WO2000024252A1 - Ftsz:ftsa multimeric proteins and their uses - Google Patents

Abstract

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

Description

FTSZ:FTSA MULTIMERIC PROTEINS AND THEIR USES

RELATED APPLICATIONS

This application claims benefit of US Provisional Patent Application Number 60/105,315, filed October 23 1998 US Provisional Patent Application Number 60/129,965 filed Apnl 19, 1999, and US Provisional Patent Application Number 60/132.333. filed May 3, 1999

FIELD OF THE INVENTION

This invention relates to newly identified higher order structures of FtsZ FtsA polypeptides, such as dimers tnmer tetramers and larger aggregations and their production and uses, as well as their vanants, their agonists and antagonists, and their uses In particular, the invention relates to multimenc polypeptides comprising FtsZ and FtsA subunits and their use in screening for antimicrobial compounds

BACKGROUND OF THE INVENTION In the prokaryotic cell cycle, the FtsZ (filamentation temperature sensitive) protein functions early in the septation process FtsZ polymerizes in a dynamic ring structure and localizes to the midpoint of the dividing cell This polymeric structure is believed to act as a scaffold for other important cell division proteins Among the proteins that are essential for cell division, FtsA functions bs localizing on the growth septum in an FtsZ -dependent manner The FtsA-FtsZ interaction is essential for bacterial cell division

Both FtsA and FtsZ are essential for cell division Mutants in E coli fail to form division septae and filament (Lutkenhaus J and A Mukherjee Cell Division in Eschenchia cob and Salmonella, Cellular and Molecular Biologv, F C Neidhardt ed Washington D C . ASM Press 1996, pp 1615-1626)

ClearK there is a need for factors, such as the compounds of the invention, that have a present benefit of bemg useful to screen compounds for antibiotic activity Such factors are also useful to determine their role in pathogenesis of infection, disfunction 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

SUMMARY OF THE INVENTION

The present invention relates to FtsZ FtsA multimeric and higher order forms of FtsZ and FtsA, particularh FtsZ and FtsA from Staphylococcus aureu and/or Eschenchia cob, Streptococcus pneumυniae and Eschenchia cob In another aspect, the invention relates to methods for using such pohpeptides including the treatment of icrobial diseases amongst others In a further aspect, the invention relates to methods for identifying agonists and antagonists using the materials provided by the invention and for treating microbial infections and conditions associated with such infections with the identified 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 the presence or activity of multimeric FtsZ FtsA polypeptides

DETAILED DESCRIPTION OF THE INVENTION The Applicants have demonstrated, using both the yeast two-hybrid assay and ELISA, that FtsA and FtsZ from both E co and S aureus are able to interact with specificity An extreme C- ternunus domain with 19 residues of S aureus FtsZ is required for this interaction with FtsA and Era, another protein involved in cell division This domain is believed to be an overlapping site involved in interaction with multiple proteins in the cell division cycle A fluorescence-based ELISA-type assay (DELF1A) can be used to screen for compounds that interfere with FtsA-FtsZ interaction Such compounds are expected to be broad spectrum antibacterial agents since both FtsA and FtsZ are essential bacterial cell division proteins that are highly conserved bacterial species The invention relates to multimenc FtsZ FtsA polypeptides as descπbed in greater detail below In particular the invention relates to polypeptides of a multimenc FtsZ FtsA of Staphylococcus aureus, Streptococcus pneumoniae and or Eschenchia cob Polynucleotide and polypeptide sequences of Streptococcus pneumoniae are disclosed in European Patent Publication Number 0899334, published March 3 1999 Polynucleotide and polypeptide sequences of Staphylococcus aureus are disclosed in US Provisional Patent Application Number 60/091,680, filed July 2, 1998 The invention relates especially to multimenc FtsZ FtsA each subumt compnsing the amino acid sequences set out in Table 1 as SEQ ID NO 2. 4 6 or 8

TABLE 1 Monomeric FtsZ Polypeptide Sequences

(A) Staphy lococcus aureus monomeric FtsZ polypeptide [SEQ ID NO 2]

MLEFEQGFNHI-ATLKVI GVGGGGNNAλΛJRMI DHGMNNVEFIAINTDGQALNLSKAESKIQI GEKLT GLGAGANP EI GKKAAEESREQI EDAI QGADMVFVTSGMGGGTGTGAAPWAKIAKEMGALTVGW RPFS FEGRKRQTQAAAG VEAMKAAVDTLIVI PNDRLLDIVDKSTP ^F-AFK--ADNVLRQGVQGI SDLIAVSGEVNLDFADVKTIMSNQGSAL MGIGVSSGENRAVF-AAKKAISSPLLETSIVGAQGVLMNITGGESLSLFEAQEAADIVQDAADEDVNMIFGTVINP E QDEIVλΛTVIATGFDDKPTSHGRKSGSTGFGTSVNTSSNATSKDESFTSNSSNAQATDSVSERTHTTKEDDIPS FI RNREERRSRRTRR

(B) Staphylococcus aureus polynucleotide encoding the monomeric FtsZ polypeptide of SEQ ID NO 2 [SEQ ID NO 1] ATGTTAGAATTTGAACAAGGATTTAATCATTTAGCGACTTTAAAGGTCATTGGTGTAGGTGGTGGCGGTAA CAACGCCGTAAACCGAATGATTGACCACGGAATGAATAATGTTGAATTTATCGCTATCAACACAGACGGTCAAG CTTTAAACTTATCTAAAGCTGAATCTAAAATCCAAATCGGTGAAAAATTAACACGTGGTTTAGGAGCAGGAGCT AACCCTGAAATCGGTAAAAAAGCTGCAGAGGAATCTCGTGAACAAATTGAAGATGCAATCCAAGGTGCAGACAT GGTATTTGTTACTTCTGGTATGGGTGGCGGAACTGGTACTGGTGCAGCACCAGTCGTTGCTAAAATTGCAAAAG AAATGGGCGCATTAACTGTTGGTGTTGTAACTCGTCCATTTAGTTTTGAAGGACGTAAACGTCAAACTCAAGCT GCTGCTGGAGTAGAAGCTATGAAAGCTGCAGTAGATACATTAATCGTTATACCAAATGACCGTTTATTAGATAT CGTTGACAAATCTACGCCAATGATGGAAGCATTTAAAGAAGCTGATAACGTGTTACGCCAAGGTGTACAAGGTA TCTCAGACTTAATCGCTGTTTCTGGTGAAGTAAACTTAGACTTTGCAGACGTTAAGACAATTATGTCTAACCAA GGTTCTGCATTAATGGGTATTGGTGTTTCTTCTGGTGAAAATAGAGCGGTAGAAGCTGCTAAAAAAGCAATCTC TTCTCCATTACTTGAAACATCTATCGTTGGTGCACAAGGTGTGCTTATGAATATTACTGGTGGCGAGTCATTGT CATTATTTGAAGCACAAGAGGCTGCTGATATTGTCCAAGATGCTGCAGATGAAGACGTTAATATGATTTTCGGT ACAGTTATTAATCCTGAATTACAAGATGAGATTGTTGTAACAGTTATTGCAACTGGTTTTGATGACAAACCAAC ATCACATGGTCGTAAATCTGGTAGCACTGGATTCGGAACAAGCGTAAATACTTCTAGCAATGCAACTTCTAAAG ATGAATCATTCACTTCAAATTCATCAAATGCACAAGCAACTGATAGTGTAAGTGAAAGAACGCATACAACTAAA GAAGATGATATTCCAAGCTTCATTAGAAATAGAGAAGAAAGACGTTCAAGAAGAACAAGACGTTAA

Monomeric FtsA Polypeptide Sequences

(C) Staphylococcus aureus monomeric FtsA polypeptide [SEQ ID NO:4].

MEEHYYVSIDIGSSSVKTIVGEKFHNGINVIGTGQTYTSGIKNG IDDFDIARQAIKDTIKKASIASGVDIKEVF LKLPIIGTEVYDESNEIDFYEDTEINGSHIEKVLEGIREKNDVQETEVINVFPIRFIVDKENEVSDPKELIARHS LKVEAGVIAlQKSILINMIKCVEACGVDVLDVYSDAYNYGSILTATEKELGACVIDIGEDVTQVAFYERGELVDA DSIEMAGRDITDDIAQGLNTSYETAEKVKHQYGHAFYDSASDQDIFTVEQVDSDETVQYTQKDLSDFIEARVEEI FFEVFDVLQDLGLTKVNGGFIVTGGSTNLLGVKELLSDMVSEKVRIHTPSQMGIRKPEFSSAISTISSSIAFDEL LDYVTINYHDNEETEEDVIDVKDKDNESKLGGFD FKRKTNKKDTHENEVESTDEEIYQSEDNHQEHKQNHEHVQ DKDKDKEESKFKKLMKSLFE

(D) Staphylococcus aureus polynucleotide encoding the monomeric FtsA polypeptide of SEQ ID N0 4 [SEQ ID NO:3].

ATGGAAGAACATTACTACGTAAGTATTGATATTGGATCATCAAGCGTAAAAACAATAGTAGGCGAGAAATTTCAC AATGGTATAAATGTGATAGGTACAGGACAAACCTACACGAGCGGTATAAAAAATGGTTTAATTGATGATTTTGAT ATTGCGCGACAAGCAATCAAAGACACAATTAAAAAGGCATCAATCGCTTCGGGTGTTGATATTAAAGAAGTTTTC CTGAAATTACCTATCATTGGAACGGAAGTTTATGATGAATCAAATGAAATCGACTTTTATGAGGATACAGAAATC AACGGTTCACATATCGAAAAAGTATTAGAAGGTATTAGAGAAAAAAATGATGTGCAAGAAACAGAAGTAATTAAT GTGTTCCCGATTCGTTTTATAGTCGATAAAGAAAATGAGGTTTCAGACCCTAAAGAATTAATTGCCAGACATTCA TTAAAGGTTGAAGCAGGCGTAATTGCTATTCAAAAATCGATTTTAATTAATATGATTAAATGCGTAGAAGCATGT GGTGTTGATGTATTAGATGTTTACTCTGATGCATATAACTATGGTTCAATCCTAACAGCTACTGAAAAAGAGTTA GGTGCATGTGTCATTGATATTGGTGAAGACGTTACGCAAGTTGCTTTTTATGAACGCGGTGAATTAGTAGATGCT GATTCTATCGAAATGGCAGGGCGTGATATTACAGACGATATTGCACAAGGATTAAACACTTCTTATGAAACTGCT GAAAAAGTTAAACACCAATATGGTCATGCATTCTATGATTCTGCTTCAGATCAAGATATCTTCACTGTTGAAC AGGTTGATAGTGATGAAACAGTACAGTATACTCAAAAAGATTTGAGTGACTTTATTGAAGCGCGTGTAGAAGAAA TATTCTTCGAAGTATTTGATGTTTTACAAGATTTAGGATTAACAAAAGTAAATGGTGGGTTTATTGTAACTGGTG GATCTACAAACTTACTTGGCGTAAAAGAATTATTATCAGATATGGTAAGTGAAAAAGTTAGAATTCACACGCCAT CACAAATGGGAATTAGAAAACCTGAATTTTCTTCAGCAATTTCTACAATTTCTAGTAGTATCGCTTTTGATGAGT TATTAGATTATGTTACAATTAATTATCATGATAATGAAGAAACTGAAGAAGATGTTATTGATGTGAAAGACAAAG ATAACGAATCTAAATTAGGCGGATTTGATTGGTTTAAACGTAAAACAAACAAAAAAGATACTCATGAAAATGAAG TAGAGTCAACAGATGAAGAAATTTATCAATCAGAAGATAATCATCAGGAACATAAACAGAATCATGAACATGTTC AAGACAAAGATAAAGATAAAGAAGAAAGTAAATTCAAAAAACTAATGAAATCTCTATTTGAATGA

Monomeric FtsZ Polypeptide Sequences of Eschenchia coli

(E) E chenchia coli monomeric FtsZ polypeptide [SEQ ID NO 6]

MFEPMELTNDAVIKVIGVGGGGGNAVEHMVRERIEGVEFFAVNTDAQALRKTAVGQTIQIGSGITKGLGAGANPE VGRNAADEDRDALRAALEGADMVFIAAGMGGGTGTGAAPVVAEVAKDLGILTVAVVTKPFNFEGKKRMAFAEQGI TELSKHVDSLITIPNDKLLKVLGRGISLLDAFGAANDVLKGAVQGIAELITRPGLIWVDFADVRTVMSEMGYAM GSGVASGEDRAEEAAEMAISSPLLEDIDLSGARGVLVNITAGFDLRLDEFETVGNTIRAFASDNATWIGTSLDP D^WDELRVTVVATGIGMDKRPEITLVTNKQVQQPVMDRYQQHGMAPL QEQKPVAKVVNDNAPQTAKEPDY DIP AFLRKQAD

(F) Eschenchia coli polynucleotide encoding the monomeric FtsZ polypeptide of SEQ ID NO 2

[SEQ ID NO 5]

ATGTTTGAACCAATGGAACTTACCAATGACGCGGTGATTAAAGTCATCGGCGTCGGCGGCGGCGGCGGTAATGCT GTTGAACACATGGTGCGCGAGCGCATTGAAGGTGTTGAATTCTTCGCGGTAAATACCGATGCACAAGCGCTGCGT AAAACAGCGGTTGGACAGACGATTCAAATCGGTAGCGGTATCACCAAAGGACTGGGCGCTGGCGCTAATCCAGAA GTTGGCCGCAATGCGGCTGATGAGGATCGCGATGCATTGCGTGCGGCGCTGGAAGGTGCAGACATGGTCTTTATT GCTGCGGGTATGGGTGGTGGTACCGGTACAGGTGCAGCACCAGTCGTCGCTGAAGTGGCAAAAGATTTGGGTATC CTGACCGTTGCTGTCGTCACTAAGCCTTTCAACTTTGAAGGCAAGAAGCGTATGGCATTCGCGGAGCAGGGGATC ACTGAACTGTCCAAGCATGTGGACTCTCTGATCACTATCCCGAACGACAAACTGCTGAAAGTTCTGGGCCGCGGT ATCTCCCTGCTGGATGCGTTTGGCGCAGCGAACGATGTACTGAAAGGCGCTGTGCAAGGTATCGCTGAACTGATT ACTCGTCCGGGTTTGATGAACGTGGACTTTGCAGACGTACGCACCGTAATGTCTGAGATGGGCTACGCAATGATG GGTTCTGGCGTGGCGAGCGGTGAAGACCGTGCGGAAGAAGCTGCTGAAATGGCTATCTCTTCTCCGCTGCTGGAA GAT^TCGACCTGTCTGGCGCGCGCGGCGTGCTGGTTAACATCACGGCGGGCTTCGACCTGCGTCTGGATGAGTTC GAAACGGTAGGTAACACCATCCGTGCATTTGCTTCCGACAACGCGACTGTGGTTATCGGTACTTCTCTTGACCCG GATATGAATGACGAGCTGCGCGTAACCGTTGTTGCGACAGGTATCGGCATGGACAAACGT CCTGAAATCACTCTGGTGACCAATAAGCAGGTTCAGCAGCCAGTGATGGATCGCTACCAGCAGCATGGGATGGCT CCGCTGACCCAGGAGCAGAAGCCGGTTGCTAAAGTCGTGAATGACAATGCGCCGCAAACTGCGAAAGAGCCGGAT TATCTGGATATCCCAGCATTCCTGCGTAAGCAAGCTGATTAA

Monomeric FtsA Polypeptide Sequences Eschenchia coli

(G) Eschenchia coli monomeric FtsA polypeptide [SEQ ID NO 8] MI KATDRKLVVGLEIGTAKVAALVGEVLPDGMVNIIGVGSCPSRGMDKGGVNDLESVVKCVQRAIDQAELMAD CQISSVYLALSGKHISCQNEIGMVPISEEEVTQEDVENVVHTAKSVRVRDEHRVLHVIPQEYAIDYQEGIKNPVG LSGVRMQAKVHLITCHNDMAKNIVKAVERCGLKVDQLIFAGLASSYSVLTEDERELGVCVVDIGGGTMDIAVYTG GALRHTKVIPYAGNWTSDIAYAFGTPPSDAF-AIKVRHGCALGSIVGKDESVEVPSVGGRPPRSLQRQTLAEVIE PRYTELLNLVNEEILQLQEKLRQQGVKHH AAGIVLTGGAAQIEGLAACAQRVFHTQVRIGAPLNITGLTDYAQE PYYSTAVGLLHYGKESHLNGEAEVEKRVTASVGS IKRLNSWLRKEF

(H) Eschenchia cob polynucleotide encoding the monomeric FtsA polypeptide of SEQ ID NO 4 |SEQ 1D N0 7] ATGATCAAGGCGACGGACAGAAAACTGGTAGTAGGACTGGAGATTGGTACCGCGAAGGTTGCCGCTTTAGTAGGGGAA GTTCTGCCCGACGGTATGGTCAATATCATTGGCGTGGGCAGCTGCCCGTCGCGTGGTATGGATAAAGGCGGGGTGAAC GACCTCGAATCCGTGGTCAAGTGCGTACAACGCGCCATTGACCAGGCAGAATTGATGGCAGATTGTCAGATCTCTTCG GTATATCTGGCGCTTTCTGGTAAGCACATCAGCTGCCAGAATGAAATTGGTATGGTGCCTATTTCTGAAGAAGAAGTG ACGCAAGAAGATGTGGAAAACGTCGTCCATACCGCGAAATCGGTGCGTGTGCGCGATGAGCATCGTGTGCTGCATGTG ATCCCGCAAGAGTATGCGATTGACTATCAGGAAGGGATCAAGAATCCGGTAGGACTTTCGGGCGTGCGGATGCAGGCA AAAGTGCACCTGATCACATGTCACAACGATATGGCGAAAAACATCGTCAAAGCGGTTGAACGTTGTGGGCTGAAAGTT GACCAACTGATATTTGCCGGACTGGCATCAAGTTATTCGGTATTGACGGAAGATGAACGTGAACTGGGTGTCTGCGTC GTCGATATCGGTGGTGGTACAATGGATATCGCCGTTTATACCGGTGGGGCATTGCGCCACACTAAGGTAATTCCTTAT GCTGGCAATGTCGTGACCAGTGATATCGCTTACGCCTTTGGCACGCCGCCAAGCGACGCCGAAGCGATTAAAGTTCGC CACGGTTGTGCGCTGGGTTCCATCGTTGGAAAAGATGAGAGCGTGGAAGTGCCGAGCGTAGGTGGTCGTCCGCCACGG AGTCTGCAACGTCAGACACTGGCAGAGGTGATCGAGCCGCGCTATACCGAGCTGCTCAACCTGGTCAACGAAGAGATA TTGCAGTTGCAGGAAAAGCTTCGCCAACAAGGGGTTAAACATCACCTGGCGGCAGGCATTGTATTAACCGGTGGCGCA GCGCAGATCGAAGGTCTTGCAGCCTGTGCTCAGCGCGTGTTTCATACGCAAGTGCGTATCGGCGCGCCGCTGAACATT ACCGGTTTAACGGATTATGCTCAGGAGCCGTATTATTCGACGGCGGTGGGATTGCTTCACTATGGGAAAGAGTCACAT CTTAACGGTGAAGCTGAAGTAGAAAAACGTGTTACAGCATCAGTTGGCTCGTGGATCAAGCGACTCAATAGTTGGCTG CGAAAAGAGTTTTAA

Deposited materials A deposit containing a Staphylococcus aureus WCUH29 strain has been deposited with the National

Collections of bdustπal and Marine Bactena Ltd (herein "NCIMB"), 23 St Machar Dπve, Aberdeen AB2 1RY. Scotland on 11 September 1995 and assigned deposit number 40771 The deposit was descπbed 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 contarns the full length FtsZ and FtsA genes The sequence of the poKτιucleotιdes contained in the deposited strain, as well as the amrno acid sequence of any polypeptide encoded thereby, are controlling in the event of any conflict with any descπption of sequences herein

The deposit of the deposited strain has been made under the terms of the Budapest Treaty on the bternational 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 derived therefrom, and no such license is hereby granted.

In one aspect of the invention there is provided an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Staphylococcus aureus WCUH29 strain, which polypeptide is contained in the deposited strain Further provided by the invention are FtsZ and FtsA polynucleotide sequences in the deposited strain, such as DNA and RNA, and amino acid sequences encoded thereby Also provided by the invention are multimenc FtsZ FtsA polypeptides compnsing the FtsZ FtsA polypeptide sequences isolated from the deposited strain and the polynucleotides that encode such seqeunces. Polypeptides

The multimenc FtsZ FtsA polypeptides of the invention is substantially phylogenetically related to other proteins ot die FtsZ and FtsA families. b one aspect of the invention there are provided polypeptides of Staphylococcus aureus, S pneumoniae and or Eschenchia cob multimeric FtsZ:FtsA polypeptides as well as biologically, diagnostically, prophylactically. clinically or therapeutically useful vanants thereof, and compositions comprising the same.

Among the particularly preferred embodiments of the invention are vanants of multimenc FtsZ:FtsA polypeptide each subunit of which is encoded by naturally occurring alleles of the FtsZ and or FtsZ genes The present invention further provides for an isolated polypeptide which:

(a) comprises or consists of a multimeric FtsZ:FtsA polypeptide each subunit of which is an ammo acid sequence which has at least 75% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% or exact identity, to that of SEQ ID NO 2. 4. 6 or 8 over the entire length of SEQ ID NO.2. 4. 6 or 8 respectively,

(b) a multimenc FtsZ FtsA polypeptide each subunit of which is a polypeptide encoded by an isolated poly nucleotide comprising or consisting of a polynucleotide sequence which has at least 75% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID NO:2, 4, 6 or 8 over the entire length of SEQ ID NO.2, 4, 6 or 8 respectively;

(c) a multimenc FtsZ FtsA polypeptide each subunit of which is a polypeptide encoded by an isolated poh nucleotide comprising or consisting of a polynucleotide sequence encoding a polypeptide which has at least 75% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99% or exact identity, to the -imino acid sequence of SEQ ID NO.2, 4, 6 or 8, over the entire length of SEQ ID NO:2, 4, 6 or 8 respectively. The multimenc FtsZ FtsA polypeptide include multimers each subunit of which compnses a polypeptide of Table 1 [SEQ ID NO 2, 4, 6 or 8] (in particular the mature polypeptide) as well as polypeptides and fragments, particularly those which have the biological activity of FtsZ or FtsA and constitute a multimenc FtsZ FtsA. preferably having the biological activity of multimenc FtsZ FtsA. and also those which have at least 75% identity to a polypeptide of Table 1 [SEQ ID NO 2, 4, 6 or 8]or the relevant portion preferably at least 80% identity to a polypeptide of Table 1 [SEQ ID NO 2, 4, 6 or 8] and more preferabK at least 90% identity to a polypeptide of Table 1 [SEQ ID NO 2. 4, 6 or 8] and still more preferably at least 95% identity to a polypeptide of Table 1 [SEQ ID NO 2, 4, 6 or 8] and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids

The invention also includes a multimenc FtsZ FtsA polypeptide each subunit of which is a poh pι-ptιde consisting of or compnsing a polypeptide of the formula

X-(R. )m-(R2MR3)n-Y wherein at the amino teπninus. X is hydrogen, a metal or any other moiety descnbed herein for modified polypeptides and at the carboxyl terminus, Y is hydrogen, a metal or any other moiety descnbed herein for modified polypeptides, R_] and R3 are any amino acid residue or modified amino 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 amino acid sequence of the imention particularh an ammo acid sequence selected from Table 1 or modified forms thereof b the formula above. R2 is onented so that its amino terminal amino acid residue is at the left, covalently bound to R_j and its carboxy terminal amino acid residue is at the nght covalently bound to R3 Any stretch of ammo acid residues denoted by either R_] or R3, where m and or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer Other preferred embodiments of the 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 preferred that a polvpeptide of the invention is deπved from Staphylococcus aureus, S pntumomae and or Eschenchia coli however it may preferably be obtained from other organisms of the same taxonomic genus as any of these organisms A polypeptide of the invention may also be obtained, for example from organisms of the same taxonomic family or order

A fragment is a vanant polypeptide having an amino acid sequence that is entirely the same as part but not all of anv amino acid sequence of any polypeptide of the invention Preferred fragments compnsing a multimenc FtsZ FtsA include, for example, truncation pol peptides ha\ mg a portion of an ammo acid sequence of Table 1 [SEQ ID NO 2, 4. 6 or 8], or of vanants thereof such as a continuous senes of residues that includes an amino- and/or carboxyl-termrnal amino acid sequence Degradation forms of the polypeptides of the invention produced by or in a host cell, particularly a Staphylococcus aureus, S pneumoniae and or Eschenchia cob, are also preferred Further preferred are fragments characteπzed by structural or functional attπbutes such as fragments that compnse alpha-helix and alpha-helix foπrung regions beta-sheet and beta-sheet-forming regions turn and tum-formmg regions coil and coil-forming regions, hvdrop lic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions Further preferred fragments include an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous ammo acids from the amino acid sequence ot SEQ ID NO 2, 4, 6 or 8, or an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated or deleted from the amino acid sequence of SEQ ID NO 2, 4, 6 or 8 A ten residue region amino acid (residues 371-380) in S aureus FtsZ is cntical for FtsA-FtsZ interaction A preferred embodiment of the invention is a polynucleotide encoding a polypeptide comprising these 10 amino acid residues preferabh encoding a polynucleotide that binds FtsA

A single residue (F376) in the above region of S aureus FtsZ is essential for the FtsA-FtsZ interaction A preferred embodiment of the invention provides a polynucleotide encodmg an S aureus FtsZ polvpeptide having a deletion of F376 or substitution of F376 with another ammo acid residue Another preferred embodiment of the invention provides an S aureus FtsZ polypeptide having a deletion of F376 or substitution of F376 with another amino acid residue

Still another preferred embodiment of the invention is a polypeptide comprising ammo acid residues 371 -380 (as set forth in S aureus FtsZ) preferably a polvpeptide that binds FtsA Also preferred are biologically active fragments which are those fragments that mediate activities of multimenc FtsZ FtsA once combined m multimenc form, including those with a similar activity or an improved activity or with a decreased undesirable activity Also mcluded are those fragments that are antigenic or immunogenic in an animal especially in a human Particularly preferred are fragments compnsing receptors or domains of enzsmes that confer a function essential for \ lability of Staphylococcus aureus S pneumoniae and/or Eschenchia cob or the ability to initiate, or maintain cause Disease in an individual, particularly a human

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 pohpeptides of the invention Vectors, Host Cells, Expression Systems

The invention also relates to vectors that compnse a polvnucleotide or polynucleotides of the invention, host cells that are genetically engineered with vectors of the mvention and the production of polypeptides of the mvention by recombinant techniques Cell-free translation systems can also be employed to produce such proteins using RNAs deπved from the DNA constructs of the mvention Recombinant polypeptides of the present invention may be prepared by processes well known in those skilled in the art from genetically engineered host cells compnsmg expression systems Accordingly, in a further aspect, the present mvention relates to expression systems which compnse a polynucleotide or pohnucleotides of the present invention, to host cells which 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 engineered to incoiporate expression systems or portions thereof or polynucleotides of the mvention btroduction of a polynucleotide into 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 Spπng Harbor Laboraton Press. Cold Spπng Harbor, N Y (1989). such as, calcium phosphate transfection, DEAE-dextran mediated transfection transvection. microinjection. cationic lipid-mediated transfection, electroporation, transduction scrape loading ballistic introduction and infection

Representative examples of appropnate hosts include bactena! cells, such as cells of streptococci, staphylococci, enterococci E cob, streptomyces, cyanobacteπa, Bacillus subtilis, and Staphylococcus aureus fungal cells such as cells of a yeast Kluveromyces. Saccharomyces, a basidiomycete, Candida alhicans and Aspergillus insect cells such as cells of Drosophύa S2 and Spodoplera Sf9, animal cells such as CHO COS HeLa, C 127. 3T3, BHK, 293, CV-1 and Bowes melanoma cells, and plant cells, such as cells of a or angiosperm A great vanety of expression systems can be used to produce the polypeptides of the invention Such vectors mclude, among others, chromosomal-, episomal- and virus-denved vectors, for example, vectors denved from bactenal plasmids, from bactenophage. from transposons, from yeast episomes, from insertion elements, from veast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as

SV40 vaccinia \ iruses adenoviruses fowl pox vimses, pseudorabies vinises. picornaviruses and retroMnises and vectors denved from combmations thereof, such as those denved from plasmid and bactenophage 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 of well-known and routine techniques such as for example, those set forth in Sambrook el al .

MOLECULAR CLONING, A LABORATORY MANUAL, (supra) b recombinant expression systems in eukaryotes, for secretion of a translated protein into the lumen of the endoplasmic reticulum, mto the penplasmic space or into the extracellular environment, appropnate secretion signals may be incorporated mto the expressed polypeptide These signals may be endogenous to the polypeptide or they may be heterologous signals Pohpeptides of the invention can be recovered and punfied from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectrn chromatography Most preferably, high performance liquid chromatography is employed for punfication Well known techmques for refolding protein ma\ be emplo ed to regenerate active conformation when the polypeptide is denatured during isolation and 01 purification

Diagnostic, Prognostic, Seroryping and Mutation Assays

This invention is also related to the use of multimenc FtsZ FtsA polypeptides of the mvention for use as diagnostic reagents Detection of multimenc FtsZ FtsA polypeptides in a eukaryote, particularly a mammal and especially a human, will provide a diagnostic method for diagnosis of disease, staging of disease or response of an infectious organism to drugs Eukarvotes particularly mammals, and especially humans, particularh those infected or suspected to be infected with an organism compnsing the multimenc FtsZ FtsA protem, may be detected by a vanety of well known techmques, for example, by non-denatunng protem gels, as well as by methods provided herein

Polypeptides for prognosis, diagnosis or other analysis may be obtained from a putatively infected and/or infected individual's bodily matenals b another embodiment an array of antibodies against multimenc FtsZ FtsA can be constructed to conduct efficient screening of for example genetic mutations serotype, taxonomic classification or identification

Thus in another aspect, the present invention relates to a diagnostic kit w ch compnses (a) a multimenc FtsZ FtsA polypeptide of the present invention, preferably a multimenc FtsZ FtsA polvpeptide comprising at least one polypeptide of SEQ ID NO 2, 4. 6 or 8 or a fragment thereof, or (b) an antibod , to a multimenc FtsZ FtsA polypeptide of the present invention, preferably a multimenc FtsZ FtsA pohpeptide comprising at least one polypeptide of SEQ ID NO 2, 4, 6 or 8

It will be appreciated that in any such kit, (a) or (b) may comprise a substantial component Such a kit will be of use in diagnosing a disease or susceptibility to a Disease, among others

Cells from an organism carrying mutations or polymorphisms (allehc vaπations) in a multimenc FtsZ FtsA polvpeptide of the invention mav also be detected at the polypeptide level by a vanety of techniques For example, a diagnostic assay in accordance with the mvention for detectmg over-expression of multimenc FtsZ FtsA polypeptide compared to normal control tissue samples may be used to detect the presence of an infection, among other diseases Assay techniques that can be used to determine levels of a multimenc FtsZ FtsA polypeptide, m a sample denved from a host, such as a bodily matenal, are well-known to those of skill in the art Such assay methods mclude radioimmunoassays, competitive-bmdmg assays, Western Blot analysis antibody sandwich assays, antibody detection and ELISA assays Antagonists and Agonists - Assays and Molecules

Multimenc FtsZ FtsA polypeptides of the mvention may also be used to assess the binding of small molecule substrates and hgands m, for example, cells, cell-free preparations, chemical hbraπes, and natural product mixtures These substrates and hgands may be natural substrates and hgands or may be structural or functional mimetics See, e g , Coligan et al , Current Protocols in Immunology 1(2) Chapter 5 ( 1991 )

Mutlimeπc FtsZ FtsA polypeptides of the present mvention are responsible for or mvolved in many biological functions, such as bactenal cell division and bacteπal cell survival h view of the fact that such mutlrmenc FtsZ FtsA polypeptides carry important bacteπal biological functions, they are therefore responsible for the mamtenance of many disease states, in particular the Diseases herembefore mentioned It is therefore desirable to devise screening methods to identify compounds which stimulate or which inhibit the function of a mutlimeπc FtsZ FtsA polypeptide and lead to slowing or stopping of bacteπal growth or bacterial killing Accordingh in a further aspect, the present mvention provides for a method of screening compounds to identify those which stimulate or which inhibit the function of a mutlrmenc FtsZ FtsA polypeptide of the mvention, as well as related polypeptides b general, agonists or antagonists may be employed for therapeutic and prophylactic purposes for such Diseases as herembefore mentioned Compounds may be identified from a vanety of sources, for example, cells, cell-free preparations, chemical libranes and natural product mixtures Such agonists, antagonists or inhibitors so-identified may be natural or modified substrates, hgands, receptors, enzymes, etc , as the case may be, of multimenc FtsZ FtsA polypeptides, 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 a mutlimenc FtsZ FtsA polypeptide or polynucleotide, or to cells or membranes bearing such polypeptide, or a fusion protein of the polypeptide bv 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 a mutlrmenc FtsZ FtsA polypeptide, using detection svstems appropriate to the cells comprising such polypeptide 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 mutlimenc FtsZ FtsA polypeptide and/or constitutively expressed polypeptides may be employed in screening methods for inverse agonists or inhibitors, in the absence of an agonist or inhibitor, by testing whether the candidate compound results in inhibition of activation of mutlimenc FtsZ FtsA polypeptide Further, the screening methods may simply comprise the steps of mixing a candidate compound with a solution containing a mutlimenc FtsZ FtsA polypeptide of the present invention, to form a mixture, measuring multimenc FtsZ FtsA poh peptide activity in the mixture and comparing the multimenc FtsZ FtsA polypeptide activity of the mixture to a standard Fusion proteins, such as those made from GST and multimenc FtsZ FtsA polypeptide, as herein before described, can also be used for high-throughput screenmg assays to identify agonists and antagonists of the polypeptide of the present invention, as well as of phylogenetically and and/or functionally related polypeptides (see D Bennett et al , J Mol Recognition, 8 52-58 (1995), and K Johanson et al , J Biol Chem, 270(16) 9459-9471 (1995)) As used herein "activity" means any functional attribute of a FtsZ or FtsA polypeptide or multimenc FtsZ FtsA polypeptide, including, for example, enzymatic activity (such as GTPase activity), binding of hgands, substrates (such as GTP) or subunits (such as FtsZ polypeptide), the formation of multimers, the disassociation of multimers, cell division cell growth ring formation and cell wall invagination, among others

Mutlrmenc FtsZ FtsA polypeptides and antibodies that bind to and or interact with such poh peptides of the present invention may also be used to configure screenmg methods for detecting the effect of added compounds on the production mutlimenc FtsZ FtsA polypeptide m cells For example, an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide usmg monoclonal and polyclonal antibodies by standard methods known in the art This can be used to discover agents which may inhibit or enhance the production of polypeptide (also called antagonist or agonist respectively) from suitably manipulated cells or tissues

The rmention also provides a method of screemng compounds to identify those which enhance (agonist) or block (antagonist) the action of multimenc FtsZ FtsA polypeptides, particularly those compounds that are bactenostatic and/or bacteπocidal The method of screemng may mvolve high-throughput techmques For example, to screen for agonists or antagonists, a synthetic reaction mix, a cellular compartment, such as a membrane cell envelope or cell wall, or a preparation of any thereof, compnsing multimenc FtsZ FtsA pohpeptide and a labeled substrate or ligand of such polypeptide is incubated in the absence or the presence of a candidate molecule that ma\ be a multimenc FtsZ FtsA agonist or antagonist The ability of the candidate molecule to agonize or antagonize the multimenc FtsZ FtsA polypeptide is reflected in decreased binding of the labeled ligand or decreased production of product from such substrate Molecules that bind gratuitously, / e , without inducing the effects of multimenc FtsZ FtsA polypeptide are most likely to be good antagonists Molecules that bind well and as the case may be mcrease the rate of product production from substrate mcrease signal transduction, or mcrease chemical channel activity are agonists Detection of the rate or level of as the case may be, production of product from substrate, signal transduction, or chemical channel activity may be enhanced by usmg a reporter system Reporter systems that may be useful in this regard include but are not limited to coloπmetnc, labeled substrate converted mto product, a reporter gene that is responsive to changes m multimenc FtsZ FtsA polypeptide activity, and binding assays known in the art A prefeπed agonist or antagonist of the mvention b d to a sequence selected from the group consistmg of a ten residue amino acid region corresponding to residues 371-380 in S aureus FtsZ, and at least a single amino acid residue corresponding to residue F376 in S aureus FtsZ

A prefened method of the mvention for detectmg agonists or antagonists of the mteraction of FtsZ and FtsA compnses a step of contactmg a compound, such as a test compound, to an FtsZ polypeptide of the mvention and detectmg if said compound bmds to a sequence selected from the group consistmg of a ten residue ammo acid region corresponding to residues 371-380 in S aureus FtsZ, and at least a single amino acid residue conespondmg to residue F376 m S aureus FtsZ

Another prefened method of the mvention for detecting agonists or antagonists of the mteraction of FtsZ and FtsA compnses a step of contactmg a compound, such as a test compound, to an FtsZ polypeptide of the invention and detecting if said compound agonizes or antagonizes at least on biological activity of FtsZ or FtsA wherein said FtsZ compnses a sequence selected from the group consistmg of a ten residue ammo acid region corresponding to residues 371-380 in S aureus FtsZ, and at least a single ammo acid residue corresponding to residue F376 in S aureus FtsZ A further preferred method is provided whereby said FtsZ lacks a sequence selected from the group consistmg of a ten residue amino acid region corresponding to residues 371-380 in S aureus FtsZ. and at least a single amino acid residue corresponding to residue F376 in S aureus FtsZ

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

A preferred assay to screen for antimicrobial compounds using multimenc FtsZ FtsA pohpeptide is to add a test compound to multimenc FtsZ FtsA and determine compounds that antagonist the FtsZ FtsA interaction by measuring the inhibition of multimenc FtsZ FtsA over after the addition of a test compound Compounds that antagonist the FtsZ FtsA interaction are believed to block the FtsA FtsZ bindmg

Preferred assay conditions for the formation of multimenc FtsZ FtsA are 140 mM NaCl, 2 7 mM KC1. 10 mMNa₂HP0₄, 1 8 mM KH₂P0₄, pH 7 3, 0 2% Triton X-100 and a blocking agent (BS A and/or Casein) A number of assays can be used to characterize compounds that block the FtsZ FtsA association The interaction between FtsZ and FtsA monomers can be monitored with techniques such as yeast two-hybrid system, ELISA, DELFIA, analytical ultracentπfugation, fluorescence polarization fluorescence energy transfer, surface plasmon resonance scintillation proximity assay and the use of ion channel switch (ICS) biosensors, among other well known detection techniques The veast two-hybrid system is a genetic approach to detect protein-protein interactions in vivo This assay can be used to monitor FtsA-FtsZ interaction m yeast cells Since both FtsA and FtsZ are expressed in yeast cells as fusion proteins and the mteraction of these protem is in solution, the interaction is expected to resemble what happens m bacterial cells Inhibition of FtsA-FtsZ interaction bv compounds can be easily detected by a sensitive reporter, among other detection methods

Another assay is the conventional ELISA, which is well documented for analysis of protein- protein binding in vitro In this assav, FtsA is coated on a plate and FtsZ with a GST tag will be applied to wells of the plate Binding of FtsZ to FtsA will be detected by an anti-GST antibody, followed by a secondary antibody-enzyme conjugate

Alternative to the ELISA, a DELFIA assay (EG&G Wallac) is available to screen compounds that block FtsA-FtsZ binding The DELFIA assay is ELISA-type assay m which GST- FtsZ binds to FtsA coated on a plate The binding is detected by the anti-GST antibody that is labeled with fluorescent dye europium Analytical ultracentπfugation is a practical and robust method for measunng molecular mass m solution It may be used to demonstrate that FtsZ and FtsA undergo a monomer-dimer-tetramer or assembly process m the absence of FtsZ and FtsA active site hgands or compounds that otherwise bind or interact with FtsZ or FtsA While not wanting to be limited to theoretical models, the Applicants believe that the midpoints of both assembly processes are at about 10 micromolar FtsZ and FtsA so the technique is ideally suited to quantitatively monitor changes m assoication that might occur as a result of the addition of small molecule inhibitors The use of interference optics is preferred and allows these experiments to be done, irrespective of the absorption or fluorescence properties of the inhibitors

The fluorescence polarization value for a fluorescently-tagged molecule depends on the rotational correlation time or tumbling rate Large protein complexes, such as multimenc FtsZ FtsA, labeled to comprise a fluorescently-labelled molecule will have higher polarization values than a fluorescently labelled monomeric protein It is preferred that this method be used to characterize small molecules that disrupt FtsZ FtsA ohgomers

Fluorescence energy transfer may be used characterize small molecules that interfere with the fonnation of FtsZ FtsA dimers trimers tetramers or higher order structures FtsZ and FtsA 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 monitor the effect of small molecules on FtsZ FtsA association FtsZ and/or FtsA can be coupled to a sensor chip at low site density such that covalently bound molecules will be monomeric Solution protein can then passed over the FtsZ- or FtsA-coated surface and specific binding can be detected in real-time by monitoring the change in resonance angle caused by a change in local refractive index This technique can be used to charactenze the effect of small molecules on kinetic rates and equilibrium binding constants for FtsZ FtsA association

A scintillation proximity assay may be used to characterize the interaction between FtsZ or FtsA monomers FtsZ and or FtsA can be coupled to a scintillation-filled bead Addition of radio- labeled FtsZ results in binding where the radioactive source molecule is m close proximity to the scintillation fluid Thus, signal is emitted upon FtZ binding and compounds that prevent FtsZ self- association will diminish signal

ICS biosensors have been described by AMBR1 (Australian Membrane Biotechnology Research Institute) They couple the self-association of macromolecules to the closing of gramacidm- facihtated ion channels in suspended membrane bilayers and hence to a measurable change in the admittance (similar to impedence) of the biosensor This approach is linear over six decades of admittance change and is ideally suited for large scale, high through-put screening of small molecule combmatoπal libraries b other embodiments of the mvention there are provided methods for identifying compounds which bind to or otherwise interact with and inhibit or activate an activity or expression of a mutlrmenc FtsZ FtsA polypeptide of the mvention compnsing contactmg a polypeptide of the mvention with a compound to be screened under conώtions to permit bmdmg to or other mteraction between the compound and the polypeptide to assess the bmdmg to or other mteraction with the compound, such binding or mteraction preferably bemg associated with a second component capable of providing a detectable signal m response to the bmdmg or mteraction of the polypeptide with the compound and determining whether the compound bmds to or otherwise interacts with and activates or inhibits an activity or expression of the polypeptide and/or pohTiucleotide by detectmg the presence or absence of a signal generated from the bmdmg or mteraction of the compound ith the polypeptide

A prefened assay embodiment compnses the step of contactmg an organism with a test comound and detectmg if FtsZ or FtsA is involved in forming a septal πng usmg an antibody of the mvention Compounds that are associated with disagregation of such πng are prefened as antimicrobial compounds Another example of an assay for multimenc FtsZ FtsA agonists is a competitive assay that combines multimenc FtsZ FtsA and a potential agonist with multimenc FtsZ FtsA-binding molecules, recombinant multimenc FtsZ FtsA bmdmg molecules, natural substrates or hgands, or substrate or ligand mimetics under appropnate conditions for a competitive inhibition assay The multimenc FtsZ FtsA molecule can be labeled such as by raώoactivity or a colonmetnc compound, such that the number of multimenc FtsZ FtsA molecules bound to a bmdmg molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist

Potential antagonists mclude, among others, small organic molecules, peptides, polypeptides and antibodies that bmd to a polypeptide of the mvention and thereby inhibit or extinguish its activity or expression Potential antagonists also may be small organic molecules, a peptide, a polypeptide such as a closeh related protem or antibody that bmds the same sites on a bmdmg molecule, such as a bmdmg molecule, without mducing multimenc FtsZ FtsA-induced activities, thereby preventing the action or expression of multimenc FtsZ FtsA polypeptides by excluding multimenc FtsZ FtsA polypeptides from bmdmg

Potential antagonists mclude a small molecule that bmds to and occupies the bmdmg site of the polypeptide thereby preventmg binding to cellular bmdmg molecules, such that normal biological activity is prevented Examples of small molecules mclude but are not limited to small organic molecules, peptides or peptide-Iike 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 descπption of these molecules) Prefened potential antagonists mclude compounds related to and vanants of multimenc FtsZ FtsA Other examples of potential polypeptide antagonists mclude antibodies or, in some cases, ohgonucleotides or proteins which are closely related to the hgands substrates, receptors, enzymes, etc , as the case may be, of the polypeptide, e g , a fragment of the hgands substrates receptors enzymes, etc or small molecules which bmd to the polypeptide of the present invention but do not elicit a response, so that the activity of the polypeptide is prevented Certain of the polypeptides of the mvention are biomimetics functional mimetics of the natural multimenc FtsZ FtsA polypeptide These functional mimetics may be used for, among other things, antagonizing the activity of multimenc FtsZ FtsA polypeptide or as a antigen or rmmunogen in a manner descnbed elsewhere herein Functional mimetics of the polypeptides of the mvention mclude but are not limited to truncated polypeptides For example prefened functional mimetics mclude, a mutlimenc FtsZ FtsA each subunit compnsing a polypeptide compnsmg the polypeptide sequence set forth m SEQ ID NO 2 4 6 or 8 lacking 20 30, 40, 50, 60, 70 or 80 ammo- or carboxy-termmal ammo acid residues, including fusion proteins compnsmg one or more of these truncated sequences Polynucleotides encoding each of these functional mimetics may be used as expression cassettes to express each mimetic polypeptide It is prefened that these cassettes compnse 5' and 3' restnction sites to allow for a convenient means to hgate the cassettes together when desired It is further prefened that these cassettes compnse gene expression signals known in the art or descnbed elsewhere herein

Thus, in another aspect, the present invention relates to a screening kit for identifying agonists, antagonists, hgands, receptors, substrates, enzymes, etc for a polypeptide and/or polynucleotide of the present invention, or compounds which decrease or enhance the production of such polypeptides and/or polynucleotides . which comprises

(a) a mutlimenc FtsZ FtsA polypeptide of the present invention,

(b) a recombinant cell expressing a mutlimenc FtsZ FtsA polypeptide of the present invention,

(c) a cell membrane expressing a mutlimenc FtsZ FtsA polypeptide of the present invention, or (d) antibody to an mutlimenc FtsZ FtsA polypeptide of the present invention, w ch polypeptide is preferably that of SEQ ID NO 2, 4, 6 or 8

It will be appreciated that in any such kit, (a), (b), (c) or (d) may compnse a substantial component

It will be readily appreciated by the skilled artisan that a polypeptide of the present invention ma\ also be used in a method for the structure-based design of an agonist, antagomst or inhibitor of a mutlimenc FtsZ FtsA polypeptide, by

(a) deteπnining in the first instance the three-dimensional structure of the polypeptide, or complexes thereof

(b) deducing the three-dimensional structure for the likely reactive sιte(s), binding sιte(s) or motιf(s) of an agonist antagonist or inhibitor,

(c) s nthesizing candidate compounds that are predicted to bind to or react with the deduced binding sιte(s), reactive sιte(s), and/or motιf(s), and

(d) testing whether the candidate compounds are indeed agonists, antagonists or inhibitors

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 b a further aspect, the present mvention provides methods of treating abnormal conώtions 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 multimenc FtsZ FtsA polypeptide

If the expression and/or activity of the polypeptide is in excess, several approaches are available One approach compnses admmistermg to an mώvidual m need thereof an inhibitor compound (antagonist) as herein descnbed optionally in combmation with a pharmaceutically acceptable earner, in an amount effective to inhibit the function and/or expression of the polypeptide. such as, for example, by blocking the binding of ligands substrates receptors, enzymes, etc or by inhibiting a second signal and thereby alleviating the abnormal condition In another approach, soluble forms of the polypeptides still capable of binding the ligand, substrate, enzymes, receptors, etc m competition with endogenous polypeptide may be administered Typical examples of such competitors include fragments of the multimenc FtsZ FtsA pohpeptide

In a further aspect the present invention relates to genetically engineered soluble fusion proteins comprising a po peptide of the present invention, or a fragment thereof, and various portions of the constant regions of heaw or light chains of lmmunoglobuhns of various subclasses (IgG, IgM, IgA, IgE) Preferred as an lmmunoglobuhn is the constant part of the heavy chain of human IgG, particularly lgG l , where fusion takes place at the hinge region In a particular embodiment, the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa Furthermore this invention relates to processes for the preparation of these fusion proteins bv genetic engineering and to the use thereof for drug screening, diagnosis and therapy A further aspect of the invention also relates to polynucleotides encoding such fusion proteins Examples of fusion protein technology can be found in International Patent Application Nos W094/29458 and W094/22914

In still another approach, expression of the gene encoding endogenous multimenc FtsZ FtsA polypeptide can be inhibited using expression blocking techniques This blocking may be targeted against any step in gene expression, but is preferably targeted against transcription and or translation An examples of a known technique of this sort involve the use of antisense sequences, either internally generated or separately administered (see, for example, O'Connor, J Neurochem (1991) 56 560 in O godeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL ( 1988)) Altemativelv ohgonucleotides which form triple helices with the gene can be supphed (see, for example Lee et al Nucleic Acids Res (1979) 6 3073, Cooney et al , Science (1988) 241 456, Dervan el al -Sc/e«ce ( 1991) 25 1 1360) These ohgomers can be administered per se or the relevant ohgomers can be expressed in vivo b accordance with yet another aspect of the mvention, there are provided multimenc FtsZ FtsA polypeptide agonists and antagonists, preferably bactenostatic or bacteπocidal agonists and antagonists

The antagonists and agonists of the mvention may be employed, for instance, to prevent, inhibit and/or treat ώseases

Hebcohacter pylon (herein "H pylori") bacteria mfect the stomachs of over one-third of the world's population causing stomach cancer, ulcers, and gastritis (International Agency for Research on Cancer ( 1994) Schistosomes, Liver Flukes and Helicobacler 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 gastric adenocarcinoma, classifying the bacterium as a Group I (definite) carcinogen Preferred antimicrobial compounds of the invention (agonists and antagonists of multimenc FtsZ FtsA poh peptides and or polynucleotides) found using screens provided by the invention, or known in the art, particularly narrow-spectrum antibiotics, should be useful in the treatment of H pylon infection Such treatment should decrease the advent of H /?y/ør/ -induced cancers, such as gastrointestinal carcinoma Such treatment should also prevent inhibit and/or cure gastric ulcers and gastritis Vaccines There are provided by the mvention. products, compositions and methods for assessing multimenc

FtsZ FtsA expression, treating disease, assaying genetic vanation, and adrnimstenng a multimenc FtsZ FtsA polypeptide and/or polynucleotide to an organism to raise an lmmunological response against a bactena, especially a Staphylococcus aureus, S pneumoniae and/or Eschenchia cob bactena

Another aspect of the invention relates to a method for inducing an lmmunological response in an individual, particularly a mammal which comprises inoculating the individual with multimenc FtsZ FtsA polypeptide or a fragment or variant thereof adequate to produce antibody and/ or T cell immune response to protect said individual from infection, particularly bacterial infection and most particularly Staphylococcus aureus, S pneumoniae or Eschenchia cob infection Also provided are methods whereby such lmmunological response slows bacteπal replication Yet another aspect of the invention relates to a method of inducing lmmunological response in an individual which compnses delivering to such individual a nucleic acid vector, sequence or πbozyme to direct expression of multrmenc FtsZ FtsA polypeptide. or a fragment or a variant thereof, for expressing multimenc FtsZ FtsA polypeptide, or a fragment or a variant thereof in vivo m order to induce an lmmunological response such as to produce antibody and/ or T cell immune response, including, for example, cytokine-producmg T cells or cytotoxic T cells, to protect said individual, preferably a human, from disease, whether that disease is already established withm the individual or not One example 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 πbozyme, a modified nucleic acid, a DNA/RNA hvbπd a DNA-protem complex or an RNA-protem complex A further aspect of the invention relates to an lmmunological composition that when introduced mto an individual preferably a human, capable of having induced within it an lmmunological response, induces an lmmunological response in such individual to a multimenc FtsZ FtsA polypeptide, wherem the composition comprises a recombinant multimenc FtsZ FtsA polypeptide The lmmunological rc ponse mav be used therapeutically or prophv lacticalh and may take the form of antibody immunity andor cellular immunity such as cellular immunity arising from CTL or CD4+ T cells

A multimenc FtsZ FtsA polypeptide or a fragment thereof may be fused with co-protem or chemical moiety which may or may not by itself produce antibodies, but which is capable of stabilizing the first protein and producing a fused or modified protein which will have antigenic and/or lmmunogemc properties, and preferably protective properties Thus fused recombinant protem, preferabh further comprises an antigenic co-protein, such as lipoprotem D from Hemophilus influenzae Glutathione-S-transferase (GST) or beta-galactosidase, or any other relatively large co- protein which solubihzes the protein and facilitates production and purification thereof Moreover, the co-protein ma\ act as an adjuvant in the sense of providing a generalized stimulation of the immune system of the organism receiving the protein The co-protem may be attached to either the ammo- or carboxv-terminus of the first protem

Prov ided b\ this invention are compositions particularh vaccine compositions, and methods comprising the poh peptides ot the invention and lmmunostimulaton DNA sequences such as those described m 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, in polynucleotide constructs used in such genetic immunization expenments in animal models of infection with Staphylococcus aureus, S pneumoniae or Eschenchia cob Such experiments will be particularh useful for identif ing 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 derived from the requisite organ of the animal successfully resisting or clearing infection, for the development of prophylactic agents or therapeutic treatments of bactenal infection particularly Staphylococcus aureus S pneumoniae and or Eschenchia cob infection, m mammals particularly humans

\ poh peptide of the inv ention mav be used as an antigen for vaccination of a host to produce specific antibodies which protect against invasion of bacteria, for example by blocking adherence of bacteria to damaged tissue Examples of tissue damage include wounds in skin or connective tissue caused, for example, by mechanical, chemical, thermal or radiation damage or by implantation of indwelling devices, or wounds in the mucous membranes, such as the mouth, throat, mammary glands, urethra or vagina The invention also includes a vaccine formulation which comprises an lmmunogenic recombinant polypeptide and/or polynucleotide of the invention together with a suitable carrier, such as a pharmaceutical h acceptable carrier Since the polypeptides and polynucleotides may be broken down in the stomach each is preferably administered parenterally, including, for example, administration that is subcutaneous, intramuscular, intravenous, or lntradermal Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti- oxidants buffers bacteπostatic compounds and solutes which render the formulation isotonic with the bodih fluid preferably the blood, of the individual, and aqueous and non-aqueous sterile suspensions which mav include suspending agents or thickening agents The formulations may be presented in unit- dose or multi-dose containers, for example, sealed ampoules and vials and may be stored m a freeze- dried condition requiring only the addition of the sterile liquid carrier immediately prior 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 activitv of the vaccine and can be readily determined by routine experimentation

While the invention has been described with reference to certain multimenc FtsZ FtsA poly peptides it is to be understood that this covers fragments of the naturally occurnng polypeptides, and similar polypeptides and polynucleotides with additions, deletions or substitutions which do not substantially affect the lmmunogenic properties of the recombinant polypeptides or polynucleotides. Compositions, kits and administration

In a further aspect of the invention there are provided compositions compnsmg a multimenc FtsZ Fts \ polypeptide for administration to a cell or to a multicellular organism

The mvention also relates to compositions compnsmg a polynucleotide and/or a polypeptides ώscussed herein or their agonists or antagonists The polypeptides and polynucleotides of the mvention may be employed in combmation with a non-stenle or stenle earner or earners for use with cells, tissues or organisms, such as a pharmaceutical earner suitable for administration to an mώvidual Such compositions compnse for mstance a meώa adώtive or a therapeutically effective amount of a polypeptide and/or pohnucleotide of the mvention and a pharmaceutically acceptable earner or excipient Such earners may mclude but are not limited to, sal e, buffered saline, dextrose, water, glycerol, ethanol and combmations thereof The formulation should suit the mode of administration The mvention further relates to diagnostic and pharmaceutical packs and kits compnsing one or more containers filled with one or more of the ingredients of the aforementioned compositions of the mvention

Polypeptides, polynucleotides and other compounds of the mvention may be employed alone or in conjunction with other compounds, such as therapeutic compounds

The pharmaceutical compositions may be admmistered m any effective, convenient manner including, for mstance, administration by topical, oral, anal, vaginal, intravenous, mtrapentoneal, intramuscular, subcutaneous, lntranasal or intradermal 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 isotomc

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 appropriate conventional additives, including, for example, preservatives, solvents to assist drug penetration, and emollients m 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 carriers 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 b a further aspect the present mvention provides for pharmaceutical compositions compnsmg a therapeutically effective amount of a polypeptide and/or polynucleotide, such as the soluble form of a polypeptide and/or polynucleotide of the present mvention. agonist or antagonist peptide or small molecule compound, in combination with a pharmaceutically acceptable earner or excipient Such earners mclude. but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof The mvention further relates to pharmaceutical packs and kits compnsmg one or more containers filled with one or more of the ingredients of the aforementioned compositions of the mvention Polypeptides, polynucleotides and other compounds of the present mvention may be employed alone or in conjunction with other compounds, such as therapeutic compounds The composition will be adapted to the route of aα-mrrustration, for instance by a systemic or an oral route Prefened forms of systemic admmistration mclude injection, typically by mtravenous injection Other injection routes such as subcutaneous intramuscular or intrapentoneal can be used Alternative means for svstemic administration mclude transmucosal and transdermal administration usmg penetrants such as bile salts or fusiώc acids or other detergents b adώtion, if a polypeptide or other compounds of the present mvention can be formulated m an entenc or an encapsulated formulation, oral administration may also be possible Admmistration of these compounds may also be topical and/or localized, m the form of salves, pastes gels and the like

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 in any event will determine the actual dosage wbch 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 merited and such are within the scope of this invention

In-dwelling devices include surgical implants, prosthetic devices and catheters, l 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, cerebrospmal fluid shunts, urinary catheters, continuous ambulatory pentoneal dialysis (CAPD) catheters

The composition of the invention may be administered by injection to achieve a systemic effect against relevant bacteria 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 bacteπal wound infections, especially

Staphylococcus aureus, S pneumoniae and or Eschenchia cob wound infections

Many orthopedic surgeons consider that humans with prosthetic joints should be considered for antibiotic prophylaxis before dental treatment that could produce a bacteremia Late deep mfection is a serious complication 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 described above, the compositions of tbs invention may be used generally as a wound treatment agent to prevent adhesion of bacteria to matrix proteins exposed wound tissue and for prophylactic use in dental treatment as an alternative to, or m 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 1 μg/ml to 1 Omg/ml for bathing of wounds or indwelling devices

A vaccine composition is conveniently m mjectable form Conventional adjuvants may be employed to enhance the immune response A suitable unit dose for vaccmation 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

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

GLOSSARY

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

"Activity" means any functional attribute of a FtsZ or FtsA polypeptide or multimenc

FtsZ FtsA polypeptide, including, for example, enzymatic activity (such as GTPase activity), bmdmg of hgands. substrates (such as GTP) or subunits (such as FtsZ polypeptide), the formation of multimers, the disassociation of multimers, cell division, cell growth ring formation and cell wall invagination, among others

"Antιbody(ιes)" as used herein includes polyclonal and monoclonal antibodies, chimenc, smgle chain and humanized antibodies, as well as Fab fragments including the products of an Fab or other lmmunoglobuhn expression library

"Antigenically equivalent deπvatιve(s)" as used herein encompasses a polypeptide, polynucleotide, or the equivalent of either which will be specifically recognized by certain antibodies which, when raised to the protein, polypeptide or polynucleotide according to the invention, interferes with the immediate physical interaction between pathogen and mammalian host

"Bispecific antιbody(ιes)" means an antibody comprising at least two antigen binding domains, each domain directed against a different epitope "Bodily mateπal(s) means any matenal denved from an mώvidual or from an organism nfectmg, infesting or inhabiting an individual, including but not limited to, cells, tissues and waste, such as, bone, blood serum, cerebrospmal fluid, semen, saliva, muscle, cartilage, organ tissue, skin, urine, stool or autopsy matenals

"Dιsease(s)" means any ώsease caused by or related to mfection by a bactena, including , for example otitis media conjunctivitis, pneumonia bacteremia meningitis sinusitis, pleural empyema and endocarditis and most particularly meningitis such as for example infection of cerebrospmal fluid, infections of the upper respiratory tract (e g , otitis meώa, bactenal tracheitis, acute epiglottitis, thyroiώtis), lower respiratory (e g empyema, lung abscess), carώac (e g , infective endocarditis), gastrointestinal (e g , secretory ώanhoea, splenic absces, retropeπtoneal abscess), CNS (e g , cerebral abscess), eye (e g , blephaπtis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital cellulitis, darcryocystitis), kidney and urinary tract (e g , epiώdymitis, intrarenal and pennephnc absces, toxic shock syndrome), skin (e g , impetigo fol culitis, cutaneous abscesses, cellulitis, wound infection, bactenal myositis) and bone and joint (e g septic arthntis, osteomyelitis)

"FtsA" means, as the case may be, FtsA polypeptides, FtsA polynucleotides, each including, for example, those from any organism defined herem

"FtsZ" means, as the case may be, FtsZ polypeptides, FtsZ polynucleotides, each including, for example those from any organism defined herem

"Fusion protem(s)" refers to a protein encoded by two, often unrelated, fused genes or fragments thereof In one example, EP-A-0464 discloses fusion proteins comprising various portions of constant region of immunoglobulm molecules together with another human protem or part thereof In many cases, employing an immunoglobulm Fc region as a part of a fusion protein is advantageous for use in therapy and diagnosis resulting in, for example, improved pharmacokmetic properties [see, e g , EP-A 0232262] On the other hand, for some uses it would be desirable to be able to delete the Fc part after the fusion protein has been expressed, detected and purified "Host cell(s)" is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous polynucleotide sequence

"Identity," as known m the art, is a relations p between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determined by comparing the sequences b the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences as the case may be, as determined by the match between strings of such sequences "Identity" can be readily calculated by known methods, including but not limited to those described (Computational Molecular Biology, Lesk, A M , ed , Oxford Umversity Press, New York, 1988, Biocomputing Informatics and Genome Projects, Smith, D W , ed , Academic Press, New York, 1993. Computer Analysis of Sequence Data, Part I, Griffin, A M , and Griffin, H G , eds , Humana Press New Jersey, 1994, Sequence Analysis in Molecular Biology, von Hemje, G . Academic Press, 1987. and Sequence Analysis Primer, Gπbskov. M and Devereux, J , eds , M Stockton Press, New York, 1991. and Caπllo, H , and Lipman, D , SIAM J Applied Math , 48 1073 (1988) Methods to determine identity are designed to give the largest match between the sequences tested Moreover, methods to determine identity are codified in publicly available computer programs Computer program methods to determine identity between two sequences 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 (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 NDH Bethesda. MD 20894, Altschul, S , et al , J Mol Biol 215 403-410 (1990) The well known Smith Waterman algorithm may also be used to determine identity

Parameters for polypeptide sequence companson include the following 1) Algorithm Needleman and Wunsch, J Mol Biol 48 443-453 (1970) Comparison matrix BLOSSUM62 from Hentikoff and Hentikoff, Proc Natl Acad Sci USA 89 10915-10919 (1992) Gap Penalty 12

Gap Length Penalty 4

A program useful with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison WI The aforementioned parameters are the default parameters for peptide comparisons (along with no penalty for end gaps) Parameters for polynucleotide comparison include the following

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

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

(1) Polynucleotide embodiments further include an isolated polynucleotide compnsmg a polynucleotide sequence having at least a 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the reference sequence of SEQ ID NO: 1 or 3, wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO: 1 or 3 or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherem said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO: 1 or 3 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleotides in SEQ ID NO: 1 or 3, or:

ⁿn ^≤ n " ( n * y).

wherein n_n is the number of nucleotide alterations, x_n is the total number of nucleotides in SEQ ID NO: l or 3, y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and • is the symbol for the multiplication operator, and wherein any non-integer product of x_n and y is rounded down to the nearest integer prior to subtracting it from x_n. Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO:2, 4, 6 or 8 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations.

(2) Polypeptide embodiments further include an isolated polypeptide comprising a polypeptide having at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO:2, 4, 6 or 8, wherein said polypeptide sequence may be identical to the reference sequence of SEQ ID NO: 2, 4, 6 or 8 or may include up to a certain integer number of amino acid alterations as compared to the reference sequence, wherem said alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non- conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy- terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of amino acid alterations is determined by multiplying the total number of amino acids in SEQ ID NO:2, 4, 6 or 8 by the mteger defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in SEQ ID NO:2, 4, 6 or 8, or: ⁿa ^{≤ x}a " (*a ^# Y)>

wherein n_a is the number of amino acid alterations, x_a is the total number of ammo acids in SEQ ID NO 2 4. 6 or 8, y is 0 50 for 50%. 0 60 for 60%, 0 70 for 70%, 0 80 for 80%, 0 85 for 85%, 0 90 for 90%, 0 95 for 95%, 0 97 for 97% or 1 00 for 100%, and • is the symbol for the multiplication operator, and wherein any non-integer product of x_a and y is rounded down to the nearest integer prior to subtracting it from x_a

"Immunologically equivalent deπvatιve(s)" as used herein encompasses a polypeptide, poh nucleotide. or the equivalent of either 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

"Immunospecific^"' means that charactenstic of an antibody whereby it possesses substantially greater affinity for the polypeptides of the mvention or the polynucleotides of the mvention than its affinity for other related polypeptides or polynucleotides respectively, particularly those polypeptides and polynucleotides in the pπor art

"bώvιdual(s)" means a multicellular eukaryote, mcludmg, but not limited to a metazoan, a mammal, an ovid, a bovid, a simian, a primate, and a human

"Isolated" means altered "by the hand of man" from its natural state, ; e , if it occurs in nature, it has been changed or removed from its ongmal environment, or both For example, a polynucleotide or a polypeptide naturally present a living organism is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting matenals of its natural state is "isolated", as the term is employed herem Moreover, a polynucleotide or polypeptide that is introduced mto an organism by transformation, genetic manipulation or by any other recombinant method is "isolated" even if it is still present in said organism, wbch organism may be living or non-living

"Multimenc FtsZ. FtsA" means a dimeπc protein, tnmeπc protein, tetramenc protein or bgher order structure protein comprising FtsZ and FtsA protein subunrts Mutl menc FtsZ FtsA may be comprised of FtsZ and FtsA proteins of different species or amino acid sequences as provided by the invention and described herein "Organιsm(s)" means a (I) prokaryote, mcludmg but not limited to, a member of the genus

Streptococcus, Staphylococcus, Bordetella, Corynebactenum, Mycobactenum, Netssena, Haemophύus, Actmomycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella, Pasturella, Moraxella, Acinetobacter, Erysipelothnx, Branhamella, Acbnobacillus, Streptobacillus, Listena, Calymmatobacterium Brucella, Bacillus, Clostndium, Treponema, Eschenchia, Salmonella, Kleibsiella, Vibrio, Proteus, Erwima, Borreba, Leptospira, Spirillum, Campylobacter, Shigella, Legionella, Pseudomυnas Ae υmonas Rickettsia, Chlamydia, Borreba 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, Staphylococcus aureus, Streptococcus pyogenes Streptococcus agalactiae Streptococcus faecahs, Streptococcus faecium, Streptococcus durans, Neissena gonυrrheae Neissena meningitidis Staphylococcus aureus, Staphylococcus epidermidis Corynebactenum dipthenae Gardnerella vagmabs Mycobaclerium tuberculosis, Mycυbaclerium bovis, Mycobactenum ulcerans, Mycobactenum leprae, Achnomyctes israebi, Listena monocytogenes, Bordetella pertusis, Bordatella parapertusis, Bordetella bronchisepbca, Eschenchia cob, Shigella dysentenae, Haemophilus tnfluenzae, Haemophilus aegypbus, Haemophilus paramfluenzae, Haemophtlus ducreyi, Bordetella Salmonella typhi, Citrobacter freundu, Proteus mirabibs, Proteus vulgans, Yersinia pestis, Kleώsiella pneumoniae Serratia marcessens Serratia bquefaciens, Vibrio cholera, Shigella dysentem, Shigella flexnen, Pseudυmυnas aeruginosa, Franscisella tularensis, Brucella aborbs, Bacillus anthracis, Bacillus cereus, Clostndium perfnngens Clost dium tetani, Clostridium botubnum, Treponema palbdum, Rickettsia nckettsii and Chlamydia trachomitts (n) an archaeon, mcludmg but not limited to Achaebacter, and (in) a unicellular or filamentous eukaryote, mcludmg but not limited to, a protozoan, a fungus, a member of the genus Saccharomyces Kluveromyces or Candida, and a member of the species Saccharomyces cenvis at Kluveromyces lactis or Candida albicam

"Polynucleotιde(s)" generally refers to any pol nbonucleotide or polydeoxnbonucleotide, wbch may be unmoώfied RNA or DNA or moώfied RNA or DNA "Polynucleotιde(s)" mclude, without limitation, smgle- and double-stranded DNA, DNA that is a mixture of smgle- and double-stranded regions or single-, double- and tnple-stranded regions, smgle- and double-stranded RNA, and RNA that is mixture of smgle- and double-stranded regions, hybπd molecules compnsmg DNA and RNA that may be single-stranded or, more typically double-stranded, or tnple-stranded regions, or a mixture of smgle- and double-stranded regions b adώtion "polynucleotide" as used herem refers to tπple-stranded regions compnsmg RNA or DNA or both RNA and DNA The strands in such regions may be from the same molecule or from different molecules The regions may mclude all of one or more of the molecules, but more typically mvolve only a region of some of the molecules One of the molecules of a tnple-hehcal region often is an oligonucleotide As used herem, the term "polynucleotιde(s)" also cludes DNAs or RNAs as descnbed above that contain one or more moώfied bases Thus DNAs or RNAs with backbones moώfied for stability or for other reasons are "polvnucleotιde(s)" as that term is mtended herem Moreover DNAs or RNAs compnsmg unusual bases, such as mosme or moώfied bases, such as tntylated bases, to name just two examples, are polynucleotides as the term is used herem It will be appreciated that a great vanety of modifications have been made to DNA and RNA that serve many useful purposes known to those of sbll in the art The term "polynucleotιde(s)" as it is employed herem embraces such chemically, enzymatically or metabohcally moώfied forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, includmg. for example, simple and complex cells. "Polynucleotide(s)" also embraces short polynucleotides often refened to as oligonucleotide(s).

"Polypeptide(s)" refers to any peptide or protem comprising two or more ammo acids jomed to each other by peptide bonds or moώfied peptide bonds. "Polypeptide(s)" refers to both short chains, commonly refened to as peptides, oligopeptides and ohgomers and to longer chains generally refened to as proteins. Polypeptides may contain amino acids other than the 20 gene encoded a ino acids. "Polypeptide(s)" include those moώfied either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques. Such moώfications are well described in basic texts and more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill in the art. It will be appreciated that the same type of modification may be present in the same or varying degree at several sites m a given polypeptide. Also, a given polypeptide may contain many types of moώfications. Moώfications can occur anywhere m a polypeptide, mcludmg the peptide backbone, the amino acid side-chains, and the ammo or carboxyl termini. Moώfications mclude, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linkmg, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, fonnation of cysteme, formation of pyroglutaπ-ate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation. myristoylation. oxidation, proteolytic processmg, phosphorylation, prenylation, racemization, giycosylation. lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation. selenoylation, sulfation, transfer-RNA mediated adώtion of amino acids to proteins, such as arg ylation, and ubiquitbation. 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 Moώfications: Perspectives 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 branc ng. Cyclic, branched and branched circular polypeptides may result from post- translational natural processes and may be made by entirely synthetic methods, as well.

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

"Subtraction set" is one or more, but preferably less than 100, polynucleotides comprising at least one polynucleotide of the invention 'Naπant(s)" as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties A typical variant of a polynucleotide differs m nucleotide sequence from another, reference polynucleotide Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result in amino acid substitutions, additions, deletions, fusion proteins and truncations in the polypeptide encoded by the reference sequence, as discussed below A typical variant of a polypeptide differs m amino acid sequence from another, reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical A variant and reference polypeptide may differ in ammo acid sequence by one or more substitutions, additions, deletions in any combination A substituted or inserted ammo acid residue may or may not be one encoded by the genetic code The present mvention also includes mclude vanants of each of 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 characteπstics Typical such substitutions are among Ala, Val, Leu and He, among Ser and Tb, among the aciώc residues Asp and Glu, among Asn and G . and among the basic residues Lys and Arg, or aromatic residues Phe and Tyr Particularly prefened are vanants m wbch several, 5-10, 1-5, 1-3, 1-2 or 1 ammo acids are substituted, deleted, or added in any combmation A variant of a polynucleotide or polypeptide may be a naturally occumng such as an allehc variant, or it may be a variant that is not known to occur naturally Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans EXAMPLES

The examples below are earned out usmg standard techmques, wbch are well known and routine to those of skill in the art, except where otherwise descnbed m detail The examples are illustrative, but do not limit the mvention

Example 1 Strain selection, Library Production and Sequencing

The polynucleotide havmg a DNA sequence given in Table 1 [SEQ ID NO 1 and 3] was obtained from a library of clones of chromosomal DNA of Staphylococcus aureus 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 1 or 3 Libraries may be prepared by routine methods, for example Methods 1 and 2 below

Total cellular DNA is isolated from Staphylococcus aureus WCUH29 accordmg to standard procedures and size-fractionated by either of two methods Method 1 Total cellular DNA is mechanically sheared by passage tbough a needle in order to size- fractionate according to standard procedures DNA fragments of up to 1 lkbp in size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added Fragments are hgated into the vector Lambda ZapII that has been cut with EcoRI, the library packaged by standard procedures and E coh infected with the packaged library The library is amplified by standard procedures

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combination of restriction enzymes appropriate to generate a series of fragments for cloning into library vectors (e g , Rsal, Pall, Alul, Bshl235I), and such fragments are size-fractionated according to standard procedures EcoRI linkers are hgated to the DNA and the fragments then hgated into the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E coli infected with the packaged library The library is amplified by standard procedures Example 2 Analysis of FtsA-FtsZ interaction The Applicants have demonstrated the specificity of FtsA-FtsZ interaction using the yeast two-hybrid system and identified a C-terminal region with 19 residues of FtsZ required for mteraction with FtsA by site-directed mutagenesis For characterisation of FtsA-FtsZ interaction and screenmg compounds that block the interaction, an ELISA and a fluorescence-based ELISA-type assay were developed Using ELISA, the Applicants determined an IC5₀ of 1 52 μM for FtsA that competes with GST-FtsZ in binding to the FtsA coated on a plate

Example 3 Analysis of interaction among bacterial cell division proteins

Genes encodmg these protems have been identified and cloned from E cob and S aureus

Using the yeast two-hybrid technology, these proteins has been analyzed to demonstrate their potential mteraction The yeast reporter strains are Y190 (MATa, leu2-3, -112, ura3-52, trp\-90l, hιs3-200, ade2-l0\, gaW, galW URA3 .GAL acZ, LYS2 GALI-HIS3, cyK) and EGY48 (MAT>, ura3, hι.s3. Irpland LexA oψ^yLEUΪ) The reporter gene lαcZ is used to detect protem-protein interaction pGBT9 and pGAD424 are plasmids that express the Gal4 DNA binding domam and activation domain, respectively pGilda and pB42AD are plasmids that express the bactenal LexA protein and an E cob peptide (B42), respectively DNAs that encode FtsA, FtsZ and the cytoplasmic domains of FtsL and FtsQ are amplified from the genomic DNA of E coli (MG1655) and S aureus

(WCUH29) by PCR and subcloned in the vectors These constructs were then transformed into yeast cells Yeast transformants were selected by culture on a synthetic medium lacking appropriate amino acids The presence and relative amount of β-galactosidase in yeast cells demonstrate both the activation of lacZ reporter and relative strength of two hybnd mteraction The filter assay uses X-gal as a substrate for β-galactosidase and the liquid culture assay uses ONPG as the substrate (Jarvis, E E et al (1988) Mol Cell Biol 8, 309-320) The yeast two-hybrid system is a approach to detect protein-protein interaction in vivo Two versions of this technology were utilized The Gal4 based assay relies on the reqmrement that the DNA binding domain (BD) and activating domain (AD) of the transcription factor be physically in proximity to activate a reporter gene (Figure 1A) The LexA based assay replaces BD with LexA and

Interaction of E co FtsA with FtsZ is summarized was demostarted using these methods A positive control was the G protein subunits β l and γ5, which form a complex in cell lines Self interaction of FtsA was observed and specificity of the interaction is demonstrated m both filter and liquid assays

A LexA based assay was used and showed that FtsA agam binds to itself specifically FtsZ, when fused to B42, specifically mteracts with FtsA

FtsA-FtsZ mteraction from S aureus was also investigated usmg the LexA based two-hybnd assay These results showed that FtsA self mteraction is undetectable Moreover, S aureus FtsZ self- interaction was demonstrated Specific interaction of FtsA with FtsZ is easily also detected in this assay These analyses demonstrate that FtsZ is able to bind to itself, that self-interaction of £ cob

FtsA was observed and that FtsA from either E cob or S aureus mteracts with FtsZ from its cognate species

Claims

What is claimed is: An isolated multimenc FtsZ FtsA polypeptide selected from the group consisting of

(1) a multimenc FtsZ FtsA polypeptide comprising an isolated polypeptide comprising an amino acid sequence selected from the group having at least

(a) 75% identity,

(b) 80% identity,

(c) 90% identity, or

(d) 95% identity to the ammo acid sequence of SEQ ID NO 2, 4, 6 or 8 over the entire length of SEQ ID

NO 2, 4, 6 or 8 respectively, (n) a multimenc FtsZ FtsA polypeptide comprising isolated polypeptide compnsing the ammo acid sequence of SEQ ID NO 2, 4, 6 or 8 or (in) a multimenc FtsZ FtsA polypeptide comprising isolated polypeptide which is the ammo acid sequence of SEQ ID NO 2, 4, 6 or 8

An antibody lmmunospecific for the polypeptide of claim 1

A method for the treatment of an individual

(I) in need of enhanced activity or expression of the polypeptide of claim 1 compnsing

(a) administering to the individual a therapeutically effective amount of an agonist to said polypeptide, and/or

(b) providing to the individual an isolated polynucleotide compnsing a nucleotide sequence encoding said polypeptide m a form so as to effect production of said polypeptide activity in vivo , or

(n) having need to inhibit activity or expression of the polypeptide of claim 1 compnsing

(a) admmistermg to the individual a therapeutically effective amount of an antagomst to said polypeptide, and/or

(b) administering to the individual a nucleic acid molecule that inhibits the expression of a nucleotide sequence encoding said polypeptide, and/or

(c) administering to the individual a therapeutically effective amount of a polypeptide that competes with said polypeptide for its ligand, substrate , or receptor 4 A process for diagnosing a disease or a susceptibility to a disease in an individual related to expression or activity of the polypeptide of claim 1 in an mώvidual compnsing

(a) determining the presence or absence of a mutation in the nucleotide sequence encodmg said polypeptide in the genome of said individual, and/or

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

5 A method for screening to identify compounds that activate or that inbbit the function of the polypeptide of claim 1 wbch compnses a method selected from the group consistmg of

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

(b) measuring the bmdmg of a candidate compound to the polypeptide (or to the cells or membranes bearing the polypeptide) or a fusion protem thereof in 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 beaπng the polypeptide,

(d) mixing a candidate compound with a solution containing 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,

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

(f) (1) contacting a composition compnsmg the polypeptide with the compound to be screened under conώtions to permit mteraction between the compound and the polypeptide to assess the mteraction of a compound, such mteraction bemg associated with a second component capable of providing a detectable signal in response to the mteraction of the polypeptide with the compound, and

(2) deteimining whether the compound mteracts with and activates or lnbbits an activity of the polypeptide by detectmg the presence or absence of a signal generated from the mteraction of the compound with the polypeptide

6 An agonist or an antagonist of the activity or expression polypeptide of claim 1

7 An expression system compnsmg a polynucleotide capable of producing a polypeptide of claim 1 when said expression system is present in a compatible host cell 8 A host cell compnsmg the expression system of claim 7 or a membrane thereof expressing a multimenc FtsZ FtsA polypeptide

9 A process for producing a multimenc FtsZ FtsA polypeptide compnsing the step of culturmg a host cell of claim 8 under conditions sufficient for the production of said polypeptide

10 A process for producing a host cell comprising a multimenc FtsZ FtsA polypeptide compnsmg the step of transforming or transfecting a cell with the expression system of claim 8 such the host cell, under appropriate culture conditions, produces a a multimenc FtsZ FtsA polypeptide polypeptide

1 1 A host cell produced by the process of claim 10 or a membrane thereof expressing a multimenc FtsZ FtsA polypeptide comprising polypeptide