EP0951296A1 - NOVEL FtsL - Google Patents

Abstract

The invention provides FtsL polypeptides and DNA (RNA) encoding FtsL polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing FtsL polypeptides to screen for antibacterial compounds.

Description

NOVEL FtsL

RELATED APPLICATIONS

This application claims benefit of GB application number 9616312.6, filed August 2,

1996 and U.S. Provisional Application Serial Number 60/027,301, filed October 1, 1996.

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, in these and in other regards, the invention relates to novel polynucleondes and polypeptides of the penicillin binding protein family, hereinafter referred to as "FtsL".

BACKGROUND OF THE INVENTION

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

The frequency of Staphylococcus aureus infections has risen dramatically in the past 20 years. This has been attributed to the emergence of multiply antibiotic resistant strains and an increasing population of people with weakened immune systems. It is no longer uncommon to isolate Staphylococcus aureus strains which are resistant to some or all of the standard antibiotics. This has created a demand for both new anti-microbial agents and diagnostic tests for this organism.

FtsL is a membrane bound protein involved in bactenal cell division. FtsL is essential for cell growth and division. An ftsL null mutation results in inhibition of cell division, the formation of long, non-septate filaments, and ultimately cessation of growth and lysis (Guzman, L-M., Barondess, J. J., and Beckwith, J. (1992) J. Bacteπol 174 : 7716-7728)

In Escheπchia coh the ftsL gene is located immediately upstream of ftsl which codes for

Penicilhn-bmdmg protein 3, a membrane protein specifically required during septation (Ishmo, F.,

& Matsuhashi, M. (1981) Biochem. Biophys. Res. Commun. 101 : 905-911). FtsL is also a cytoplasmic membrane protein with a cytoplasmic N-terminus, a membrane-spanning segment, and a penplasmic carboxy terminus.

Although there are many examples of cell division proteins in E.coli their precise functions are mostly unknown. However it is likely that membrane proteins involved in cell division will have a major role in the regulation of cell division and in septum initiation. FtsL homologues have been reported in Haemophilus lnfiuenzae (Fleischmann, R. et al

(1995) Science 269 ^• 496-512) and Bacillus subti s (Daniel, R A., Williams, A. M., Erπngton, J. (1995) Genbank Accession Z68230).

Clearly, there is a need for factors, such as the novel compounds of the invention, that have a present benefit of being useful to screen compounds for antibiotic activity. Such factors are also useful to determine their role m pathogenesis of infection, dysfunction and disease. There is also a need for identification and characterization of such factors and their antagonists and agonists which can play a role in preventing, ameliorating or correcting infections, dysfunctions or diseases.

The polypeptides of the invention have amino acid sequence homology to a known Bacillus subtihs FtsL protein.

SUMMARY OF THE INVENTION

It is an object of the invention to provide polypeptides that have been identified as novel FtsL polypeptides by homology between the ammo acid sequence set out in Table 1 [SEQ LD NO.

z 2] and a known ammo acid sequence or sequences of other proteins such as Bacillus subtihs FtsL protein

It is a further object of the invention to provide polynucleotides that encode FtsL polypepndes, particularly polynucleotides that encode the polypeptide herein designated FtsL In a particularly preferred embodiment of the invention the polynucleotide compπses a region encodmg FtsL polypeptides comprising the sequence set out in Table 1 [SEQ LD NO 1] which includes a full length gene, or a vanant thereof.

In another particularly preferred embodiment of the invention there is a novel FtsL protein from Staphylococcus aureus comprising the amino acid sequence of Table 1 [SEQ ID NO.2], or a vanant thereof

In accordance with another aspect of the invention there is provided an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Staphylococcus aureus WCUH 29 strain contained in the deposited strain

A further aspect of the invention there are provided isolated nucleic acid molecules encoding FtsL, particularly Staphylococcus aureus FtsL, including mRNAs, cDNAs, genomic

DNAs Further embodiments of the invention include biologically, diagnostically, prophylactically, clinically or therapeutically useful vaπants thereof, and compositions compπsmg the same.

In accordance with another aspect of the invention, there is provided the use of a polynucleotide of the invention for therapeutic or prophylactic purposes, in particular genetic immunization Among the particularly preferred embodiments of the invention are naturally occurring allehc vanants of FtsL and polypeptides encoded thereby

Another aspect of the invention there are provided novel polypeptides of Staphylococcus aureus referred to herein as FtsL as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful vanants thereof, and composmons compπsing the same

Among the particularly preferred embodiments of the invention are vaπants of FtsL polypeptide encoded by naturally occurring alleles of the FtsL gene.

In a preferred embodiment of the invention there are provided methods for producing the aforementioned FtsL polypeptides. In accordance with yet another aspect of the invention, there are provided inhibitors to such polypeptides, useful as antibactenal agents, including, for example, antibodies In accordance with certain preferred embodiments of the invention, there are provided products, compositions and methods for assessing FtsL expression, treating disease, for example, disease, such as, infections of the upper respiratory tract (e g., otitis media, bactenal tracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g., empyema, lung abscess), cardiac (e.g , infective endocarditis), gastrointestinal (e.g , secretory diarrhoea, splenic absces, retropeπtoneal abscess), CNS (e.g., cerebral abscess), eye (e.g., blephaπtis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital cellu tis, darcryocyshtis), kidney and unnary tract (e.g., epididymitis, intrarenal and peπnephπc absces, toxic shock syndrome), skin (e.g., impetigo, follicu tis, cutaneous abscesses, celluhtis, wound infection, bactenal myositis) bone and joint (e.g., septic arthπtis, osteomyelitis), assaying genetic vaπation, and admmisteπng a FtsL polypeptide or polynucleotide to an organism to raise an lmmunological response against a bacteπa, especially a Staphylococcus aureus bactena

In accordance with certain preferred embodiments of this and other aspects of the invention there are provided polynucleotides that hybπdize to FtsL polynucleotide sequences, particularly under stnngent conditions.

In certain preferred embodiments of the invention there are provided antibodies against FtsL polypeptides.

In other embodiments of the invention there are provided methods for identifying compounds which bind to or otherwise interact with and inhibit or activate an activity of a polypeptide or polynucleotide of the invention compπsmg: contacting a polypeptide 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 or polynucleotide to assess the binding to or other interaction with the compound, such binding or interaction being associated with a second component capable of providing a detectable signal m response to the binding or interaction of the polypeptide or polynucleotide with the compound; and determinmg whether the compound binds to or otherwise interacts with and activates or inhibits an activity of the polypeptide or polynucleotide by detecting the presence or absence of a signal generated from the binding or interaction of the compound with the polypeptide or polynucleotide.

In accordance with yet another aspect of the invention, there are provided FtsL agonists and antagonists, preferably bacteπostatic or bacteπocidal agonists and antagonists. In a further aspect of the invention there are provided compositions compnsing a FtsL polynucleotide or a FtsL polypeptide for administration to a cell or to a multicellular organism

Vaπous changes and modifications within the spiπt and scope of the disclosed invention will become readily apparent to those skilled m the art from reading the following descπptions and from reading the other parts of the present disclosure.

GLOSSARY

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

"Host cell" is a cell which has been transformed or transfected, or is capable of 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 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 stnngs of such sequences. "Identity" and "similaπty" can be readily calculated by known methods, including but not limited to those descπbed m (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988, Biocomputing Informatics and Genome Projects, Smith, D W , ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer. Gπbskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM / Applied Math , 48 1073 (1988). Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similaπty are codified m publicly available computer programs Preferred computer program methods to determine identity and similanty between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S.F. et al., J. Molec. Biol. 215. 403-410 (1990). The BLAST X program is publicly available from NCBI and other sources {BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894, Altschul, S , et al , J Mol Biol 215 403-410 (1990) As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence of SEQ ID NO 1 it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence of SEQ ID NO 1. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides m the reference sequence may be inserted into the reference sequence These mutations of the reference sequence may occur at the 5 or 3 terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides m the reference sequence or in one or more contiguous groups within the reference sequence. Analogously , by a polypeptide having an ammo acid sequence having at least, for example, 95% identity to a reference amino acid sequence of SEQ ID NO 2 is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of SEQ LD NO 2 In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference ammo acid sequence, up to 5% of the ammo acid residues in the reference sequence may be deleted or substituted with another ammo acid, or a number of amino acids up to 5% of the total ammo acid residues m the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference ammo acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or m one or more contiguous groups within the reference sequence

"Isolated" means altered "by the hand of man" from its natural state, i e , if it occurs m nature, it has been changed or removed from its oπginal environment, or both. For example, a polynucleonde or a polypeptide naturally present in a living organism is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting mateπals of its natural state is "isolated" as the term is employed herein

& "Polynucleotιde(s)" generally refers to any polyπbonucleotide or polydeoxπbonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotιde(s)" include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and tnple-stranded regions, single- and double- stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybnd molecules compnsmg DNA and RNA that may be single-stranded or, more typically, double-stranded, or tnple-stranded regions, or a mixture of single- and double-stranded regions. In addition, "polynucleotide" as used herein refers to tnple-stranded regions compπsmg RNA or DNA or both RNA and DNA. The strands m such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a tπple-helical region often is an ohgonucleotide. As used herein, the term "polynucleotιde(s)" also includes DNAs or RNAs as descnbed above that contain one or more modified bases Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotιde(s)" as that term is intended herein Moreover, DNAs or RNAs compnsmg unusual bases, such as inosine, or modified bases, such as tπtylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great vanety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term "polynucleotιde(s)" as it is employed herein embraces such chemically, enzymatically or metabolicaily modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteπstic of viruses and cells, including, for example, simple and complex cells. "Polynucleotιde(s)" also embraces short polynucleotides often referred to as ohgonucleotιde(s).

"Polypeptιde(s)" refers to any peptide or protein compnsmg two or more ammo acids joined to each other by peptide bonds or modified peptide bonds. "Polypeptιde(s)" refers to both short chains, commonly referred to as peptides, o gopeptides and oligomers and to longer chains generally referred to as proteins. Polypeptides may contain ammo acids other than the 20 gene encoded amino acids. "Polypeptιde(s)" include those modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques. Such modifications are well descnbed in basic texts and in more detailed monographs, as well as in a volummous research literature, and they are well known to those of skill in the art. It will be appreciated that the same type of modification may be present in the same or varying degree at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Modifications can occur anywhere m a polypeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide deπvative, covalent attachment of a lipid or lipid deπvative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, lodination, methylation, mynstoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-πbosylation, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins, such as arginylation, and ubiquitination. See, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993) and Wold, F., Posttranslational Protein Modifications: Perspecnves and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth Enzymol. 182:626-646 (1990) and Rattan et al., Protein Synthesis: Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663: 48-62 (1992). Polypeptides may be branched or cyclic, with or without branching. Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well.

"Variant(s)" as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential propeπies. A typical vanant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the vanant 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, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be a naturally occumng such as an allelic variant, or it may be a variant that is not known to occur naturally. Non- naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans.

DESCRIPTION OF THE INVENTION

The invention relates to novel FtsL polypeptides and polynucleotides as described in greater detail below. In particular, the invention relates to polypeptides and polynucleotides of a novel FtsL of Staphylococcus aureus, which is related by ammo acid sequence homology to Bacillus subtilis FtsL polypeptide. The invention relates especially to FtsL having the nucleotide and ammo acid sequences set out in Table 1 [SEQ LD NO: 1] and Table 1 [SEQ ID NO: 2] respectively, and to the FtsL nucleotide sequences of the DNA in the deposited strain and amino acid sequences encoded thereby.

TABLE 1 FtsL Polynucleotide and Polypeptide Sequences

(A) Sequences from Staphylococcus aureus FtsL polynucleotide sequence [SEQ ID NO: 1 ] .

5 ' - 1 ATGGCTGTAG AAAAAGTGTA CCAACCATAT GACGAACAAG TTTATAATAG

51 TATACCGAAG CAACAACCAC AAACTAAGCC CGAAAAGAAG ACTGTTTCGA

101 GAAAAGTGGT TGTACAATTA ACTAAATTTG AAAAAGTTTT ATACATAACT

151 TTGATTACTG TAATTGCTAT GTTAAGTATT TATATGCTAT CTTTAAAAAT

201 GGATGCGTAT GATACGCGAG GAAAGATTGC AGATTTAGAT TATAAAATAG

°l 251 ATAAACAATC AAGTGAAAAC AGTGCTTTAC AATCTGAAAT CAAAAAGAAT

301 TCTTCTTATG AACGCATATA CGAAAAGGCT AAGAAACAGG GGATGAGCCT

351 TGAGAACGAT AATGTAAAGG TAGTGCGTAG TAATGGCGAA GCAAAAAAT- 3 '

(B) FtsL polypeptide sequence deduced from the polynucleotide sequence in this table [SEQ ID NO:2j\

NH- - 1 MAVEKVYQPY DEQVΥNS IPK QQPQTKPEKK TVSRKWVQL TKFEKVLYIT

51 ITVIAMLSI YMLS KMDAY DTRGKIADLD YKIDKQSSEN SALQSEIKK

101 SSYERIYEKA KKQGMSLEND NVKWRSNGE AKN-COOH

(C) Polynucleotide sequence embodiments [SEQ ID NO: 1 ]. ^χ-(^Rl⁾n"¹ ATGGCTGTAG AAAAAGTGTA CCAACCATAT GACGAACAAG TTTATAATAG

51 TATACCGAAG CAACAACCAC AAACTAAGCC CGAAAAGAAG ACTGTTTCGA

101 GAAAAGTGGT TGTACAATTA ACTAAATTTG AAAAAGTTTT ATACATAACT

151 TTGATTACTG TAATTGCTAT GTTAAGTATT TATATGCTAT CTTTAAAAAT

201 GGATGCGTAT GATACGCGAG GAAAGATTGC AGATTTAGAT TATAAAATAG

251 ATAAACAATC AAGTGAAAAC AGTGCTTTAC AATCTGAAAT CAAAAAGAAT

301 TCTTCTTATG AACGCATATA CGAAAAGGCT AAGAAACAGG GGATGAGCCT

351 TGAGAACGAT AATGTAAAGG TAGTGCGTAG TAATGGCGAA GCAAAAAAT- (R₂ ) _n-

I V (D) Polypeptide sequence embodiments [SEQ ID NO:2].

X - ( R₁ ) _n - l MAVEKVYQPY DEQVYNSIPK QQPQTKPEKK TVSRKVWQL TKFEKVLYIT

51 LITVIAMLSI YMLSLKMDAY DTRGKIADLD YKIDKQSSEN SALQSEIKKN

101 SSYERIYEKA KKQGMSLEND NVKWRSNGE AKN- ( R₂ ) _n -Y

(E) Sequences from Staphylococcus aureus FtsL polynucleotide ORF sequence [SEQ ID NO:3]. 5'-l ATGACCGANC AAGTTTATAA TAGTATACCG AAGCAACAAC CACANACTAA

51 GCCCGAAAAG AAGACTGTTT CGAGAAAAGT GGTTGTACAA TTAACTAAAT

101 TTGNAAAAGT TTTATACATA ACTTTGATTA CTGTAATTGC TATGTTAAGT

151 ATTTATATGC TATCTTTAAA AATGGATGCG TATGATACGC GAGGAAAGAT

201 TGCAGATTTA GATTATAAAA TAGATAAACA ATCAAGTGAA AACAGTGCTT

251 TACAATCTGA AATCAAAAAG AATTCTTCTT ATGAACGCAT ATACGAAAAG

301 GCTAAGAAAC AGGGGATGAG CCTTGAGAAC GATAATGTAA AGGTAGTGCG

351 TAGTAATGGC GAAGCAAAAA AT-3 '

(F) FtsL polypeptide sequence deduced from the polynucleotide ORF sequence m this table [SEQ ID NO:4].

NH₂-1 MTXQVYNSIP KQQPXTKPEK KTVSRKVWQ LTKFXKVLYI TLITVIAMLS

51 IYMLSLKMDA YDTRGKIADL DYKIDKQSSE NSALQSEIKK NSSYERIYEK

101 AKKQGMSLEN DNVKWRSNG EAKN-COOH

// Deposited materials

A deposit containing a Staphylococcus aureus WCUH 29 strain has been deposited with the National Collections of Industnal and Maπne Bacteπa Ltd. (herein "NCIMB"), 23 St Machar Dnve, Aberdeen AB2 1RY, Scotland on 11 September 1995 and assigned NCIMB Deposit No 40771, and is referred to as Staphylococcus aureus WCUH29 on deposit The Staphylococcus aureus strain deposit is referred to herein as "the deposited strain" or as "the DNA of the deposited strain."

The deposited strain contains the full length FtsL gene. The sequence of the polynucleotides contained in the deposited strain, as well as the ammo acid sequence of the polypeptide encoded thereby, are controlling in the event of any conflict with any descnption of sequences herein.

The deposit of the deposited strain has been made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for Purposes of Patent Procedure The strain will be irrevocably and without restnction or condition released to the public upon the issuance of a patent The deposited strain is provided merely as 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 Polypeptides

The polypeptides of the invention include the polypeptide of Table 1 [SEQ ID NO 2] (m particular the mature polypeptide) as well as polypeptides and fragments, particularly those which have the biological activity of FtsL, and also those which have at least 70% identity to a polypeptide of Table 1 [SEQ ED NOS:2 and 4] or the relevant portion, preferably at least 80% identity to a polypeptide of Table 1 [SEQ ID NOS.2 and 4], and more preferably at least 90% similaπty (more preferably at least 90% identity) to a polypeptide of Table 1 [SEQ ID NOS.2 and 4] and still more preferably at least 95% similaπty (still more preferably at least 95% identity) to a polypeptide of Table 1 [SEQ ED NOS:2 and 4] and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 ammo acids and more preferably at least 50 amino acids

lλ The invention also includes polypeptides of the formula set forth in Table 1 (D) [SEQ LD

NO:2] wherein, at the ammo terminus, X is hydrogen, and at the carboxyl terminus, Y is hydrogen or a metal, R\ and R2 is any ammo acid residue, and n is an integer between 1 and

1000. Any stretch of ammo acid residues denoted by either R group, where R is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer.

A fragment is a vanant polypeptide having an ammo acid sequence that entirely is the same as part but not all of the amino acid sequence of the aforementioned polypeptides As with FtsL 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 continuous region, a single larger polypeptide.

Preferred fragments include, for example, truncation polypeptides having a portion of an ammo acid sequence of Table 1 [SEQ ID NOS:2 and 4], or of vaπants thereof, such as a continuous senes of residues that includes the ammo terminus, or a continuous senes of residues that includes the carboxyl terminus. Degradation forms of the polypeptides of the invention in a host cell, particularly a Staphylococcus aureus, are also preferred. Further preferred are fragments characteπzed by structural or functional attributes such as fragments that compnse alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn- forming regions, coil and coil-forming regions, hydrophi c regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions.

Also preferred are biologically active fragments which are those fragments that mediate activities of FtsL, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those fragments that are antigenic or lmmunogenic in an animal, especially in a human. Particularly preferred are fragments compnsmg receptors or domains of enzymes that confer a function essential for viability of Staphylococcus aureus or the ability to initiate, or maintain cause disease in an individual, particularly a human.

Vaπants that are fragments of the polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, these vanants may be employed as intermediates for producing the full-length polypeptides of the invention.

lό Polynucleotides

Another aspect of the invention relates to isolated polynucleotides, including the full length gene, that encode the FtsL polypeptide having a deduced amino acid sequence of Table 1 [SEQ ID NOS:2 and 4] and polynucleotides closely related thereto and vaπants thereof. Using the information provided herein, such as a polynucleotide sequence set out m Table

1 [SEQ ID NOS:l and 3], a polynucleotide of the invention encoding FtsL polypeptide may be obtained using standard cloning and screening methods, such as those for cloning and sequencing chromosomal DNA fragments from bacteπa using Staphylococcus aureus WCUH 29 cells as starting matenal, followed by obtaining a full length clone. For example, to obtain a polynucleotide sequence of the invention, such as a sequence given in Table 1 [SEQ ID NOS. l and 3], typically a library of clones of chromosomal DNA of Staphylococcus aureus WCUH 29 in E coli or some other suitable host is probed with a radiolabeled ohgonucleotide, preferably a 17-mer or longer, derived from a partial sequence. Clones carrying DNA identical to that of the probe can then be distinguished using stnngent conditions. By sequencing the individual clones thus identified with sequencing primers designed from the ongmal sequence it is then possible to extend the sequence m both directions to determine the full gene sequence. Conveniently, such sequencing is performed using denatured double stranded DNA prepared from a plasmid clone. Suitable techniques are descnbed by Mamatis, T., Fntsch, E.F and Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed., Cold Spπng Harbor Laboratory Press, Cold Spπng Harbor, New York (1989) (see in particular Screening By Hybπdization 1.90 and Sequencing Denatured Double-Stranded DNA Templates 13 70) Illustrative of the invention, the polynucleotide set out m Table 1 [SEQ ID NO 1] was discovered in a DNA library deπved from Staphylococcus aureus WCUH 29.

The DNA sequence set out m Table 1 [SEQ ED NOS:l] contains an open reading frame encoding a protein having about the number of ammo acid residues set forth in Table 1 [SEQ ID NO:2] with a deduced molecular weight that can be calculated using ammo acid residue molecular weight values well known in the art. The polynucleotide of SEQ D NO. 1, between nucleotide number 1 through number 399 encodes the polypeptide of SEQ ED NO:2

FtsL of the invention is structurally related to other proteins of the penicillin binding protein family, as shown by the results of sequencing the DNA encoding FtsL of the deposited strain. The protein exhibits greatest homology to Bacillus subtilis FtsL protein among known

π proteins FtsL of Table 1 [SEQ ED NO 2] has about 26% identity over its entire length and about 41% similanty over its entire length with the ammo acid sequence of Bacillus subtihs FtsL polypeptide

The invention provides a polynucleotide sequence identical over its entire length to the coding sequence in Table 1 [SEQ ID NO 1] Also provided by the invention is the coding sequence for the mature polypeptide or a fragment thereof, by itself as well as the coding sequence for the mature polypeptide or a fragment in reading frame with other coding sequence, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence The polynucleotide may also contain non-coding sequences, including for example, but not limited to non-codmg 5' and 3' sequences, such as the transcnbed, non-translated sequences, termination signals, πbosome binding sites, sequences that stabilize mRNA, introns, polyadenylation signals, and additional coding sequence which encode additional ammo acids For example, a marker sequence that facilitates punfication of the fused polypeptide can be encoded In certain embodiments of the invention, the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc ) and descnbed m Gentz et al , Proc Natl Acad. Sci , USA 86 821-824 (1989), or an HA tag (Wilson et al , Cell 37 767 (1984) Polynucleotides of the invention also include, but are not limited to, polynucleotides compnsmg a structural gene and its naturally associated sequences that control gene expression

A preferred embodiment of the invention is a polynucleotide of compnsmg nucleotide 1 to 399 set forth in SEQ ED NO: 1 of Table 1 which encode the FtsL polypeptide

The invention also includes polynucleotides of the formula set forth in Table 1 (C)[SEQ ED NO 1] wherein, at the 5' end of the molecule, X is hydrogen, and at the 3¹ end of the molecule, Y is hydrogen or a metal, Rj and R2 is any nucleic acid residue, and n is an mteger between 1 and 1000. Any stretch of nucleic acid residues denoted by either R group, where R is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer

The term "polynucleotide encoding a polypeptide" as used herein encompasses polynucleotides that include a sequence encoding a polypeptide of the invention, particularly a bactenal polypeptide and more particularly a polypeptide of the Staphylococcus aureus FtsL having the ammo 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, interrupted by integrated phage or an insertion sequence or editing) together with additional regions, that also may contain coding and/or non-coding sequences

The invention further relates to vaπants of the polynucleotides descnbed herein that encode for vanants of the polypeptide having the deduced ammo acid sequence of Table 1 [SEQ ED NO.2]. Vanants that are fragments of the polynucleotides of the invention may be used to synthesize full-length polynucleotides of the invention.

Further particularly preferred embodiments are polynucleotides encoding FtsL vanants, that have the am o acid sequence of FtsL polypeptide of Table 1 [SEQ ED NO 2] in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no ammo acid residues are substituted, deleted or added, in any combination. Especially preferred among these are silent substitutions, additions and deletions, that do not alter the properties and activities of FtsL.

Further preferred embodiments of the invention are polynucleotides that are at least 70% identical over their entire length to a polynucleotide encoding FtsL polypeptide having an amino acid sequence set out m Table 1 [SEQ ID NOS:2 and 4], and polynucleotides that are complementary to such polynucleotides. Alternatively, most highly preferred are polynucleotides that compnse a region that is at least 80% identical over its entire length to a polynucleotide encoding FtsL polypeptide of the deposited strain and polynucleotides complementary thereto In this regard, polynucleotides at least 90% identical over their entire length to the same are particularly preferred, and among these particularly preferred polynucleotides, those with at least 95% are especially preferred. Furthermore, those with at least 97% are highly preferred among those with at least 95%, and among these those with at least 98% and at least 99% are particularly highly preferred, with at least 99% being the more preferred

Preferred embodiments are polynucleotides that encode polypeptides that retain substantially the same biological function or activity as the mature polypeptide encoded by the DNA of Table 1 [SEQ ED NO:l],

The invention further relates to polynucleotides that hybndize to the herem above- descnbed sequences. In this regard, the invention especially relates to polynucleotides that hybndize under stπngent conditions to the herein above-descnbed polynucleotides. As herein used, the terms "stπngent conditions" and "stnngent hybπdization conditions" mean hybπdization will occur only if there is at least 95% and preferably at least 97% identity between the sequences An example of stπngent hybπdization conditions is overnight incubation at 42°C in a solution compnsmg 50% formamide, 5x SSC (150mM NaCl, 15mM tnsodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml denatured, sheared salmon sperm DNA, followed by washing the hybπdization support in 0 lx SSC at about 65°C Hybndization and wash conditions are well known and exemplified in Sambrook, et al., Molecular Cloning A Laboratory Manual, Second Edition, Cold Spnng Harbor, N Y., (1989), particularly Chapter 11 therein.

The invention also provides a polynucleotide consisting essentially of a polynucleotide sequence obtainable by screening an appropnate library containing the complete gene for a polynucleotide sequence set forth in SEQ ID NO:l or SEQ ID NO.3 under stπngent hybπdization 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 DNA sequence. Fragments useful for obtaining such a polynucleotide include, for example, probes and pnmers descnbed elsewhere herein.

As discussed additionally herein regarding polynucleotide assays of the invention, for instance, polynucleotides of the invention as discussed above, may be used as a hybndization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding FtsL and to isolate cDNA and genomic clones of other genes that have a high sequence similaπty to the FtsL gene Such probes generally will compnse at least 15 bases Preferably, such probes will have at least 30 bases and may have at least 50 bases. Particularly preferred probes will have at least 30 bases and will have 50 bases or less.

For example, the coding region of the FtsL gene may be isolated by screening using the DNA sequence provided in SEQ ED NO: 1 to synthesize an oligonucleotide probe. A labeled ohgonucleotide having a sequence complementary to that of a gene of the mvention is then used to screen a library of cDNA, genomic DNA or mRNA to determine which members of the library the probe hybπdizes to.

The polynucleotides and polypeptides of the invention may be employed, for example, as research reagents and matenals for discovery of treatments of and diagnostics for disease, particularly human disease, as further discussed herein relating to polynucleotide assays

Polynucleotides of the invention that are o gonucleotides deπved from the sequences of SEQ ID NOS: 1 and/or 2 may be used in the processes herein as descnbed, but preferably for PCR, to determine whether or not the polynucleotides identified herein in whole or in part are

n transcnbed in bactena 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 may encode a polypeptide that is the mature protein plus additional ammo or carboxyl-terminal amino acids, or amino acids intenor to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance). Such sequences may play a role m processing of a protein from precursor to a mature form, may allow protein transport, may lengthen or shorten protein half-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 the mature protein by cellular enzymes.

A precursor protein, having the 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. In sum, a polynucleotide of the invention may encode a mature protein, a mature protein plus a leader sequence (which may be referred to as a preprotein), a precursor of a mature protein having one or more prosequences that are not the leader sequences of a preprotein, or a preproprotem, which is a precursor to a proprotein, having a leader sequence and one or more prosequences, which generally are removed duπng processing steps that produce active and mature forms of the polypeptide

Vectors, host cells, expression

The invention also relates to vectors that compπse a polynucleotide or polynucleotides of the invention, host cells that are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques. Cell-free translation systems can also be employed to produce such proteins using RNAs deπved from the DNA constructs of the invention.

For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the invention. Introduction of a polynucleotide mto the host cell can be effected by methods descnbed in many standard laboratory manuals, such as Davis et al , BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook et al., MOLECULAR CLONING A LABORATORY MANUAL, 2nd Ed., Cold

t Spnng Harbor Laboratory Press, Cold Spnng Harbor, N.Y. (1989), such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, catiomc lipid- mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection Representative examples of appropnate hosts include bactenal cells, such as streptococci, staphylococci, enterococci E coli, streptomyces and Bacillus subtύis cells; fungal cells, such as yeast cells and Aspergύlus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells, animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells; and plant cells A great vanety of expression systems can be used to produce the polypeptides of the invention Such vectors include, among others, chromosomal, episomal and virus-denved vectors, e g , vectors deπved from bactenal plasmids, from bacteπophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors deπved from combinations thereof, such as those deπved from plasmid and bacteπophage genetic elements, such as cosmids and phagemids. The expression system constructs may contain 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 appropnate DNA sequence may be inserted into the expression system by any of a vanety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL, (supra).

For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the peπplasmic space or into the extracellular environment, appropnate 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 puπfied 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

t l chromatography is employed for puπfication. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured dunng isolation and or punfication. Diagnostic Assays This invention is also related to the use of the FtsL polynucleotides of the invention for use as diagnostic reagents. Detection of FtsL in a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of a disease. Eukaryotes (herein also "mdιvιdual(s)"), particularly mammals, and especially humans, particularly those infected or suspected to be infected with an organism compnsmg the FtsL gene may be detected at the nucleic acid level by a vanety of techniques.

Nucleic acids for diagnosis may be obtained from an infected individual's cells and tissues, such as bone, blood, muscle, cartilage, and skin. Genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification technique pπor to analysis. RNA or cDNA may also be used in the same ways. Using amplification, charactenzation of the species and strain of prokaryote present in an individual, may be made by an analysis of the genotype of the prokaryote gene. Deletions and insertions can be detected by a change in size of the amplified product m compaπson to the genotype of a reference sequence Point mutations can be identified by hybπdizing amplified DNA to labeled FtsL polynucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures. DNA sequence differences may also be detected by alterations in the electrophoretic mobility of the DNA fragments in gels, with or without denatunng agents, or by direct DNA sequencing. See, e.g , Myers et al., Science, 230 1242 (1985). Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase and SI protection or a chemical cleavage method. See, e.g , Cotton et al., Proc Natl. Acad. Sci., USA, 85. 4397-4401 (1985).

Cells carrying mutations or polymorphisms in the gene of the invention may also be detected at the DNA level by a vanety of techniques, to allow for serotypmg, for example. For example, RT-PCR can be used to detect mutations. It is particularly preferred to used RT-PCR in conjunction with automated detection systems, such as, for example, GeneScan. RNA or cDNA

2 o may also be used for the same purpose, PCR or RT-PCR. As an example, PCR pnmers complementary to a nucleic acid encoding FtsL can be used to identify and analyze mutations.

The invention further provides these pnmers with 1, 2, 3 or 4 nucleotides removed from the 5' and/or the 3' end. These pnmers may be used for, among other things, amplifying FtsL DNA isolated from a sample derived from an individual. The pnmers may be used to amplify the gene isolated from an infected individual such that the gene may then be subject to vanous techniques for elucidation of the DNA sequence. In this way, mutations m the DNA sequence may be detected and used to diagnose infection and to serotype and/or classify the infectious agent. The invention further provides a process for diagnosing, disease, preferably bactenal infections, more preferably infections by Staphylococcus aureus, and most preferably disease, such as, infections of the upper respiratory tract (e.g., otitis media, bactenal tracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g., empyema, lung abscess), cardiac (e.g., infective endocarditis), gastrointestinal (e.g., secretory diarrhoea, splenic absces, retropentoneal abscess), CNS (e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital celluhtis, darcryocystitis), kidney and urinary tract (e.g., epididymitis, intrarenal and pennephπc absces, toxic shock syndrome), skm (e.g., impetigo, folhculitis, cutaneous abscesses, celluhtis, wound infection, bactenal myositis) bone and joint (e.g., septic arthritis, osteomyelitis), comprising determinmg from a sample derived from an individual a increased level of expression of polynucleotide having the sequence of Table 1 [SEQ ID NO: 1]. Increased or decreased expression of FtsL polynucleotide can be measured using any on of the methods well known in the art for the quantation of polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNase protection, Northern blotting and other hybπdization methods. In addition, a diagnostic assay in accordance with the invention for detecting over- expression of FtsL protein 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 FtsL protein, in a sample deπved from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays. Antibodies

The polypeptides of the invention or vaπants thereof, or cells expressing them can be used as an lmmunogen to produce antibodies lmmunospecific for such polypeptides

"Antibodies" as used herein includes monoclonal and polyclonal antibodies, chimeπc, single chain, simia zed antibodies and humanized antibodies, as well as Fab fragments, including the products of an Fab immunolglobu n expression library.

Antibodies generated against the polypeptides of the invention can be obtained by admimstenng the polypeptides or epitope-beaπng fragments, analogues or cells to an animal, preferably a nonhuman, using routine protocols. For preparation of monoclonal antibodies, any technique known m the art that provides antibodies produced by continuous cell line cultures can be used. Examples include vanous techniques, such as those in Kohler, G. and Milstein, C, Nature 256 495-497 (1975); Kozbor et al , Immunology Today 4 72 (1983), Cole et al , pg 77- 96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc (1985)

Techniques for the production of single chain antibodies (U.S Patent No 4,946.778) can be adapted to produce single chain antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized antibodies.

Alternatively phage display technology may be utilized to select antibody genes with binding activities towards the polypeptide either from repertoires of PCR amplified v-genes of lymphocytes from humans screened for possessing anti-FtsL or from naive hbraπes (McCafferty, J. et al., (1990), Nature 348, 552-554; Marks, J. et al., (1992) Biotechnology 10, 779-783). The affinity of these antibodies can also be improved by chain shuffling (Clackson, T. et al., (1991) Nature 352, 624-628).

If two antigen binding domains are present each domain may be directed against a different epitope - termed 'bispecific' antibodies The above-descnbed antibodies may be employed to isolate or to identify clones expressing the polypeptides to punfy the polypeptides by affinity chromatography.

Thus, among others, antibodies against FtsL- polypeptide may be employed to treat infections, particularly bactenal infections and especially disease, such as, mfections of the upper respiratory tract (e.g., otitis media, bactenal tracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g., empyema, lung abscess), cardiac (e.g., infective endocarditis), gastrointestinal

(e.g., secretory diarrhoea, splenic absces, retropentoneal abscess), CNS (e.g., cerebral abscess),

lλ eye (e.g., blephantis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital celluhtis, darcryocystitis), kidney and unnary tract (e.g., epididymitis, lntrarenal and peπnephnc absces, toxic shock syndrome), skin (e.g., impetigo, folhcu tis, cutaneous abscesses, celluhtis, wound infection, bactenal myositis) bone and joint (e g., septic arthntis, osteomyelitis) Polypeptide variants include antigenically, epitopically or immunologically equivalent vanants that form a particular aspect of this invention. The term "antigenically equivalent denvative" as used herein encompasses a polypeptide or its equivalent which will be specifically recognized by certain antibodies which, when raised to the protein or polypeptide according to the invention, interfere with the immediate physical interaction between pathogen and mammalian host. The term "immunologically equivalent deπvative" as used herein encompasses a peptide or its equivalent which when used in a suitable formulation to raise antibodies in a vertebrate, the antibodies act to interfere with the immediate physical interaction between pathogen and mammalian host.

The polypeptide, such as an antigenically or immunologically equivalent denvative or a fusion protein thereof is used as an antigen to immunize a mouse or other animal such as a rat or chicken. The fusion protein may provide stability to the polypeptide The antigen may be associated, for example by conjugation, with an immunoge c earner protein for example bovine serum albumin (BSA) or keyhole limpet haemocyan (KLH). Alternatively a multiple antigenic peptide compnsmg multiple copies of the protein or polypeptide, or an antigenically or immunologically equivalent polypeptide thereof may be sufficiently antigenic to improve lmmunogemcity so as to obviate the use of a earner.

Preferably, the antibody or vanant thereof is modified to make it less immunogemc m the individual. For example, if the individual is human the antibody may most preferably be "humanized", where the complimentaπty determining regιon(s) of the hybπdoma-deπved antibody has been transplanted into a human monoclonal antibody , for example as described in Jones, P. et al. (1986), Nature 321, 522-525 or Tempest et al., (1991) Biotechnology 9, 266- 273.

The use of a polynucleotide of the invention m genetic immunization will preferably employ a suitable delivery method such as direct injection of plasmid DNA into muscles (Wolff et al , Hum Mol Genet 1992, 1:363, Manthorpe et al, Hum. Gene Ther. 1963-4, 419), delivery of DNA complexed with specific protein earners (Wu et al , J Biol Chem 1989-

ϊ 264,16985), coprecφitation of DNA with calcium phosphate (Benvemsty & Reshef. PNAS USA, 1986:83,9551), encapsulation of DNA in various forms of posomes (Kaneda et al , Science 1989:243,375), particle bombardment (Tang et al , Nature 1992, 356: 152, Eisenbraun et al , DNA Cell Biol 1993, 12.791) and in vivo infection using cloned retroviral vectors (Seeger et al., PNAS USA 1984 81 ,5849)

Antagonists and agonists - assays and molecules

Polypeptides of the invention may also be used to assess the binding of small molecule substrates and hgands in, for example, cells, cell-free preparations, chemical branes, and natural product mixtures. These substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics. See, e g , Coligan et al , Current Protocols in Immunology 1(2) Chapter s (1991)

The invention also provides a method of screening compounds to identify those which enhance (agonist) or block (antagonist) the action of FtsL polypeptides or polynucleotides, particularly those compounds that are bacteπostatic and/or bactenocidal The method of screening may involve high-throughput techniques. For example, to screen for agonists or antagoists, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, compnsmg FtsL polypeptide and a labeled substrate or ligand of such polypeptide is incubated m the absence or the presence of a candidate molecule that may be a FtsL agonist or antagonist The ability of the candidate molecule to agonize or antagonize the FtsL polypeptide is reflected in decreased bindmg of the labeled ligand or decreased production of product from such substrate. Molecules that bind gratuitously, i e , without inducing the effects of FtsL polypeptide are most likely to be good antagonists Molecules that bind well and increase the rate of product production from substrate are agonists. Detection of the rate or level of production of product from substrate may be enhanced by using a reporter system. Reporter systems that may be useful in this regard include but are not limited to colonmetπc labeled substrate converted into product, a reporter gene that is responsive to changes in FtsL polynucleotide or polypeptide activity, and binding assays known m the art

Another example of an assay for FtsL antagonists is a competitive assay that combines

FtsL and a potential antagonist with FtsL-bmd g molecules, recombinant FtsL binding molecules, natural substrates or ligands, or substrate or ligand mimetics, under appropnate conditions for a competitive inhibition assay FtsL can be labeled, such as by radioactivity or a

d Y colonmetπc compound, such that the number of FtsL molecules bound to a binding molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist

Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to a polynucleotide or polypeptide of the invention and thereby inhibit or extinguish its activity Potential antagonists also may be small organic molecules, a peptide, a polypeptide such as a closely related protein or antibody that binds the same sites on a binding molecule, such as a binding molecule, without inducing FtsL-induced activities, thereby preventing the action of FtsL by excluding FtsL from binding Potential antagonists mclude a small molecule that binds to and occupies the binding site of the polypeptide thereby preventing binding to cellular binding molecules, such that normal biological activity is prevented. Examples of small molecules include but are not limited to small organic molecules, peptides or peptide- ke molecules. Other potential antagonists mclude antisense molecules (see Okano, J Neurochem 56. 560 (1991), OUGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, FL ( 1988), for a descnption of these molecules) Preferred potential antagonists include compounds related to and vaπants of FtsL

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

The invention also provides the use of the polypeptide, polynucleotide or inhibitor of the invention to interfere with the initial physical interaction between a pathogen and mammalian host responsible for sequelae of infection. In particular the molecules of the invention may be used: in the prevention of adhesion of bactena, in particular gram positive bacteria, to mammalian extracellular matπx proteins on m-dwelling devices or to extracellular matπx proteins in wounds, to block FtsL protem-mediated mammalian cell invasion by, for example, initiating phosphorylation of mammalian tyrosine kmases (Rosenshine et al , Infect Immun 60 221 1 (1992), to block bacterial adhesion between mammalian extracellular matπx

' O proteins and bactenal FtsL proteins that mediate tissue damage and; to block the normal progression of pathogenesis in infections initiated other than by the implantation of in-dwelling devices or by other surgical techniques.

The antagonists and agonists of the invention may be employed, for instance, to inhibit and treat disease, such as, infections of the upper respiratory tract (e.g., otitis media, bactenal tracheitis, acute epiglottitis, thyroiditis), lower respiratory (e g , empyema, lung abscess), cardiac (e.g., infective endocarditis), gastrointestinal (e.g., secretory diarrhoea, splenic absces, retropentoneal abscess), CNS (e.g., cerebral abscess), eye (e.g., blephaπtis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital celluhtis, darcryocystitis), kidney and uπnary tract (e.g., epididymitis, mtrarenal and peπnephπc absces, toxic shock syndrome), skm (e.g., impetigo, follicu tis, cutaneous abscesses, celluhtis, wound infection, bactenal myositis) bone and joint (e.g., septic arthπtis, osteomyelitis).

Helicobacter pylori (herein H pylori) bacteria infect the stomachs of over one-third of the world's population causing stomach cancer, ulcers, and gastπtis (International Agency for Research on Cancer (1994) Schistosomes, Liver Flukes and Helicobacter Pylon (International Agency for Research on Cancer, Lyon, France; http://www.uicc.ch/ecp/ecp2904.htm). Moreover, the international Agency for Research on Cancer recently recognized a cause-and- effect relationship between H. pylori and gastπc adenocarcmoma, classifying the bacteπum as a Group I (definite) carcinogen. Preferred antimicrobial compounds of the invention (agonists and antagonists of FtsL) found using screens provided by the invention, particularly broad- spectrum antibiotics, should be useful in the treatment of H. pylori infection Such treatment should decrease the advent of H. cancers, such as gastrointestinal carcinoma. Such treatment should also cure gastπc ulcers and gastπtis. Vaccines Another aspect of the invention relates to a method for inducing an immunological response m an individual, particularly a mammal which compnses inoculating the individual with FtsL, or a fragment or variant thereof, adequate to produce antibody and/ or T cell immune response to protect said individual from infection, particularly bactenal infection and most particularly Staphylococcus aureus infection. Also provided are methods whereby such immunological response slows bactenal replication. Yet another aspect of the invention relates to a method of inducing immunological response in an individual which compnses dehveπng

øt & to such individual a nucleic acid vector to direct expression of FtsL, or a fragment or a variant thereof, for expressing FtsL, or a fragment or a variant thereof in vivo in order to induce an immunological response, such as, to produce antibody and or T cell immune response, including, for example, cytokine-producing T cells or cytotoxic T cells, to protect said individual from disease, whether that disease is already established within the individual or not. One way of administering the gene is by accelerating it into the desired cells as a coating on particles or otherwise. Such nucleic acid vector may comprise DNA, RNA, a modified nucleic acid, or a DNA/RNA hybrid.

A further aspect of the invention relates to an immunological composition which, when introduced into an individual capable or having induced within it an immunological response, induces an immunological response in such individual to a FtsL or protein coded therefrom, wherein the composition comprises a recombinant FtsL or protein coded therefrom compnsing DNA which codes for and expresses an antigen of said FtsL or protein coded therefrom. The immunological response may be used therapeutically or prophylactically and may take the form of antibody immunity or cellular immunity such as that arising from CTL or CD4+ T cells.

A FtsL polypeptide or a fragment thereof may be fused with co-protein which may not by itself produce antibodies, but is capable of stabilizing the first protein and producing a fused protein which will have immunogenic and protective properties. Thus fused recombinant protein, preferably further comprises an antigenic co-protein, such as lipoprotein D from Hemophilus influenzae, Glutathione-S-transferase (GST) or beta-galactosidase, relatively large co-protems which solubilize the protein and facilitate production and purification thereof. Moreover, the co-protein may act as an adjuvant in the sense of providing a generalized stimulation of the immune system. The co-protein may be attached to either the ammo or carboxy terminus of the first protein.

Provided by this invention are compositions, particularly vaccine compositions, and methods comprising the polypeptides or polynucleotides of the invention and immunostimulatory DNA sequences, such as those described in Sato, Y. et al. Science 273: 352 (1996). Also, provided by this invention are methods using the described polynucleotide or particular fragments thereof which have been shown to encode non-variable regions of

- bacterial cell surface proteins m DNA constructs used in such genetic immunization expeπments m animal models of infection with Staphylococcus aureus will be particularly useful for identifying protein epitopes able to provoke a prophylactic or therapeutic immune response. It is believed that this approach will allow for the subsequent preparation of monoclonal antibodies of particular value from the requisite organ of the animal successfully resisting or cleanng infection for the development of prophylactic agents or therapeutic treatments of bacterial infection, particularly Staphylococcus aureus infection, in mammals, particularly humans.

The polypeptide may be used as an antigen for vaccination of a host to produce specific antibodies which protect against invasion of bacteπa, for example by blocking adherence of bacteria to damaged tissue. Examples of tissue damage include wounds m skm or connective tissue caused, e.g., by mechanical, chemical or thermal damage or by implantation of indwelling devices, or wounds in the mucous membranes, such as the mouth, mammary glands, urethra or vagma. The invention also includes a vaccine formulation which compnses an immunogemc recombinant protein of the invention together with a suitable earner. Since the protein may be broken down in the stomach, it is preferably administered parenterally, including, for example, administration that is subcutaneous, intramuscular, intravenous, or mtradermal. Formulations suitable for parenteral administration include aqueous and non-aqueous stenle injection solutions which may contain anti-oxidants, buffers, bactenostats and solutes which render the formulation lnsoto c with the bodily fluid, preferably the blood, of the individual, and aqueous and non-aqueous steπle suspensions which may include suspending agents or thickening agents The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials and may be stored in a freeze-dπed condition requinng only the addition of the stenle liquid earner immediately pnor to use. The vaccine formulation may also include adjuvant systems for enhancing the lmmunogenicity of the formulation, such as oil-in water systems and other systems known in the art The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation

While the invention has been descnbed with reference to certain FtsL protein, it is to be understood that this covers fragments of the naturally occurnng protein and similar proteins

1 3* with additions, deletions or substitutions which do not substantially affect the immunogemc properties of the recombinant protein

Compositions, kits and administration

The invention also relates to compositions compnsmg the polynucleotide or the polypeptides discussed above or their agonists or antagonists The polypeptides of the invention may be employed m combination with a non-steπle or steπle earner or earners for use with cells, tissues or organisms, such as a pharmaceutical earner suitable for administration to a subject. Such compositions compnse, for instance, a media additive or a therapeutically effective amount of a polypeptide of the invention and a pharmaceutically acceptable earner or excipient Such earners may include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof. The formulation should suit the mode of administration The invention further relates to diagnostic and pharmaceutical packs and kits compnsmg one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention Polypeptides and other compounds of the invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.

The pharmaceutical compositions may be administered in any effective, convenient manner including, for instance, administration by topical, oral, anal, vaginal, intravenous, intrapentoneal, intramuscular, subcutaneous, mtranasal or mtradermal routes among others In therapy or as a prophylactic, the active agent may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion, preferably isoto c

Alternatively the composition may be formulated for topical application for example in the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, mouthwash, impregnated dressings and sutures and aerosols, and may contain appropnate conventional additives, including, for example, preservatives, solvents to assist drug penetration, and emollients in ointments and creams. Such topical formulations may also contain compatible conventional earners, for example cream or ointment bases, and ethanol or oleyl alcohol for lotions. Such earners may constitute from about 1% to about 98% by weight of the formulation; more usually they will constitute up to about 80% by weight of the formulation

λ-1 For administration to mammals, and particularly humans, it is expected that the daily dosage level of the active agent will be from 0 01 mg kg to 10 mg/kg, typically around 1 mg/kg. The physician m any event will determine the actual dosage which will be most suitable for an individual and will vary with the age, weight and response of the particular individual. The above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are mented, and such are within the scope of this invention.

In-dwelling devices include surgical implants, prosthetic devices and catheters, i.e., devices that are introduced to the body of an individual and remain in position for an extended time. Such devices include, for example, artificial joints, heart valves, pacemakers, vascular grafts, vascular catheters, cerebrosp al fluid shunts, unnary catheters, continuous ambulatory pentoneal dialysis (CAPD) catheters.

The composition of the invention may be administered by injection to achieve a systemic effect against relevant bacteπa shortly before insertion of an in-dwelling device. Treatment may be continued after surgery during the m-body time of the device. In addition, the composition could also be used to broaden peπoperative cover for any surgical technique to prevent bactenal wound infections, especially Staphylococcus aureus wound infections.

Many orthopaedic surgeons consider that humans with prosthetic joints should be considered for antibiotic prophylaxis before dental treatment that could produce a bacteremia. Late deep infection is a senous comphcation sometimes leading to loss of the prosthetic joint and is accompanied by significant morbidity and mortality. It may therefore be possible to extend the use of the active agent as a replacement for prophylactic antibiotics in this situation

In addition to the therapy descnbed above, the compositions of this invention may be used generally as a wound treatment agent to prevent adhesion of bactena to matnx proteins exposed in wound tissue and for prophylactic use in dental treatment as an alternative to, or in conjunction with, antibiotic prophylaxis.

Alternatively, the composition of the invention may be used to bathe an indwelling device immediately before insertion. The active agent will preferably be present at a concentration of lμg/ml to lOmg/ml for bathing of wounds or indwelling devices A vaccine composition is conveniently in mjectable form. Conventional adjuvants may be employed to enhance the immune response A suitable unit dose for vaccination is 0 5-5 microgram kg of antigen, and such dose is preferably administered 1-3 times and with an interval of 1-3 weeks. With the indicated dose range, no adverse toxicological effects will be observed with the compounds of the invention which would preclude their administration to suitable individuals.

Each reference disclosed herein is incorporated by reference herein in its entirety. Any patent application to which this application claims priority is also incorporated by reference herein in its entirety.

EXAMPLES

The examples below are carried out using standard techniques, which are well known and routine to those of skill in the art, except where otherwise described in detail. The examples are illustrative, but do not limit the invention.

Example 1 Strain selection, Library Production and Sequencing The polynucleotide having the DNA sequence given in SEQ ED NO: l was obtained from a library of clones of chromosomal DNA of Staphylococcus aureus in E. coli. The sequencing data from two or more clones containing overlapping Staphylococcus aureus DNAs was used to construct the contiguous DNA sequence in SEQ ID NO: l. Libraries may be prepared by routine methods, for example: Methods 1 and 2 below.

Total cellular DNA is isolated from Staphylococcus aureus WCUH 29 according to standard procedures and size-fractionated by either of two methods. Method 1

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

3 / Method 2

Total cellular DNA is partially hydrolyzed with a one or a combination of restπction enzymes appropnate to generate a series of fragments for cloning into library vectors (e.g., Rsal, Pall, Alul, Bshl235I), and such fragments are size-fractionated according to standard procedures. EcoRI linkers are ligated to the DNA and the fragments then ligated into the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E.coli infected with the packaged library. The library is amplified by standard procedures.

3λ SEQUENCE LISTING (1) GENERAL INFORMATION (i) APPLICANT: SmithKline Beecham Corp. (ii) TITLE OF THE INVENTION: NOVEL FtsL

(iϋ) NUMBER OF SEQUENCES: 4

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Dechert Price & Rhoads

(B) STREET: 997 Lenox Drive, BuUding 3, Suite 210

(C) CTTY: LawrenceviUe

(D) STATE: NJ

(E) COUNTRY: USA

(F) ZIP: 08543

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette

(B) COMPUTER: D3M Compatible ( OPERATING SYSTEM: DOS

CD) SOFTWARE: FastSEQ for Windows Version 2.0

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Bloom, Allen

(B) REGISTRATION NUMBER: 29,135

( REFERENCE/DOCKET NUMBER: GM10060

(ix) TELECOMMUNICATION INFORMATION:

21 (A) TELEPHONE: 609-520-3214

(B) TELEFAX: 609-520-3259

(C) TELEX:

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 399 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

ATGGCTGTAG AAAAAGTGTA CCAACCATAT GACGAACAAG TTTATAATAG TATACCGAAG 60

CAACAACCAC AAACTAAGCC CGAAAAGAAG ACTGTTTCGA GAAAAGTGGT TGTACAATTA 120

ACTAAATTTG AAAAAGTTTT ATACATAACT TTGATTACTG TAATTGCTAT GTTAAGTATT 180

TATATGCTAT CTTTAAAAAT GGATGCGTAT GATACGCGAG GAAAGATTGC AGATTTAGAT 240

TATAAAATAG ATAAACAATC AAGTGAAAAC AGTGCTTTAC AATCTGAAAT CAAAAAGAAT 300

TCTTCTTATG AACGCATATA CGAAAAGGCT AAGAAACAGG GGATGAGCCT TGAGAACGAT 360

AATGTAAAGG TAGTGCGTAG TAATGGCGAA GCAAAAAAT 399

(2) INFORMATION FOR SEQ H) NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 135 amino acids

(B) TYPE: amino acid

(O STRANDEDNESS: single (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Ala Val Glu Lys Val Tyr Gin Pro Tyr Asp Glu Gin Val Tyr Asn 1 5 10 15

3 V Ser He Pro Lys Gin Gin Pro Gin Thr Lys Pro Glu Lys Lys Thr Val

20 25 30

Ser Arg Lys Val Val Val Gin Leu Thr Lys Phe Glu Lys Val Leu Tyr

35 40 45

He Thr Leu He Thr Val He Ala Met Leu Ser De Tyr Met Leu Ser

50 55 60

Leu Lys Met Asp Ala Tyr Asp Thr Arg Gly Lys Be Ala Asp Leu Asp 65 70 75 80

Tyr Lys Be Asp Lys Gin Ser Ser Glu Asn Ser Ala Leu Gin Ser Glu

85 90 95

Be Lys Lys Asn Ser Ser Tyr Glu Arg Be Tyr Glu Lys Ala Lys Lys

100 105 110

Gin Gly Met Ser Leu Glu Asn Asp Asn Val Lys Val Val Arg Ser Asn

115 120 125

Gly Glu Ala Lys Asn 130

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 372 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRD7TION: SEQ ID NO:3:

ATGACCGANC AAGTTTATAA TAGTATACCG AAGCAACAAC CACANACTAA GCCCGAAAAG 60

AAGACTGTTT CGAGAAAAGT GGTTGTACAA TTAACTAAAT TTGNAAAAGT TTTATACATA 120

ACTTTGATTA CTGTAATTGC TATGTTAAGT ATTTATATGC TATCTTTAAA AATGGATGCG 180

TATGATACGC GAGGAAAGAT TGCAGATTTA GATTATAAAA TAGATAAACA ATCAAGTGAA 240

AACAGTGCTT TACAATCTGA AATCAAAAAG AATTCTTCTT ATGAACGCAT ATACGAAAAG 300

GCTAAGAAAC AGGGGATGAG CCTTGAGAAC GATAATGTAA AGGTAGTGCG TAGTAATGGC 360 GAAGCAAAAA AT 372

(2) INFORMATION FOR SEQ H) NO:4:

/3 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 124 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Met Thr Xaa Gin Val Tyr Asn Ser Be Pro Lys Gin Gin Pro Xaa Thr 1 5 10 15

Lys Pro Glu Lys Lys Thr Val Ser Arg Lys Val Val Val Gin Leu Thr

20 25 30

Lys Phe Xaa Lys Val Leu Tyr Be Thr Leu Be Thr Val Be Ala Met

35 40 45

Leu Ser Be Tyr Met Leu Ser Leu Lys Met Asp Ala Tyr Asp Thr Arg

50 55 60

Gly Lys Be Ala Asp Leu Asp Tyr Lys Be Asp Lys Gin Ser Ser Glu 65 70 75 80

Asn Ser Ala Leu Gin Ser Glu Be Lys Lys Asn Ser Ser Tyr Glu Arg

85 90 95

Be Tyr Glu Lys Ala Lys Lys Gin Gly Met Ser Leu Glu Asn Asp Asn

100 105 110

Val Lys Val Val Arg Ser Asn Gly Glu Ala Lys Asn 115 120

3

Claims

What is claimed is:

1 An isolated polynucleotide compnsmg a polynucleotide sequence selected from the group consisting of

(a) a polynucleotide having at least a 70% identity to a polynucleotide encoding a polypeptide compnsmg the amino acid sequence of SEQ ID NO.2;

(b) a polynucleotide having at least a 70% identity to a polynucleotide encoding die same mature polypeptide expressed by the FtsL gene contamed the Staphylococcus aureus of the deposited strain,

(c) a polynucleotide encoding a polypeptide compnsmg an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ED NO:2;

(d) a polynucleotide which is complementary to the polynucleotide of (a), (b) or (c); and

(e) a polynucleotide compnsmg at least 15 sequential bases of the polynucleotide of (a), (b), (c) or (d)

2 The polynucleotide of Claim 1 wherein the polynucleotide is DNA.

3 The polynucleotide of Claim 1 wherein the polynucleotide is RNA.

4 The polynucleotide of Claim 2 compnsmg the nucleic acid sequence set forth in SEQ ED NO 1

5 The polynucleotide of Claim 2 compnsmg nucleotide 1 to 399 set forth in SEQ ED NO.l

6 The polynucleotide of Claim 2 which encodes a polypeptide compnsmg the ammo acid sequence of SEQ ID NO:2

7 A vector compnsmg the polynucleotide of Claim 1

8. A host cell compnsmg the vector of Claim 7.

9 A process for producing a polypeptide compnsmg: expressing from the host cell of Claim 8 a polypeptide encoded by said DNA.

10 A process for producing a FtsL polypeptide or fragment compnsmg cultuπng a host of claim 8 under conditions sufficient for the production of said polypeptide or fragment

11. A polypeptide compnsmg an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID NO.2.

12. A polypeptide compnsmg an ammo acid sequence as set forth in SEQ ED NO.2.

3?

13. An antibody against the polypeptide of claim 11.

14. An antagonist which inhibits the activity or expression of the polypeptide of claim 11.

15. A method for the treatment of an individual in need of FtsL polypeptide comprising: administenng to the individual a therapeutically effective amount of the polypeptide of claim 11.

16. A method for the treatment of an individual having need to inhibit FtsL polypeptide comprising: administenng to the individual a therapeutically effective amount of the antagonist of Claim 14.

17. A process for diagnosing a disease related to expression or activity of the polypeptide of claim 11 in an individual compπsing:

(a) determining a nucleic acid sequence encoding said polypeptide, and/or

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

18. A method for identifying compounds which interact with and inhibit or activate an activity of the polypeptide of claim 11 compnsmg: contacting a composition compπsing the polypeptide with the compound to be screened under conditions to permit interaction between the compound and the polypeptide to assess the interaction of a compound, such interaction being associated with a second component capable of providing a detectable signal in response to the interaction of the polypeptide with the compound; and determining whether the compound interacts with and activates or inhibits an activity of the polypeptide by detecting die presence or absence of a signal generated from the interaction of the compound with the polypeptide.

19. A method for inducing an immunological response in a mammal which compnses inoculating the mammal with FtsL polypeptide of claim 11 , or a fragment or vanant thereof, adequate to produce antibody and or T cell immune response to protect said animal from disease.

20. A method of inducing immunological response in a mammal which comprises delivenng a nucleic acid vector to direct expression of FtsL polypeptide of claim 11, or fragment or a vanant thereof, for expressing said FtsL polypeptide, or a fragment or a vanant

33 thereof in vivo m order to induce an immunological response to produce antibody and or T cell immune response to protect said animal from disease.

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