples of appropπate hosts include bacteπal cells, such as cells of streptococci, staphylococci, enterococci E coh, streptomyces, cyanobacteπa, Bacillus subtihs, and Streptococcus pneumomae, fungal cells, such as cells of a yeast, Kluveromyces, Saccharomyces, a basidiomycete, Candida albtcans and Aspergtllus, insect cells such as cells oϊDrosophila 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 variety of expression systems can be used to produce the polypeptides of the invention. Such vectors include, among others, chromosomal-, episomal- and virus-derived vectors, for example, vectors derived from bacterial plasmids, from bacteriophage, 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 relroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The expression system constructs may comprise control regions that regulate as well as engender expression. Generally, any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide in a host may be used for expression in this regard. The appropriate DNA sequence may be inserted into the expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al, MOLECULAR CLONING, A LABORATORY MANUAL, (supra).

In recombinant expression systems in eukaryotes, for secretion of a translated protein into the lumen of the endoplasmic reticulurn, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals.

Polypeptides of the invention can be recovered and purified from recombinant cell cultures by well- known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification.

Diagnostic, Prognostic, Serotyping and Mutation Assays This invention is also related to the use of yybQ polynucleotides and polypeptides of the invention for use as diagnostic reagents. Detection of yybQ polynucleotides and/or polypeptides in a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of disease, staging of disease or response of an infectious organism to drugs. Eukaryotes, particularly mammals, and especially humans, particularly those infected or suspected to be infected with an organism comprising the yybQ gene or protein, may be detected at the nucleic acid or amino acid level by a variety of well known techniques as well as by methods provided herein.

Polypeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtained from a putatively infected and/or infected individual's bodily materials. Polynucleotides from any of these sources, particularly DNA or RNN may be used directly for detection or may be amplified enzymatically by using PCR or any other amplification technique pπor to analysis RNN particularly mRΝA, cDΝA and genomic DΝA may also be used in the same ways Us g amplification, characterization of the species and strain of infectious or resident organism present in an individual, may be made by an analysis of the genotype of a selected polynucleotide of the organism Deletions and insertions can be detected by a change in size of the amplified product m compaπson to a genotype of a reference sequence selected from a related organism, preferably a different species of the same genus or a different strain of the same species Point mutations can be identified by hybπdizing amplified DΝA to labeled yybQ polynucleotide sequences Perfectly or significantly matched sequences can be distinguished from imperfectly or more significantly mismatched duplexes by DΝase or RΝase digestion, for DΝA or RΝA respectively, or by detecting differences in melting temperatures or renaturation kmetics Polynucleotide sequence differences may also be detected by alterations m the electrophoretic mobility of polynucleotide fragments in gels as compared to a reference sequence This may be earned out with or without denaturing agents Polynucleotide differences may also be detected by direct DΝA or RΝA sequencing 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 RΝase, VI and SI protection assay or a chemical cleavage method See, for example, Cotton et al , Proc Natl Acad Set , USA, 85 4397-4401 (1985)

In another embodiment, an aπay of ohgonucleotides probes compπsmg yybQ 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 vaπety of questions m molecular genetics mcludmg gene expression, genetic linkage, and genetic vaπabihty (see, for example, Chee et al , Science, 274 610 (1996))

Thus m another aspect, the present mvention relates to a diagnostic kit that comprises (a) a polynucleotide of the present mvention, preferably the nucleotide sequence of SEQ ID NO 1 , or a fragment thereof, (b) a nucleotide sequence complementary to that of (a), (c) a polypeptide of the present mvention, preferably the polypeptide of SEQ ID NO 2 or a fragment thereof, or (d) an antibody to a polypeptide of the present mvention, 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 m diagnosing a disease or susceptibility to a Disease, among others This mvention also relates to the use of polynucleotides of the present mvention as diagnostic reagents Detection of a mutated form of a polynucleotide of the mvention, preferable, SEQ ID NO 1 , that is associated with a disease or pathogenicity will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, a prognosis of a course of disease, a determination of a stage of disease, or a susceptibility to a disease, that results fromunder-expression, over-expression or altered expression of the polynucleotide. Organisms, particularly infectious organisms, carrying mutations in such polynucleotide may be detected at the polynucleotide level by a variety of techniques, such as those described elsewhere herein.

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

Cells from an organism carrying mutations or polymorphisms (allelic variations) in a polynucleotide and/or polypeptide of the invention may also be detected at the polynucleotide or polypeptide level by a variety of techniques, to allow for serotyping, for example. For example, RT-PCR can be used to detect mutations in the RNA. It is particularly prefeπed to use RT-PCR in conjunction with automated detection systems, such as, for example, GeneScan. RNA, cDNA or genomic DNA may also be used for the same purpose, PCR. As an example, PCR primers complementary to a polynucleotide encoding yybQ polypeptide can be used to identify and analyze mutations. The invention 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 yybQ DNA and/or RNA isolated from a sample derived from an individual, such as a bodily material. The primers may be used to amplify a polynucleotide isolated from an infected individual, such that the polynucleotide may then be subject to various techniques for elucidation of the polynucleotide sequence. In this way, mutations in the polynucleotide sequence may be detected and used to diagnose and/or prognose the infection or its stage or course, or to serotype and/or classify the infectious agent.

The invention further provides a process for diagnosing, disease, preferably bacterial infections, more preferably infections caused by Streptococcus pneumoniae, comprising determining from a sample derived from an individual, such as a bodily material, an increased level of expression of polynucleotide having a sequence of Table 1 [SEQ ID NO: 1]. Increased or decreased expression of a yybQ polynucleotide can be measured using any on of the methods well known in the art for the quantitation of polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNase protection, Northern blotting, spectrometry and other hybridization methods.

In addition, a diagnostic assay in accordance with the invention for detecting over- expression of yybQ polypeptide compared to normal control tissue samples may be used to detect the presence of an infection, for example. Assay techniques that can be used to determine levels of a yybQ polypeptide, in a sample derived from a host, such as a bodily material, are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis, antibody sandwich assays, antibody detection and ELISA assays. Antagonists and Agonists - Assays and Molecules

Polypeptides and polynucleotides of the invention may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures. These substrates and Ugands may be natural substrates and Ugands or may be structural or functional mimetics. See, e.g., CoUgan et α/., Current Protocols in Immunology 1 (2) : Chapter 5 (1991).

Polypeptides and polynucleotides of the present invention are responsible for many biological functions, including many disease states, in particular the Diseases herein mentioned. It is therefore desirable to devise screening methods to identify compounds that agonize (e.g. , stimulate) or that antagonize (e.g. ,inhibit) the function of the polypeptide or polynucleotide. Accordingly, in a further aspect, the present invention provides for a method of screening compounds to identify those that agonize or that antagonize the function of a polypeptide or polynucleotide of the invention, as weU as related polypeptides and polynucleotides. In general, agonists or antagonists (e.g. , inhibitors) may be employed for therapeutic and prophylactic purposes for such Diseases as herein mentioned. Compounds may be identified from a variety of sources, for example, cells, cell-free preparations, chemical Ubraries, and natural product mixtures. Such agonists and antagonists so-identified may be natural or modified substrates, Ugands, receptors, enzymes, etc., as the case may be, of yybQ 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 protein of the polypeptide by means of a label directly or indirectly associated with the candidate compound. Alternatively, the screening method may involve competition with a labeled competitor. Further, these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide or polynucleotide, using detection systems appropriate to the cells comprising the polypeptide or polynucleotide. Inhibitors of activation are generally assayed 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 in screening methods for inverse agonists, in the absence of an agonist or antagonist, by testing whether the candidate compound results in inhibition of activation of the polypeptide or polynucleotide, as the case may be. Further, the screening methods may simply comprise the steps of mixing a candidate compound with a solution comprising a polypeptide or polynucleotide of the present mvention, to form a mixture, measuring yybQ polypeptide and/or polynucleotide activity in the mixture, and comparing the yybQ polypeptide and/or polynucleotide activity of the mixture to a standard. Fusion proteins, such as those made from Fc portion and yybQ polypeptide, as herem descnbed, can also be used for high-throughput screening assays to identify antagomsts of the polypeptide of the present mvention, as well as of phylogenetically and and/or functionally related polypeptides (see D Bennett et al , ] Mol Recognition, 8 52-58 (1995), and K Johanson et al , ] Biol Chem, 270(16) 9459-9471 (1995)) The polynucleotides, polypeptides and antibodies that bmd to and/or mteract with a polypeptide of the present mvention may also be used to configure 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 using monoclonal and polyclonal antibodies by standard methods known m the art This can be used to discover agents that may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues

The mvention also provides a method of screening compounds to identify those that enhance (agonist) or block (antagonist) the action of yybQ polypeptides or polynucleotides, particularly those compounds that are bacteπstatic and/or bacteπcidal The method of screening may volve high-throughput techmques For example, to screen for agomsts or antagomsts, a synthetic reaction mix, a ceUular compartment, such as a membrane, ceU envelope or ceU wall, or a preparation of any thereof, compπsmg yybQ polypeptide and a labeled substrate or Ugand of such polypeptide is incubated m the absence or the presence of a candidate molecule that may be a yybQ agonist or antagonist The abiUty of the candidate molecule to agonize or antagonize the yybQ polypeptide is reflected in decreased binding of the labeled Ugand or decreased production of product from such substrate Molecules that bmd gratuitously, / e , without inducing the effects of yybQ polypeptide are most likely to be good antagomsts Molecules that bmd weU and, as the case may be, mcrease the rate of product production from substrate, mcrease signal transduction, or mcrease chemical channel activity are agomsts Detection of the rate or level of, as the case may be, production of product from substrate, signal transduction, or chemical channel activity may be enhanced by us g a reporter system Reporter systems that may be useful in this regard mclude but are not limited to coloπmetπc, labeled substrate converted mto product, a reporter gene that is responsive to changes in yybQ polynucleotide or polypeptide activity, and binding assays known in the art

Polypeptides of the mvention may be used to identify membrane bound or soluble receptors, if any, for such polypeptide, through standard receptor bindmg techmques known in the art These techniques mclude, but are not limited to, hgand bmdmg and crosslinking assays m which the polypeptide is labeled with a radioactive isotope (for instance, ^^1), chemically modified (for instance, biotinylated), or fused to a peptide sequence suitable for detection or puπfication, and mcubated with a source of the putative receptor (e g , cells, cell membranes, cell supernatants, tissue extracts, bodily mateπals) Other methods mclude biophysical techmques such as surface plasmon resonance and spectroscopy These screening methods may also be used to identify agomsts and antagomsts of the polypeptide that compete with the bmdmg of the polypeptide to its receptor(s), if any Standard methods for conducting such assays are well understood m the art

The fluorescence polaπzation value for a fluorescently-tagged molecule depends on the rotational correlation time or tumbling rate Protem complexes, such as formed by yybQ polypeptide associatmg with another yybQ polypeptide or other polypeptide, labeled to comprise a fluorescently-labeled molecule will have higher polaπzation values than a fluorescently labeled monomenc protem It is prefeπed that this method be used to characterize small molecules that disrupt polypeptide complexes Fluorescence energy transfer may also be used characteπze small molecules that interfere with the formation of yybQ polypeptide d mers, tπmers, tetramers or higher order structures, or structures formed by yybQ polypeptide bound to another polypeptide YybQ polypeptide can be labeled with both a donor and acceptor fluorophore Upon mixing of the two labeled species and excitation of the donor fluorophore, fluorescence energy transfer can be detected by observing fluorescence of the acceptor Compounds that block dimeπzation will inhibit fluorescence energy transfer

Surface plasmon resonance can be used to momtor the effect of small molecules on yybQ polypeptide self-association as well as an association of yybQ polypeptide and another polypeptide or small molecule YybQ polypeptide can be coupled to a sensor chip at low site density such that covalently bound molecules will be monomeric Solution protem can then passed over the yybQ polypeptide -coated surface and specific bmdmg can be detected m real-time by momtormg the change m resonance angle caused by a change m local refractive mdex This technique can be used to characterize the effect of small molecules on kinetic rates and equihbnum bind g constants for yybQ polypeptide self-association as well as an association of yybQ polypeptide and another polypeptide or small molecule

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

In other embodiments of the mvention there are provided methods for identifying compounds that bmd to or otherwise interact with and inhibit or activate an activity or expression of a polypeptide and/or polynucleotide of the invention comprising: contacting a polypeptide and/or polynucleotide of the invention with a compound to be screened under conditions to permit binding to or other interaction between the compound and the polypeptide and/or polynucleotide to assess the binding to or other interaction with the compound, such binding or interaction preferably being associated with a second component capable of providing a detectable signal in response to the binding or interaction of the polypeptide and/or polynucleotide with the compound; and determining whether the compound binds to or otherwise interacts with and activates or inhibits an activity or expression of the polypeptide and/or polynucleotide by detecting the presence or absence of a signal generated from the binding or interaction of the compound with the polypeptide and/or polynucleotide. Another example of an assay for yybQ agonists is a competitive assay that combines yybQ and a potential agonist with yybQ-binding molecules, recombinant yybQ binding molecules, natural substrates or Ugands, or substrate or Ugand mimetics, under appropriate conditions for a competitive inhibition assay. YybQ can be labeled, such as by radioactivity or a colorimetric compound, such that the number of yybQ molecules bound to a binding molecule or converted to product can be deteπriined accurately to assess the effectiveness of the potential antagonist.

It will be readily appreciated by the skilled artisan that a polypeptide and/or polynucleotide of the present invention may also be used in a method for the structure-based design of an agonist or antagonist of the polypeptide and/or polynucleotide, by: (a) deteimining in the first instance the three- dimensional structure of the polypeptide and/or polynucleotide, or complexes thereof; (b) deducing the three-dimensional structure for the likely reactive site(s), binding site(s) or motif(s) of an agonist or antagonist; (c) synthesizing candidate compounds that are predicted to bind to or react with the deduced binding site(s), reactive site(s), and/or motif(s); and (d) testing whether the candidate compounds are indeed agomsts or antagonists.

It will be further appreciated that this will normally be an iterative process, and this iterative process may be performed using automated and computer-controlled steps.

In a further aspect, the present invention provides methods of treating abnormal conditions such as, for instance, a Disease, related to either an excess of, an under-expression of, an elevated activity of, or a decreased activity of yybQ 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 comprises administering to an individual in need thereof an inhibitor compound (antagonist) as herein described, optionally in combination with a pharmaceutically acceptable carrier, in an amount effective to inhibit the function and/or expression of the polypeptide and/or polynucleotide, such as, for example, by blocking the binding of Ugands, substrates, receptors, enzymes, etc., or by inhibiting a second signal, and thereby aUeviating 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 mclude fragments of the yybQ polypeptide and/or polypeptide

In still another approach, expression of the gene encodmg endogenous yybQ polypeptide can be inhibited usmg expression blocking techniques This blocking may be targeted agamst any step in gene expression, but is preferably targeted agamst transcnption 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, JNeurochem (1991) 56 560 in Ohgodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)) Alternatively, oligonucleotides that form tπple helices with the gene can be supplied (see, for example, Lee et al , Nucleic Acids Res (1979) 6 3073, Cooney et al , Science (1988) 241 456, Dervan et α/ , Science (1991) 251 1360) These ohgomers can be administered per se or the relevant ohgomers can be expressed in vivo

Each of the polynucleotide sequences provided herem may be used m the discovery and development of antibacteπal compounds The encoded protem, upon expression, can be used as a target for the screening of antibacterial drugs Additionally, the polynucleotide sequences encoding the ammo terminal regions of the encoded protem or Shine-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the codmg sequence of mterest The mvention also provides the use of the polypeptide, polynucleotide, agomst or antagonist of the mvention to interfere with the initial physical mteraction between a pathogen or pathogens and a eukaryotic, preferably mammalian, host responsible for sequelae of infection In particular, the molecules of the mvention may be used m the prevention of adhesion of bacteria, in particular gram positive and/or gram negative bacteπa, to eukaryotic, preferably mammalian, extracellular matrix protems on in-dwelling devices or to extracellular matrix protems in wounds, to block bacterial adhesion between eukaryotic, preferably mammalian, extracellular matπx protems and bacterial yybQ protems that mediate tissue damage and/or, to block the normal progression of pathogenesis m infections initiated other than by the implantation of m-dwellmg devices or by other surgical techmques In accordance with yet another aspect of the mvention, there are provided yybQ agomsts and antagomsts, preferably bacteπstatic or bacteπcidal 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") bactena infect the stomachs of over one-third of the world's population causmg stomach cancer, ulcers, and gastntis (International Agency for Research on Cancer (1994) Schistosomes, Liver Flukes and Hehcobacter Pylori (International Agency for Research on Cancer, Lyon, France, http //www nice ch/ecp/ecp2904 htm) Moreover, the International Agency for Research on Cancer recently recognized a cause-and-effect relationship between H pylori and gastric adenocarcmoma, classifying the bacterium as a Group I (definite) carcinogen Prefeπed antimicrobial compounds of the mvention (agomsts and antagomsts of yybQ polypeptides and/or polynucleotides) found usmg screens provided by the mvention, or known in the art, particularly narrow-spectrum antibiotics, should be useful m the treatment of H pylori infection Such treatment should decrease the advent of H pylori -mduced cancers, such as gastrointestinal carcmoma Such treatment should also prevent, inhibit and/or cure gastric ulcers and gastritis

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

GLOSSARY The foUowing definitions are provided to facilitate understanding of certam terms used frequently herem

"Bodily mateπal(s) means any mateπal deπved from an individual or from an organism infecting, infesting or inhabiting an dividual, mcludmg but not limited to, cells, tissues and waste, such as, bone, blood, serum, cerebrospinal fluid, semen, sahva, muscle, cartilage, organ tissue, skm, 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, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid

"Host cell(s)" is a cell that has been mtroduced (e g , transformed or transfected) or is capable of introduction (e g , transformation or transfection) by an exogenous polynucleotide sequence

"Identity," as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determined by comparing the sequences In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences "Identity" can be readily calculated by known methods, mcludmg but not limited to those descπbed in (Computational Molecular Biology, Lesk, A M , ed , Oxford University Press, New York, 1988, Biocomputing Informatics and Genome Projects, Smith, D W , ed , Academic Press, New York, 1993, Computer Analysis of Sequence Data, Part I, Gπffin, 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, Gnbskov, M and Devereux, J , eds , M Stockton Press, New York, 1991, and Canllo, H , and Lipman, D , SIAM J Applied Math , 48 1073 (1988) Methods to determme identity are designed to give the largest match between the sequences tested Moreover, methods to determine identity are codified m publicly available computer programs Computer program methods to determme identity between two sequences mclude, 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 NTH Bethesda, MD 20894, Altschul, S , et al , J Mol Biol 215 403-410 (1990) The well known Smith Waterman algoπthm may also be used to determine identity

Parameters for polypeptide sequence comparison mclude the following Algonthm Needleman and Wunsch, J Mol Biol 48 443-453 (1970)

Companson 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 compaπsons (along with no penalty for end gaps) Parameters for polynucleotide comparison mclude the following Algorithm Needleman and

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

A preferred meaning for "identity" for polynucleotides and polypeptides, as the case may be, are provided m (1) and (2) below (1) Polynucleotide embodiments further mclude an isolated polynucleotide comprismg a polynucleotide sequence having at least a 95, 97 or 100% identity to the reference sequence of SEQ ID NO 1, wherem said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO 1 or may mclude up to a certam mteger number of nucleotide alterations as compared to the reference sequence, wherem said alterations are selected from the group consistmg of at least one nucleotide deletion, substitution, mcludmg transition and transversion, or insertion, and wherem said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, mterspersed either individually among the nucleotides in the reference sequence or m one or more contiguous groups within the reference sequence, and wherem said number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO 1 by the mteger defining the percent identity divided by 100 and then subtracting that product from said total number of nucleotides m SEQ ID NO 1, or

n_n < x_n (^χ _n • y)>

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

(2) Polypeptide embodiments further mclude an isolated polypeptide comprismg a polypeptide havmg at least a 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO 2, wherem said polypeptide sequence may be identical to the reference sequence of SEQ ID NO 2 or may mclude up to a certam mteger number of ammo acid alterations as compared to the reference sequence, wherem said alterations are selected from the group consistmg of at least one amino acid deletion, substitution, mcludmg conservative and non-conservative substitution, or insertion, and wherem said alterations may occur at the ammo- or carboxy-termmal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either mdividuall} among the ammo acids m the reference sequence or in one or more contiguous groups within the reference sequence, and wherem said number of ammo acid alterations is determined by multiplying the total number of ammo acids m SEQ ID NO 2 by the mteger defmmg the percent identity divided by 100 and then subtractmg that product from said total number of ammo acids in SEQ ID NO 2, or ⁿa ≤ ^xa " (^xa • ),

wherem n_a is the number of ammo acid alterations, x_a is the total number of ammo acids in SEQ ID NO 2, y is 0 95 for 95%, 0 97 for 97% or 1 00 for 100%, and • is the symbol for the multiplication operator, and wherem any non-mteger product of x_a and y is rounded down to the nearest integer prior to subtracting it from x_a

"Indιvιdual(s)" means a multiceUular 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, i e , if it occurs in nature, it has been changed or removed from its onginal environment, or both For example, a polynucleotide or a polypeptide naturally present m a Uvmg 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 mtroduced to an organism by transformation, genetic manipulation or by any other recombinant method is "isolated" even if it is still present m said organism, which organism may be living or non-Uving

"Organιsm(s)" means a (l) prokaryote, mcludmg but not limited to, a member of the genus Streptococcus, Staphylococcus, Bordetella, Corynebactenum, Mycobactenum, Ne ssena, Haemophilus, Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersima, Fancisella, Pasturella, Moraxella, Acinetobacter, Erysipelothnx, Branhamella, Actinobacillus, Streptobacillus, Listena, Calymmatobactenum, Brucella, Bacillus, Clostndium, Treponema, Eschenchia, Salmonella, Kleibsiella, Vibno, Proteus, Erwinia, Borrelia, Leptospira, Spinllum, Campylobacter, Shigella Legionella Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borrelia and Mycoplasma, and further mcludmg, but not limited to, a member of the species or group, Group A Streptococcus, Group B Streptococcus, Group C Streptococcus, Group D Streptococcus, Group G Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus faecahs, Streptococcus faecium Streptococcus durans, Neissena gonorrheae, Neissena memngitidis, Staphylococcus aureus Staphylococcus epidermidis, Corynebactenum dipthenae, Gardnerella vagina s Mycobactenum tuberculosis, Mycobactenum bovis, Mycobactenum ulcerans, Mycobactenum leprae, Acttnomyctes israelu, Listena monocytogenes, Bordetella pertusis, Bordatella parapertusis, Bordetella bronchiseptica Eschenchia coli, Shigella dysentenae, Haemophilus influenzae, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonella typhi, Citrobacter freundu, Proteus mirabihs, Proteus vulgans, Yersima pestis, Kleibsiella pneumoniae, Serratia marcessens Serratia hquefaciens, Vibno cholera, Shigella dysenteni, Shigella flexnen, Pseudomonas aeruginosa, Franscisella tularensis, Brucella abortis, Bacillus anthracis, Bacillus cereus, Clostndium perfhngens, Clostndium tetani, Clostndium botuhnum, Treponema palhdum, Rickettsia nckettsn and Chlamydia trachomitis, (n) an archaeon, mcludmg but not limited to Archaebacter, and (in) a uniceUular 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)" generaUy refers to any polyπbonucleotide or polydeoxyπbonucleotide, that may be unmodified RNA or DNA or modified RNA or DNA "Polynucleotide(s)" mclude, without limitation, single- and double-stranded DNN DΝA that is a mixture of smgle- and double-stranded regions or single-, double- and tπple-stranded regions, smgle- and double-stranded RΝN and RΝA that is mixture of single- and double-stranded regions, hybnd molecules compnsing DΝA and RΝA that may be single-stranded or, more typically, double-stranded, or ti ple-stranded regions, or a mixture of smgle- and double-stranded regions In addition, "polynucleotide" as used herem refers to tnple-stranded regions compπsmg RΝA or DΝA or both RΝA and DΝA The strands m such regions may be from the same molecule or from different molecules The regions may mclude aU of one or more of the molecules, but more typically mvolve only a region of some of the molecules One of the molecules of a tnple-heUcal region often is an oUgonucleotide As used herein, the term "polynucleotide(s)" also mcludes DΝAs or RΝAs as descnbed above that compπse one or more modified bases Thus, DΝAs or RΝAs with backbones modified for stabiUty or for other reasons are "polynucleotide(s)" as that term is intended herem Moreover, DΝAs or RΝAs compπsmg 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 variety of modifications have been made to DΝA and RΝA that serve many useful purposes known to those of skill in the art The term "polynucleotide(s)" as it is employed herem embraces such chemically, enzymatically or metaboUcaUy modified forms of polynucleotides, as weU as the chemical forms of DΝA and RΝA charactenstic of viruses and ceUs, mcludmg, for example, simple and complex ceUs "Polynucleotide(s)" also embraces short polynucleotides often refeπed to as olιgonucleotιde(s)

"Polypeptide(s)" refers to any peptide or protem compπsmg two or more ammo acids joined to each other by peptide bonds or modified peptide bonds "Polypeptιde(s)" refers to both short chains, commonly refeπed to as peptides, ohgopeptides and ohgomers and to longer chains generally refeπed to as proteins Polypeptides may compπse ammo acids other than the 20 gene encoded ammo acids "Polypeptide(s)" mclude those modified either by natural processes, such as processmg and other post-translational modifications, but also by chemical modification techmques Such modifications are well descnbed in basic texts and m more detailed monographs, as weU as m a voluminous research Uterature, and they are well known to those of skύl m the art It wiU be appreciated that the same type of modification may be present m the same or varying degree at several sites m a given polypeptide Also, a given polypeptide may compnse many types of modifications Modifications can occur anywhere m a polypeptide, mcludmg the peptide backbone, the ammo acid side-chains, and the ammo or carboxyl termini Modifications mclude, for example, acetylation, acylation, ADP-nbosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide denvative, covalent attachment of a hpid or Upid denvative, covalent attachment of phosphotidyhnositol, cross-hnkmg, cycUzation, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteme, formation of pyroglutamate, formylation, gamma-carboxylation, GPI anchor formation, hydroxylation, lodination, methylation, myπstoylation, oxidation, proteolytic processmg, phosphorylation, prenylation, racemization, glycosylation, Upid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-nbosylation, selenoylation, sulfation, transfer-RNA mediated addition of ammo acids to proteins, such as argrnylation, and ubiquitination See, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed , T E Creighton, W H Freeman and Company, New York (1993) and Wold, F , Posttranslational Protem Modifications Perspectives and Prospects, pgs 1-12 m POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B C Johnson, Ed , Academic Press, New York (1983), Serfter et al , Meth Enzymol 182 626-646 (1990) and Rattan et al , Protein Synthesis Posttranslational Modifications and Aging, Ana N Y Acad Sci 663 48- 62 (1992) Polypeptides may be branched or cyclic, with or without branching Cychc, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as weU "Recombinant 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

'Naπant(s)" as the term is used herem, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties A typical variant of a polynucleotide differs m nucleotide sequence from another, reference polynucleotide Changes in the nucleotide sequence of the vaπant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result in ammo acid substitutions, additions, deletions, fusion protems and truncations m the polypeptide encoded by the reference sequence, as discussed below A typical vanant of a polypeptide differs in ammo acid sequence from another, reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the vaπant are closely similar overall and, m many regions, identical A vaπant and reference polypeptide may differ m ammo acid sequence by one or more substitutions, additions, deletions m any combmation A substituted or inserted ammo acid residue may or may not be one encoded by the genetic code The present mvention also mcludes mclude vanants of each of the polypeptides of the mvention, that is polypeptides that vary from the referents by conservative ammo acid substitutions, whereby a residue is substituted by another with like charactenstics Typical such substitutions are among Ala, Val, Leu and lie, 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 prefeπed are vaπants m which several, 5-10, 1-5, 1-3, 1-2 or 1 ammo acids are substituted, deleted, or added in any combination A vanant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic vanant, or it may be a vanant that is not known to occur naturally Non- naturally occurring vanants of polynucleotides and polypeptides may be made by mutagenesis techmques, by direct synthesis, and by other recombinant methods known to skilled artisans

EXAMPLES

The examples below are earned out usmg standard techmques, that are well known and routme to those of skill in the art, except where otherwise descπbed m detail The examples are lUustrative, but do not limit the mvention

Example 1 Strain selection, Library Production and Sequencing

The polynucleotide having a DNA sequence given m Table 1 [SEQ ID NO 1] was obtained from a library of clones of chromosomal DNA of Streptococcus pneumoniae in E coll The sequencmg data from two or more clones compπsmg overlappmg Streptococcus pneumoniae DNAs was used to construct the contiguous DNA sequence m 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 pneumoniae 0100993 according to standard procedures and size-fractionated by either of two methods Method 1

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

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combmation of restriction enzymes appropriate to generate a series of fragments for cloning mto library vectors (e g , Rsal, Pall, Alul, Bshl235I), and such fragments are size-fractionated accordmg to standard procedures EcoRI linkers are hgated to the DNA and the fragments then hgated mto the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E coh mfected with the packaged library The library is amplified by standard procedures

Example 2 yybQ Characterization

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 processmg to give stable preparations and high yields of bacterial RNA are followed by the use of hybπdisation to a radiolabelled oligonucleotide specific to Streptococcus pneumoniae 16S RNA on Northern blots The RNAase free, DNAase free, DNA and protem free preparations of RNA obtamed are suitable for Reverse Transcription PCR (RT-PCR) usmg unique primer pairs designed from the sequence of each gene of Streptococcus pneumoniae 0100993 Using this procedure it was possible to demonstrate that yybQ is transcibed during infection.

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

Streptococcus pneumoniae 0100993 is grown either on TSN5%horse blood plates or m AGCH medium overnight, 37°C, 5%C0₂ Bacteria are then collected and resuspended m phosphate-buffered salme to an Noo of approximately 0 4 Mice a e anaesthetized with isofluorane and 50ml of bactenal suspension (approximately 2 x 10⁵ bacteria) is administered intianasally using a pipetman Mice are allowed to recover and have food and water ad libitum After 48 hours, the mice are euthanized by carbon dioxide overdose, and lungs are aseptically removed and snap-frozen m liquid nitrogen

b) Isolation of Streptococcus pneumoniae 0100993 RNA from infected tissue samples Infected tissue samples, m 2-ml cryo-strorage tubes, are removed from -80°C storage into a dry ice ethanol bath In a microbiological safety cabinet the samples are disrupted up to eight at a time while the remaining samples are kept frozen m the dry ice ethanol bath To disrupt the bacteria within the tissue sample, 50-100 mg of the tissue is t ansfered to a FastRNA tube containing a silica/ceramic matπx (BIO 101) Immediately, 1 ml of extraction reagents (FastRNA reagents, BIO101) are added to give a sample to reagent volume ratio of approximately 1 to 20 The tubes are shaken m a reciprocatmg shaker (FastPrep FP120, BIO101) at 6000 rpm 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 in this isopropanol solution at -80°C if necessary The RNA is pelleted (12,000g for 10 mm ), washed with 75% ethanol (v/v m DEPC-treated water), air-dπed for 5-10 mm, and resuspended m 0 1 ml of DEPC-treated water, followed by 5-10 minutes at 55 °C Finally, afte at least 1 minute on ice, 200 units of Rnasin (Promega) is added

RNA preparations are stored at -80 °C 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°C

Quality of the RNA isolated is assessed by running samples on 1% agarose gels 1 x TBE gels stained with ethidmm 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 pneumoniae The size of the hybridising band is compared to that of control RNA isolated from in vitro grown Streptococcus pneumoniae 0100993 in the Northern blot Correct sized bacterial 16S rRNA bands can be detected m total RNA samples which show degradation of the mammalian RNA when visualised on TBE gels

c) The removal of DNA from Streptococcus pneumoniae-derw^' ed RNA

DNA was removed from 50 microgram samples of RNA by a 30 minute treatment at 37°C with 20 umts of RNAase-free DNAasel (GenHunter) in the buffer supplied in a final volume of 57 microhters

The DNAase was mactivated and removed by treatment with TRIzol LS Reagent (Gibco BRL, Life Technologies) according to the manufacturers protocol

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

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

3 microgram samples of DNAase treated RNA are reverse transcnbed usmg a SuperScπpt Preamplification System for First Strand cDNA Synthesis kit (Gibco BRL, Life Technologies) accordmg to the manufacturers instructions 150 nanogram of random hexamers is used to prune each reaction Controls without the addition of SuperScπptll reverse transcπptase are also run Both +/-RT samples are treated with RNaseH before proceedmg to the PCR reaction

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

PCR reactions are run on a Perk n Elmer GeneAmp PCR System 9600 as follows 2 mmutes at 94 °C, then 50 cycles of 30 seconds each at 94 °C, 50 °C and 72 °C followed by 7 mmutes at 72 °C and then a hold temperature of 20 °C (the number of cycles is optimally 30-50 to determine the appearance or lack of a PCR product and optimally 8-30 cycles if an estimation of the starting quantity of cDNA from the RT reaction is to be made), 10 microhtre ahquots are then run out on 1% 1 x TBE gels stained with ethidium bromide, with PCR product, if present, sizes estimated by comparison to a 100 bp DNA Ladder (Gibco BRL, Life Technologies) Alternatively if the PCR products are convemently 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 usmg a suitable gel scanning system (e g ABI Prism 377 Sequencer usmg GeneScan™ software as supplied by Perkrn Elmer) RT/PCR controls may mclude +/- reverse transcnptase reactions, 16S rRNA primers or

DNA specific primer pairs designed to produce PCR products from non-transcribed Streptococcus pneumoniae 0100993 genomic sequences

To test the efficiency of the primer pairs they are used m DNA PCR with Streptococcus pneumoniae 0100993 total DNA PCR reactions are set up and run as descπbed above using approx 1 microgram of DNA m 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 umnformative Of those which give the correct size product with DNA PCR two classes are distinguished m RT/PCR 1 Genes which are not transcribed in vivo reproducibly fail to give a product m RT/PCR, and 2 Genes which are transcribed in vivo reproducibly give the coπect size product m RT/PCR and show a stronger signal in the +RT samples than the signal (if at all present) m -RT controls

Example 3 Demonstration of gene essentiality to bacterial viability An allelic replacement cassette was generated using PCR technology. The cassette consisted of a pair of 500bp chromosomal DNA fragments flanking an erythromycin resistance gene. The chromosomal DNA sequences are the 500bp preceding and following the DNA sequence encoding the NDP contained in SEQ ID NO.l The allelic replacement cassette was introduced into S. pneumoniae R6 by transformation. Competent cells were prepared according to published protocols. DNA was introduced into the cells by incubation of ng quantities of allelic 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 erythromycin resistance gene. Cells were plated in agar containing lug erythromycin 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 allelic replacement are obtained. If no transformants are obtained in three separate transformation experiments as was the case for this gene yybQ, then the gene is considered as being essential in vitro.

Claims

What is claimed is:

1 An isolated polypeptide selected from the group consistmg of

(1) an isolated polypeptide compπsmg an ammo 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 compπsmg 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 consistmg of

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

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

NO 2,

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

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

ID NO 2,

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

(vt) an isolated polynucleotide of at least 30 nucleotides m length obtainable by screening an appropπate library under stringent hybπdization conditions with a p obe having the sequence of

SEQ ID NO 1 o a fragment thereof of of at least 30 nucleotides in length,

(vn) an isolated polynucleotide encoding a mature polypeptide expressed by the yybQ gene compπsed in the Streptococcus pneumoniae, and

(vm) a polynucleotide sequence complementary to said isolated polynucleotide of (I), (u), (in), (iv) (v), (vi) or (vn)

3 A method for the treatment of an individual (i) in need of enhanced activity or expression of or immunological response to the polypeptide of claim 1 comprising the step of: admimsterrng to the individual a the apeutically effective amount of an antagonist to said polypeptide; or

(ii) having need to inhibit activity or expression of the polypeptide of claim 1 comprising:

(a) admimsterrng to the individual a therapeutically effective amount of an antagonist to said polypeptide; or

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

(c) administering to the individual a theiapeutically effective amount of a polypeptide that competes with said polypeptide for its Ugand, substrate, or receptor; or

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

4. A process for diagnosing or prognosing a disease or a susceptibility to a disease in an individual related to expression or activity of the polypeptide of claim 1 in 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 amino acid sequence selected from the group having at least 95 % identity to the amino acid sequence of SEQ ID NO:2 ove the entire length of SEQ ID N0.2;

(ii) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:2;

(iii) an isolated polypeptide that is the amino acid sequence of SEQ ID NO:2, and

(iv) a polypeptide that is encoded by a recombinant polynucleotide comprising the polynucleotide sequence of SEQ ID NO:l, comprising the step of culturing a host cell under conditions sufficient for the production of the polypeptide.

6 A process for producmg a host cell compπsing an expression system or a membrane thereof expressmg a polypeptide selected from the group consistmg of

(1) an isolated polypeptide compnsmg an ammo acid sequence selected from the group having at least 95% identity to the ammo acid sequence of SEQ ID NO 2 over the entire length of SEQ ID NO 2, (n) an isolated polypeptide compnsmg 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 compnsmg the polynucleotide sequence of SEQ ID NO 1, said process compnsmg the step of transforming or transferring a cell with an expression system compnsmg a polynucleotide capable of producmg said polypeptide of (l), (u), (m) or (iv) when said expression system is present m a compatible host cell such the host cell, under appropriate culture conditions, produces said polypeptide of (I), (n), (in) or (iv)

7 A host cell or a membrane expressmg a polypeptide selected from the group consistmg of (l) an isolated polypeptide compπsmg 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 compnsmg the ammo acid sequence of SEQ ID NO 2, (m) an isolated polypeptide that is the ammo acid sequence of SEQ ID NO 2, and (iv) a polypeptide that is encoded by a recombinant polynucleotide comprising the polynucleotide sequence of SEQ ID NO 1

8 An antibody rmmunospecific for the polypeptide of claim 1

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

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

(b) measuring the bmding of a candidate compound to the polypeptide (or to the cells or membranes bearing the polypeptide) or a fusion protem thereof m the presence of a labeled competitor, (c) testing whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems appropriate to the cells or cell membranes bearing the polypeptide;

(d) mixing a candidate compound with a solution comprising a polypeptide of claim 1, to form a mixture, 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.