WO2000023611A9 - FabG - Google Patents

Abstract

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

Description

FabG

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides and polypeptides, and their production and uses, as well as their vanants, agonists and antagonists, and their uses In particular, the invention relates to polynucleotides and polypeptides of the fabG (3-ketoacyl-ACP reductase) family, as well as their vanants, hereinafter referred to as "fabG," "fabG polynucleotιde(s)," and "fabG polypeptιde(s)" as the case may be

BACKGROUND OF THE INVENTION

The -frequency of Pseudomonas aeruginosa infections has nsen dramatically in the past few decades This has been attributed to the emergence of multiply antibiotic resistant strains and an increasing population of people with weakened immune systems It is no longer uncommon to isolate Pseudomonas aeruginosa strains which are resistant to some or all of the standard antibiotics This phenomenon has created an unmet medical need and demand for new anti-microbial agents, vaccines, drug screening methods and diagnostic tests for this organism

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

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

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

Vanous changes and modifications within the spint and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following descnptons and from reading the other parts of the present disclosure

DESCRIPTION OF THE INVENTION

The invention relates to fabG polypeptides and polynucleotides as descnbed in greater detail below In particular the invention relates to polypeptides and polynucleotides of a fabG of Pseudomonas aerugmo a, which is related by a-m-ino acid sequence homology to E cob FabG polypeptide The invention relates especially to fabG having the nucleotide and ammo acid sequences set out in Table 1 as SEQ ED NO 1 and SEQ ID NO 2 respectively Note that sequences recited in the Sequence Listing below as ' DNA^" represent an exemplification of the invention, smce those of ordinary skill will recognize that such sequences can be usefully employed m polynucleotides in general, including πbopolynucleotides

TABLE 1 fabG Polynucleotide and Polypeptide Sequences

(A) Pseudomonas aeruginosa fabG polynucleotide sequence [SEQ ID NO 1]

5 ' -ATGAGTCTGCAAGGTAAGGTCGCATTGGTAACCGGCGCCAGCCGTGGCATCGGCCAGGCG ATTGCGCTGGAACTGGGGCGCCTGGGTGCCGTGGTCATCGGCACCGCGACCAGCGCGTCC GGCGCCGAGAAGATCGCCGAAACCCTCAAGGCCAATGGCGTCGAGGGTGCGGGCCTGGTC CTGGACGTTTCCAGCGACGAATCCGTAGCCGCGACCCTGGAGCACATCCAGCAGCATCTC GGCCAACCGCTGATCGTGGTCAATAACGCCGGCATCACCCGCGATAATCTGCTGGTGCGC ATGAAAGACGACGAGTGGTTCGATGTGGTCAACACCAACCTGAACAGTCTCTACCGTCTG TCGAAAGCCGTTCTGCGCGGTATGACCAAGGCCCGCTGGGGGCGCATCATCAACATCGGT TCCGTGGTCGGCGCCATGGGCAATGCCGGGCAAACCAACTATGCCGCGGCGAAGGCCGGC CTGGAGGGCTTCACCCGTGCGCTGGCCCGGGAAGTCGGTTCGCGTGCCATTACCGTGAAT GCGGTGGCGCCGGGCTTCATCGACACCGACATGACCCGCGAGCTGCCGGAAGCCCAGCGC GAAGCGCTGCTGGGCCAGATTCCGCTGGGTCGCCTGGGGCAGGCGGAAGAGATCGCCAAG GTGGTCGGCTTCCTCGCTTCGGACGGCGCAGCCTATGTGACCGGGGCCACCGTGCCGGTC AATGGTGGGATGTACATGAGCTGA-3' (B) Pseudomonas aerugmosa fabG polypeptide sequence deduced from a polynucleotide sequence in this table [SEQ ID NO 2]

NH_^ -MSLQGKVALVTGASRGIG AIALELGRLGAVVIGTATSASGAEKIAETLKANGVEGAGLV LDVSSDESVAATLEHIQQHLGQPLIVVNNAGITRDNLLVRMKDDE FDVVNTNLNSLYRL SKAVLRGMTKARWGRI INIGSVVGA.MGNAGQTNYAAAKAGLEGFTRALAREVGSRAITVN AVAPG FI DTDMTRELPEAQREALLGQI PLGRLGQAEE IAKVVGFLASDGAAYVTGATVPV NGGMYMS-COOH

Polypeptides

FabG polypeptide of the invention is substantially phylogeneucally related to other proteins of the fabG (3-ketoacyl-ACP reductase) family

In one aspect of the invention there are provided polypeptides of Pseudomonas aerugmosa referred to herein as "fabG" and "fabG polypeptides" as well as biologically, diagnostcally, prophvlactically clinically or therapeutically useful vanants thereof, and compositions compnsing the same Among the particularly preferred embodiments of the invention are vanants of fabG polypeptide encoded by naturally occurring alleles of the fabG gene The present invention further provides for an isolated polypeptide which (a) comprises or consists of an am o acid sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferabl> at least 95% identity, most preferably at least 97-99% or exact identity to that of SEQ ID NO 2 over the entire length of SEQ ID NO 2, (b) a polypeptide encoded bv an isolated polynucleotide comprising or consisting of a polynucleotide sequence which has at least 70% 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 1 over the entire length of SEQ ID NO 1, (c) a polypeptide encoded by an isolated poh nucleotide comprising or consisting of a polynucleotide sequence encoding a polypeptide which has at least 70% identity, preferably at least 80% identity, more preferabh at least 90% identity, yet more preferablv at least 95% identity, even more preferably at least 97-99% or exact identity, to the ammo acid sequence of SEQ ID NO 2, over the entire length of SEQ ID NO 2

The polypeptides of the invention include a polypeptide of Table 1 [SEQ D NO 2] (in particular the mature polypeptide) as well as polypeptides and fragments, particularly those which have the biological actnits of fabG and also those which have at least 70% identity to a polypeptide of Table 1 [SEQ ED NO 1 ]or the relevant portion preferabh at least 80% identity to a polypeptide of Table 1 [SEQ ID NO 2and more preferably at least 90% identity to a polypeptide of Table 1 [SEQ ID NO 2] and still more preferably at least 95% identity to a polypeptide of Table 1 [SEQ ID NO 2] and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 ammo acids and more preferably at least 50 ammo acids The invention also includes a polypeptide consisting of or compnsmg a polypeptide of the formula

X-(R₁)_m-(R₂)-(R₃)_n-Y wherem at the ammo terminus, 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 herem for modified polypeptides, R_j and R3 are any ammo acid residue or modified ammo acid residue, m is an integer between 1 and 1000 or zero, n is an mteger between 1 and 1000 or zero, and R₂ is an ammo acid sequence of the invention, particularly an ammo acid sequence selected from Table 1 or modified forms thereof In the formula above, R₂ is onented so that its ammo terminal ammo acid residue is at the left, covalently bound to Rj and its carbox} terminal ammo acid residue is at the nght, covalently bound to R3 Am 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 mteger between 1 and 50, 100 or 500, and n is an mteger between 1 and 50 100, or 500

It is most preferred that a polypeptide of the mvention is denved from Pseudomonas aerugmosa, however, it may preferably be obtained from other organisms of the same taxonomic genus A polypeptide of the invention may also be obtained for example, from organisms of the same taxonomic family or order A fragment is a vaπant polypeptide havmg an ammo acid sequence that is entirely the same as part but not all of any ammo acid sequence of any polypeptide of the mvention As with fabG polypeptides, fragments may be "free-standing," or compnsed within a larger polypeptide of which they form a part or region most preferably as a smgle continuous region m a s gle larger polypeptide Preferred fragments mclude, for example, truncation polypeptides havmg a portion of an ammo acid sequence of Table 1 [SEQ ID NO 2]. or of vanants thereof, such as a continuous senes of residues that uicludes an ammo- and/or carboxyl-terminal ammo acid sequence Degradation forms of the polypeptides of the invention produced by or m a host cell, particularly a Pseudomonas aerugmosa, are also preferred Further preferred are fragments characterized by structural or functional attnbutes such as fragments that compnse alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-formmg regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region and high antige c 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 amino acids from the amino acid sequence of SEQ ID NO:2, or an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated or deleted from the amino acid sequence of SEQ ID NO:2.

Also preferred are biologically active fragments which are those fragments that mediate activities of fabG, including those with a similar activity or an improved activity, or with a decreased undesirable activity- Also included are those fragments that are antigenic or immunogenic in an animal, especially in a human- Particularly preferred are fragments comprising receptors or domains of enzymes that confer a function essential for viabibty of Pseudomonas aeruginosa 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 variants may be employed as intermediates for producing the full-length polypeptides of the invention. In addition to the standard single and triple letter representations for amino acids, the term

"X" or "Xaa" may also be used in describing certain polypeptides of the invention. "X" and "Xaa" mean that any of the twenty naturally occurring amino acids may appear at such a designated position in the polypeptide sequence- Polynucleotides It is an object of the invention to provide polynucleotides that encode fabG polypeptides, particularly polynucleotides that encode the polypeptide herein designated fabG.

In a particularly preferred embodiment of the invention the polynucleotide comprises a region encoding fabG polypeptides comprising a sequence set out in Table 1 [SEQ ID NO:l] which includes a full length gene, or a variant thereof. The Applicants believe that this full length gene is essential to the growth and/or survival of an organism which possesses it, such as Pseudomonas aeruginosa.

As a further aspect of the invention there are provided isolated nucleic acid molecules encoding and/or expressmg fabG polypeptides and polynucleotides, particularly Pseudomonas aeruginosa fabG poh eptides and polynucleotides. including, for example, unprocessed RNAs, ribozyme RNAs, mRNAs, cDNAs. genomic DNAs, B- and Z-DNAs. Further embodiments of the invention include biologically, diagnostically. prophylactically, clinically or therapeutically useful polynucleotides and polypeptides, and variants thereof, and compositions comprising the same. Another aspect of the invention relates to isolated polynucleotides, including at least one fiill length gene, that encodes a fabG polypeptide having a deduced amino acid sequence of Table 1 [SEQ ID NO:2] and polynucleotides closely related thereto and variants thereof.

In another particularly preferred embodiment of the invention there is a fabG polypeptide from Pseudomonas aeruginosa comprising or consisting of an amino acid sequence of Table 1 [SEQ ID NO: 2], or a variant thereof-

Using the information provided herein, such as a polynucleotide sequence set out in Table 1 [SEQ ID NO:l], a polynucleotide of the invention encoding fabG polypeptide may be obtained using standard cloning and screening methods, such as those for cloning and sequencing chromosomal DNA fragments from bacteria using Pseudomonas aeruginosa strain 4 cells as starting material, followed by obtaining a full length clone- For example, to obtain a polynucleotide sequence of the invention, such as a polynucleotide sequence given in Table 1 [SEQ ID NO:l], typically a library of clones of chromosomal DNA of Pseudomonas aeruginosa strain 4 in E.coli or some other suitable host is probed with a radiolabeled oligonucleotide, preferably a 17-mer or longer, derived from a partial sequence. Clones carrying DNA identical to that of the probe can then be distinguished using stringent hybridization conditions. By sequencing the individual clones thus identified by hybridization with sequencing primers designed from the original polypeptide or polynucleotide sequence it is then possible to extend the polynucleotide sequence in both directions to determine a full length gene sequence. Conveniently, such sequencing is performed, for example, using denatured double stranded DNA prepared from a plasmid clone. Suitable techniques are described by Maniatis, T., Fritsch, E.F. and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989). (see in particular Screening By Hybridization 1.90 and Sequencing Denatured Double-Stranded DNA Templates 13.70). Direct genomic DNA sequencing may also be performed to obtain a full length gene sequence. Illustrative of the invention, each polynucleotide set out in Table 1 [SEQ ID NO:l] was discovered in a DNA library derived from Pseudomonas aeruginosa strain 4.

Moreover, each DNA sequence set out in Table 1 [SEQ ID NO:l] contains an open reading frame encoding a protein having about the number of amino acid residues set forth in Table 1 [SEQ ID NO:2] with a deduced molecular weight that can be calculated using amino acid residue molecular weight values well known to those skilled in the art. The polynucleotide of SEQ ID NO:l, between nucleotide number 1 and the stop codon which begins at nucleotide number 742 of SEQ ID NO:l, encodes the polypeptide of SEQ ID NO.2. In a further aspect, the present mvention provides for an isolated polynucleotide compπsing or consisting of (a) a polynucleotide sequence which has at least 70% identity, preferably at least 80% identity more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferabh at least 97-99% or exact identity to SEQ ID NO 1 over the entire length of SEQ ID NO 1, (b) a polynucleotide sequence encoding a polypeptide which has at least 70% 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 100% exact, to the ammo acid -sequence of SEQ ID NO 2, over the entire length of SEQ ID NO 2

A polynucleotide encoding a polypeptide of the present mvention, including homologs and orthologs from species other than Pseudomonas aeruginosa, may be obtained by a process which compnses the steps of screening an appropnate library under stringent hybndization conditions with a labeled or detectable probe consisting of or compnsmg the sequence of SEQ ED NO 1 or a fragment thereof, and isolating a full- length gene and/or genomic clones containing said polynucleotide sequence

The mvention provides a polynucleotide sequence identical over its entire length to a cod g sequence (open readmg frame) in Table 1 [SEQ ID NO 1] Also provided by the mvention is a codmg sequence for a mature polypeptide or a fragment thereof, by itself as well as a codmg sequence for a mature pohpeptide or a fragment in readmg frame with another codmg sequence, such as a sequence encoding a leader or secretory sequence, a pre- or pro- or prepro-protem sequence The polynucleotide of the mvention may also contain at least one non-codmg sequence, including for example, but not limited to at least one non-codmg 5 and 3 sequence, such as the transcnbed but non-translated sequences, termination signals (such as rho-dependent and rho- ndependent termination signals), nbosome binding sites, Kozak sequences, sequences that stabilize m-RNA, mtrons and polyadenylation signals The polynucleotide sequence may also compnse additional codmg sequence encoding additional ammo acids For example, a marker sequence that facilitates punfication of the fused polypeptide can be encoded In certain embodiments of the mvention, the marker sequence is a hexa-histid-ine peptide, as provided in the pQE vector (Qiagen, Inc ) and descnbed m Gentz et al , Proc Natl Acad Sci , USA 86 821-824 (1989), or an HA peptide tag (Wilson et al Cell 37 767 (1984) both of which may be useful m puπfying polypeptide sequence fused to them Polynucleotides of the invention also include but are not limited to, polynucleotides compnsmg a structural gene and its naturally associated sequences that control gene expression A preferred embodiment of the invention is a polynucleotide of consisting of or compnsmg nucleotide 1 to the nucleotide immediately upstream of or including nucleotide 742 set forth m SEQ ID NO 1 of Table 1, both of which encode the fabG polypeptide The mvention also mcludes a polynucleotide consisting of or compnsmg a polynucleotide of the formula

X-(R₁)_m-(R₂)-(R₃)_n-Y wherein at the 5' end of the molecule X is hydrogen, a metal or a modified nucleotide residue, or together with Y defines a covalent bond, and at the 3' end of the molecule, Y is hydrogen, a metal, or a modified nucleotide residue or together with X defines the covalent bond, each occurrence of R_j and R3 is independently any nucleic acid residue or modified nucleic acid residue, m is an integer between 1 and 3000 or zero , n is an mteger between 1 and 3000 or zero, and R₂ is a nucleic acid sequence or modified nucleic acid sequence of the mvention, particularly a nucleic acid sequence selected from Table 1 or a modified nucleic acid sequence thereof In the polynucleotide formula above, R₂ is oriented so that its 5' end nucleic acid residue is at the left, bound to Ri and its 3' end nucleic acid residue is at the right bound to R3 Any stretch of nucleic acid residues denoted by either R\ and/or R where m and/or n is greater than 1 may be either a heteropolymer or a homopolymer, preferably a heteropolymer Where, in a preferred embodiment, X and Y together define a covalent bond, the polynucleotide of the above formula is a closed, circular polynucleotide, which can be a double- stranded polynucleotide wherein the formula shows a first strand to which the second strand is complementary In another preferred embodiment m and/or n is an integer between 1 and 1000. Other 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 polynucleotide of the mvention is denved from Pseudomonas aerugmosa however it may preferably be obtained from other organisms of the same taxonomic genus A polynucleotide of the invention may also be obtained, for example, from organisms of the same taxonomic family or order

The term "polynucleotide encodmg a polypeptide" as used herein encompasses polynucleotides that include a sequence encoding a polypeptide of the mvention, particularly a bactenal polypeptide and more particularly a polypeptide of the Pseudomonas aeruginosa fabG havmg an ammo acid sequence set out m Table 1 [SEQ ID NO 2] The term also encompasses polynucleotides that mclude a smgle continuous region or discontinuous regions encodmg the polypeptide (for example, polynucleotides interrupted by integrated phage an integrated insertion sequence, an integrated vector sequence, an integrated transposon sequence or due to RN A editmg or genomic DNA reorganization) together with additional regions, that also ma contain codmg and/or non-codmg sequences

The invention further relates to vanants of the polynucleotides descnbed herem that encode vanants of a polypeptide having a deduced ammo acid sequence of Table 1 [SEQ ID NO 2] Fragments of a polynucleotides of the mvention may be used, for example, to synthesize full-length polynucleotides of the invention

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

Further preferred embodiments of the mvention are polynucleotides that are at least 70% identical over their entire length to a polynucleotide encodmg fabG polypeptide havmg an ammo acid sequence set out in Table 1 [SEQ ID NO 2], and polynucleotides that are complementary to such polynucleotides

Alternatively most highly preferred are polynucleotides that compnse a region that is at least 80% identical over its entire length to a polynucleotide encodmg fabG polypeptide and polynucleotides complementary thereto In this regard, polynucleotides at least 90% identical over their entire length to the same are particularly preferred, and among these particularly preferred polynucleotides, those with at least 95% are especially preferred Furthermore, those with at least 97% are highly preferred among those with at least

95% and among these those with at least 98% and at least 99%> are particularly highly preferred, with at least 99% being the more preferred

Preferred embodiments are polynucleotides encodmg polypeptides that retain substantially the same biological function or activity as the mature polypeptide encoded by a DNA of Table 1 [SEQ ID NO 1] In accordance with certain preferred embodiments of this mvention there are provided polynucleotides that hybndize, particularly under strmgent conditions, to fabG polynucleotide sequences, such as those polynucleotides in Table 1

The invention further relates to polynucleotides that hybndize to the polynucleotide sequences provided herein In this regard, the invention especially relates to polynucleotides that hybndize under strmgent conditions to the polynucleotides descnbed herem As herem used, the terms "strmgent conditions" and "strmgent hybndization conditions" mean hybndization occurring only if there is at least 95% and preferably at least 97% identity between the sequences A specific example of strmgent hybndization conditions is overnight incubation at 42°C m a solution compnsmg 50% formamide, 5x SSC

( 150mM NaCl 15mM tπsodium citrate) 50 mM sodium phosphate (pH7 6), 5x Denhardt's solution, 10% dextran sulfate and 20 micrograms/ml of denatured, sheared salmon sperm DNA, followed by washing the hybridization support in 0 lx SSC at about 65°C Hybridization and wash conditions are well known and exemplified in Sambrook, et al , Molecular Clonmg A Laboratory Manual, Second Edition, Cold Spring Harbor, N Y , (1989), particularly Chapter 11 therein Solution hybndization may also be used with the polynucleotide sequences provided by the mvention

The invention also provides a polynucleotide consisting of or compnsmg a polynucleotide sequence obtained by screening an appropnate library containing the complete gene for a polynucleotide sequence set forth m SEQ ID NO 1 under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence set forth in SEQ ID NO 1 or a fragment thereof, and isolating said polynucleotide sequence Fragments useful for obtaining such a polynucleotide include, for example, probes and pnmers fully descnbed elsewhere herem

As discussed elsewhere herem regarding polynucleotide assays of the mvention, for instance, the polynucleotides of the mvention, may be used as a hybndization probe for R-N- , cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encodmg fabG and to isolate cDNA and genomic clones of other genes that have a high identity, particularly high sequence identit} to the fabG gene Such probes generally will compnse at least 15 nucleotide residues or base pairs Preferably, such probes will have at least 30 nucleotide residues or base pairs and may have at least 50 nucleotide residues or base pairs Particularly preferred probes will have at least 20 nucleotide residues or base pairs and will have lee than 30 nucleotide residues or base pairs

A codmg region of a fabG gene may be isolated by screening usmg a DNA sequence provided m Tablt 1 [SEQ ID NO 1J to synthesize an ohgonucleotide probe A labeled ohgonucleotide havmg a sequence complementary to that of a gene of the mvention is then used to screen a library of cDNA, genomic DNA or m-RNA to determine which members of the library the probe hybndizes to

There are several methods available and well known to those skilled in the art to obtain full- length DNAs, or extend short DNAs. for example those based on the method of Rapid Amplification of cD A ends (RACE) (see for example Froh an, et al , PNAS USA 85 8998-9002, 1988) Recent modifications of the technique exemplified by the Marathon™ technology (Clontech Laboratories Inc ) for example, have significantly simplified the search for longer cDNAs In the Marathon™ technology, cDNAs have been prepared from m-RNA extracted from a chosen tissue and an 'adaptor' sequence ligated onto each end Nucleic acid amplification (PCR) is then earned out to amplify the "missing" 5' end of the DNA using a combination of gene specific and adaptor specific ohgonucleotide primers The PCR reaction is then repeated usmg "nested" primers, that is, primers designed to anneal within the amplified product (typically an adaptor specific primer that anneals further 3' m the adaptor sequence and a gene specific primer that anneals further 5' in the selected gene sequence) The products of this reaction can then be analyzed by DNA sequencing and a full-length DNA constructed either by joining the product directly to the existing DNA to give a complete sequence, or carrying out a separate full-length PCR using the neyv sequence information for the design of the 5' primer.

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

The polynucleotides of the invention that are oligonucleotides derived from a sequence of

Table 1 [SEQ ID NOS: l or 2] may be used in the processes herein as described, but preferably for

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

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

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

A precursor protein, having a mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide- When prosequences are removed such inactive precursors generally are activated. Some or all of the prosequences may be removed before activation. Generally, such precursors are called proproteins. In addition to the standard A, G, C, T/U representations for nucleotides, the term "N" may also be used in describing certain polynucleotides of the invention- "N" means that any of the four DNA or RNA nucleotides may appear at such a designated position in the DNA or RNA sequence, except it is preferred that N is not a nucleic acid that when taken in combination with adjacent nucleotide positions, when read in the correct reading frame, would have the effect of generating a premature termination codon in such reading frame.

In sum, a polynucleotide of the mvention may encode a mature protein, a mature protein plus a leader sequence (which may be referred to as a preprotein), a precursor of a mature protein having one or more prosequences that are not the leader sequences of a preprotein, or a preproprotern, which is a precursor to a proprotein, having a leader sequence and one or more prosequences, which generally are removed during processmg steps that produce active and mature forms of the polypeptide Vectors, Host Cells, Expression Systems

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

Recombinant polypeptides of the present mvention may be prepared by processes well known m those skilled m the art from genetically engineered host cells compnsmg expression systems Accordingly, in a further aspect, the present invention relates to expression systems which compnse a polynucleotide or polynucleotides of the present mvention. to host cells which are genetically engineered with such expression systems, and to the production of polypeptides of the mvention by recombinant techniques

For recombinant production of the polypeptides of the mvention, host cells can be genetically engmeered to incorporate expression systems or portions thereof or polynucleotides of the mvention Introduction of a polynucleotide mto the host cell can be effected by methods descnbed 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 Laboratory Press. Cold Spring Harbor, N Y (1989), such as, calcium phosphate transfection, DEAE- dextran mediated transfection, transvection, micromjection, cationic lipid-mediated transfection, electroporation transduction scrape loading, ballistic mtroduction and infection

Representative examples of appropnate hosts include bactenal cells, such as cells of streptococci, staphylococci enterococci E coli, streptomyces, cyanobactena, Bacillus subtihs, and Pseudomonas aerugmosa fungal cells such as cells of a yeast. Kluveromyces , Saccharomyces , a basidiomycete, Candida albicans and Aspergillus, insect cells such as cells of Drosop la S2 and Spodoptera Sf9, animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-1 and Bowes melanoma cells, and plant cells, such as cells of a gymnosperm or angiosperm

A great variety of expression systems can be used to produce the polypeptides of the mvention Such vectors include, 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 yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses, picornaviruses and retroviruses. and vectors denved from 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 appropriate DNA sequence may be inserted into the expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sa brook et al.. MOLECULAR CLONING, A LABORATORY MANUAL, (supra).

In recombinant expression systems in eukaryotes, for secretion of a translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals. Polypeptides of the invention can be recovered and purified from recombinant cell cultures by well- known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification.

Diagnostic, Prognostic, Serotyping and Mutation Assays

This invention is also related to the use of fabG polynucleotides and polypeptides of the invention for use as diagnostic reagents. Detection of fabG polynucleotides and/or polypeptides in a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of disease, staging of disease or response of an infectious organism to drugs. Eukaryotes, particularly mammals, and especially humans, particularly those infected or suspected to be infected with an organism comprising the fabG gene or protein, may be detected at the nucleic acid or amino acid level by a variety of well known techniques as well as by methods provided herein. Polypeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtained from a putatively infected and/or infected individual's bodily materials. Polynucleotides from any of these sources, particularly DNA or RNA, may be used directly for detection or may be amplified enzymatically by using PCR or any other amplification technique prior to analysis. RNA, particularly mRNA, cDNA and genomic DNA may also be used in the same ways. Using amplification, characterization of the species and strain of infectious or resident organism present in an individual, may be made by an analysis of the genotype of a selected polynucleotide of the organism. Deletions and insertions can be detected by a change in size of the amplified product in comparison to a genotype of a reference sequence selected from a related organism, preferably a different species of the same genus or a different strain of the same species. Point mutations can be identified by hybridizing amplified DNA to labeled fabG polynucleotide sequences. Perfectly or significantly matched sequences can be distinguished from imperfectly or more significantly mismatched duplexes by DNase or R-Nase digestion, for DNA or RNA respectively, or by detecting differences in melting temperatures or renaturation kinetics. Polynucleotide sequence differences may also be detected by alterations in the electrophoretic mobihty of polynucleotide fragments in gels as compared to a reference sequence- This may be carried out with or without denaturing agents. Polynucleotide differences may also be detected by direct DNA or RNA sequencing. See. for example, Myers et al., Science, 230: 1242 (1985). Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase, VI and S I protection assay or a chemical cleavage method. See, for example, Cotton et al, Proc. Natl. Acad S i.. USA. 85: 4397-4401 (1985).

In another embodiment, an array of oligonucleotides probes comprising fabG nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of, for example, genetic mutations, serotype, taxonomic classification or identification. Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability (see, for example, Chee et al., Science, 274: 610 (1996)).

Thus in another aspect, the present invention relates to a diagnostic kit which comprises: (a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NO:l, or a fragment thereof ; (b) a nucleotide sequence complementary to that of (a); (c) a polypeptide of the present invention, preferably the polypeptide of SEQ ID NO:2 or a fragment thereof; or (d) an antibody to a polypeptide of the present invention, preferably to the polypeptide of SEQ ID NO:2.

It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component- Such a kit will be of use in diagnosing a disease or susceptibility to a Disease, among others- This invention also relates to the use of polynucleotides of the present invention as diagnostic reagents. Detection of a mutated form of a polynucleotide of the invention, preferable, SEQ ID NO:l, which is associated with a disease or pathogenicity will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, a prognosis of a course of disease, a determination of a stage of disease, or a susceptibility to a disease, which results from under-expression, over-expression or altered expression of the polynucleotide. Organisms, particularly infectious organisms, carrying mutations in such polynucleotide may be detected at the polynucleotide level by a variety of techniques, such as those described elsewhere herein. The nucleotide sequences of the present invention are also valuable for organism chromosome identification The sequence is specifically targeted to, and can hybndize with, a particular location on an organism's chromosome, particularly to a Pseudomonas aeruginosa chromosome The mapping of relevant sequences to chromot-omes according to the present invention may be an important step in conelating those sequences with pathogenic potential and/or an ecological niche of an organism and/or drug resistance of an organism as well as the essentiality of the gene to the organism Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data Such data may be found on-lme m a sequence database The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through known genetic methods for example through linkage analysis (coinhentance of physically adjacent genes) or mating studies, such as by conjugation

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

Cells from an organism carry mg mutations or polymorphisms (allehc vanations) in a polynucleotide and/or polypeptide of the mvention may also be detected at the polynucleotide or polypeptide level by a vanety of techniques, to alloyv for serotyping, for example For example, RT-PCR can be used to detect mutations in the RNA It is particularly preferred to use RT-PCR m conjunction with automated detection systems such as for example GeneScan RNA cDNA or genomic DNA may also be used for the same purpose PCR As an example PCR primers complementary to a polynucleotide encodmg fabG polypeptide can be used to identify and analyze mutations These primers may be used for, among other things amplifying fabG DNA and/or RNA isolated from a sample denved from an mdividual, such as a bodily matenal The primers may be used to amphfy a polynucleotide isolated from an infected mdividual, such that the polynucleotide may then be subject to vanous techniques for elucidation of the polynucleotide sequence In this way, mutations in the polynucleotide sequence may be detected and used to diagnose and/or prognose the infection or its stage or course, or to serotype and/or classify the infectious agent

The invention further provides a process for diagnosing, disease, preferably bacterial infections, more preferably infections caused by Pseudomonas aeruginosa, compnsmg determining from a sample derived from an individual, such as a bodily matenal, an mcreased level of expression of polynucleotide having a sequence of Table 1 [SEQ ID NO 1] Increased or decreased expression of a fabG polynucleotide can be measured using anv on of the methods well known m the art for the quantitation of polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNase protection Northern blotting spectrometry and other hybridization methods

In addition a diagnostic assay in accordance with the mvention for detecting over- cxpression of fabG polypeptide compared to nomial control tissue samples may be used to detect the presence of an infection, for example Assay techniques that can be used to determine levels of a fabG polypeptide in a sample denved from a host such as a bodily matenal, are well-known to those of skill m the art Such assay methods mclude radioimmunoassays, competitive-bmdmg assays, Western Blot analy sis antibody sandwich assays antibody detection and ELISA assays Difiei ential Expression The polynucleotides and polynucleotides of the invention may be used as reagents for differential screening methods There are many differential screening and differential display methods known m the art in which the polynucleotides and polypeptides of the mvention may be used For example, the differential display technique is descnbed by Chuang et al , J Bacterwl 175 2026-2036 (1993) This method identifies those genes which are expressed m an organism by identifying mRNA present usmg randomh -primed RT-PCR Bv companng pre-mfection and post infection profiles, genes up and down regulated during infection can be identified and the RT-PCR product sequenced and matched to ORF 'unknowns "

In Vivo Expression Technology (IVET) is described by Camilh et al , Proc Nat 'I Acad Sci USA 91 2634-2638 (1994) IVET identifies genes up-regulated dunng infection when compared to laboratory cultivation, implying an important role in infection ORFs identified by this techmque are impli d to a significant role in infection establishment and/or maintenance In this techmque random chromosomal fragments of target organism are cloned upstream of a promoter-less rυcombinas gene in a plasmid vector This construct is introduced into the target organism which carries an antibiotic resistance gene flanked by resolvase sites Growth in the presence of the antibiotic removes from the population those fragments cloned mto the plasmid vector capable of supporting transcription of the recombmase gene and therefore have caused loss of antibiotic resistance The resistant pool is introduced into a host and at various times after infection bacteria may be recovered and assessed for the presence of antibiotic resistance The chromosomal fragment carried by each antibiotic sensitive bacterium should carry a promoter or portion of a gene normally upregulated during infection Sequencing upstream of the recombmase gene allows identification of the up regulated gene

RT-PCR may also be used to analyze gene expression patterns For RT PCR using the polynucleotides of the invention, messenger RNA is isolated from bacterial mfected tissue, e g , 48 hour muπne lung infections, and the amount of each mRNA species assessed by reverse transcnption of the RNA sample primed with random hexanucleotides followed by PCR with gene specific primer pairs The determination of the presence and amount of a particular m-RNA species by quantification of the resultant PCR product provides information on the bacterial genes which are transcribed in the infected tissue Analysis of gene transcription can be earned out at different times of infection to gam a detailed knowledge of gene regulation in bacterial pathogenesis allowing for a clearer understanding of which gene products represent targets for screens for antibactenals Because of the gene specific nature of the PCR primers employed it should be understood that the bactenal mRNA preparation need not be free of mammalian RNA This allows the investigator to carry out a simple and quick RNA preparation from infected tissue to obtain bacterial mRNA species which are very short lived m the bacterium (in the order of 2 minute halflives) Optimally the bacterial m-RNA is prepared from infected muπne lung tissue by mechanical disruption in the presence of TRIzole (GIBCO-BRL) for very short periods of time, subsequent processing according to the manufacturers of TRIzole reagent and DNAase treatment to remove contaminating DNA Preferably the process is optimized by finding those conditions which give a maximum amount of Pseudomonas aeruginosa 16S nbosomal RNA as detected by probing Northerns with a suitably labeled sequence specific ohgonucleotide probe Typically a 5' dye labeled primer is used in each PCR primer pair m a PCR reaction which is terminated optimally between 8 and 25 cycles The PCR products are separated on 6% polyacrylamide gels with detection and quantification usmg GeneScanner (manufactured by ABI) Gridding and Polynucleotide Subtraction

Methods have been described for obtaining information about gene expression and identity using so called "high density DNA arrays" or gnds See, e g , M Chee et al , Science, 274 610-614 ( 1996) and other references cited therein Such gnddmg assays have been employed to identify certain novel gene sequences, referred to as Expressed Sequence Tags (EST) (Adams et a , Science, 252 165 1 -1656 (1991)) A variety of techniques have also been descnbed for identifying particular gene sequences on the basis of their gene products For example, see International Patent Application No W091/07087, published May 30, 1991 In addition, methods have been described for the amplification of desired sequences For example, see International Patent Application No W091/17271, published November 14, 1991 The polynucleotides of the invention mav be used as components of polynucleotide arrays, preferably high density arrays or grids These high density arrays are particularly useful for diagnostic and prognostic purposes For example, a set of spots each compnsmg a different gene, and further comprising a polynucleotide or polynucleotides of the invention, may be used for probing such as using hybridization or nucleic acid amplification, usmg a probes obtained or derived from a bodily sample to determine the presence of a particular polynucleotide sequence or related sequence in an individual Such a presence may indicate the presence of a pathogen, particularly Pseudomonas aeruginosa and may be useful in diagnosing and/or prognosing disease or a course of disease A grid comprising a number of variants of the polynucleotide sequence of SEQ ID NO 1 are preferred Also preferred is a comprising a number of variants of a polynucleotide sequence encoding the polypeptide sequence of SEQ ID NO 2 Antibodies

The polypeptides and polynucleotides of the mvention or vanants thereof, or cells expressmg the same can be used as lmmunogens to produce antibodies immunospecific for such polypeptides or polynucleotides respectively

In certain prefened embodiments of the mvention there are provided antibodies against fabG polypeptides or polynucleotides

Antibodies generated against the polypeptides or polynucleotides of the mvention can be obtained bv administering the polypeptides and/or polynucleotides of the mvention-, or epitope-beanng fragments of either or both, analogues of either or both, or cells expressmg either or both, to an animal, preferably a nonhuman usmg routine protocols For preparation of monoclonal antibodies, any techmque known m the art that provides antibodies produced by continuous cell line cultures can be used Examples mclude vaπous techniques such as those in Kohler, G and Milstein, C , Nature 256 495-497 (1975), Kozbor et al , Immunology Today 4 72 (1983), Cole et al , pg 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R Liss, Inc (1985)

Techniques for the production of smgle chain antibodies (U S Patent No 4,946,778) can be adapted to produce smgle cham antibodies to polypeptides or polynucleotides of this mvention Also, transgeruc mice or other organisms such as other mammals, may be used to express humanized antibodies lmmunospecific to the polypeptides or polynucleotides of the mvention

Alternatively, phage display technology may be utilized to select antibody genes with binding activities towards a polypeptide of the invention either from repertoires of PCR amplified v-genes of lymphocytes from humans screened for possessmg anti-fabG or from naive hbranes (McCafferty, et al ( 1990) Nature 348 552-554 Marks, et al , ( 1992) Biotechnology 10, 779-783) The affinity of these antibodies can also be improved by, for example, cham shuffling (Clackson et al , (1991) Nature 352 628) The above-descnbed antibodies may be employed to isolate or to identify clones expressmg the polypeptides or polynucleotides of the mvention to punfy the polypeptides or polynucleotides by, for example, affinity chromatography

Thus, among others antibodies against fabG-polypeptide or fabG-polynucleotide may be employed to treat infections, particularly bactenal infections

Poh peptide variants include antigenically, epitopically or rmmunologically equivalent variants form a particular aspect of this invention

A polypeptide or polynucleotide of the invention, such as an antigenically or rmmunologically equivalent derivative or a fusion protem of the polypeptide is used as an antigen to immunize a mouse or other animal such as a rat or chicken The fusion protein may provide stability to the polypeptide The antigen may be associated, for example by conjugation, with an lmmunogenic carrier protem for example bovine serum albumin keyhole limpet haemocyanin or tetanus toxoid Alternatively, a multiple antigemc polypeptide compnsmg multiple copies of the polypeptide, or an antigenically or immunologically equivalent polypeptide thereof may be sufficiently antigemc to improve lmmunogenicity so as to obviate the use of a carrier

Preferably the antibody or vanant thereof is modified to make it less lmmunogemc in the ldual For example if the individual is human the antibody may most preferably be "humanized," where the complimentaπty determining region or regions of the hybndoma-denved antibody has been transplanted into a human monoclonal antibody, for example as descnbed in Jones et al (1986), Nature 321 522-525 or Tempest et al , (1991) Biotechnology 9, 266-273

In accordance with an aspect of the mvention, there is provided the use of a polynucleotide of the invention for therapeutic or prophylactic purposes, in particular genetic immunization Among the particularK preferred embodiments of the mvention are naturally occurring allehc vanants of fabG polynucleotides and polypeptides encoded thereby The use of a polynucleotide of the invention in genetic immunization will preferably employ a suitable delivery method such as direct injection of plasmid DNA mto muscles (Wolff et al , Hum Mol Genet (1992) 1 363, Manthorpe et α/ , Hum Gene Ther (1983) 4 419), delivery of DNA complexed with specific protein earners (Wu et al , J Biol Chem (1989) 264 16985), coprecipitation of DNA with calcium phosphate (Benvenisty & Reshef, PNAS USA, (1986) 83 9551), encapsulation of DNA in various forms of liposomes (Kaneda et al , Science (1989) 243 375), particle bombardment (Tang et al Nature ( 1992) 356 152, Eisenbraun et o/ DNA Cell Biol (1993) 12 791) and in vivo infection using cloned retroviral vectors (Seeger et al , PNAS USA (1984) 81 5849) Antagonists and Agonists - Assays and Molecules Polypeptides and polynucleotides of the invention may also be used to assess the binding of small molecule substrates and hgands in. for example, cells, cell-free preparations, chemical libranes, and natural product mixtures These substrates and hgands may be natural substrates and ligands or may be structural or functional mimetics. See, e.g., Cohgan et al., Current Protocols in Immunology 1(2): Chapter 5 (1991). Polypeptides and polynucleotides of the present invention are responsible for many biological functions, including many disease states, in particular the Diseases hereinbefore mentioned. It is therefore desirable to devise screening methods to identify compounds which stimulate or which inhibit the function of the polypeptide or polynucleotide Accordingly, m a further aspect, the present invention provides for a method of screening compounds to identify those which stimulate or which inhibit the function of a polypeptide or polynucleotide of the mvention, as well as related polypeptides and polynucleotides. In general, agonists or antagonists may be employed for therapeutic and prophylactic purposes for such Diseases as hereinbefore mentioned. Compounds may be identified from a variety 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, ligands, receptors, enzymes, etc., as the case may be. of fabG polypeptides and polynucleotides, or may be structural or functional mimetics thereof (see Cohgan et al , Current Protocols in Immunology 1(2): Chapter 5 (1991)).

The screening methods may simply measure the binding of a candidate compound to the polypeptide or polynucleotide, or to cells or membranes bearing the polypeptide or polynucleotide, or a fusion protein of the polypeptide by means of a label directly or indirectly associated with the candidate compound Alternatively, the screening method may involve competition with a labeled competitor Further, these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide or polynucleotide, using detection systems appropriate to the cells comprising the polypeptide or polynucleotide. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed. Constitutively active polypeptide and/or constitutively expressed polypeptides and polynucleotides may be employed in screening methods for inverse agonists or inhibitors, in the absence of an agonist or inhibitor, by testing whether the candidate compound results in inhibition of activation of the polypeptide or polynucleotide, as the case may be Further, the screening methods may simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide or polynucleotide of the present invention, to form a mixture, measuring fabG polypeptide and/or polynucleotide activity in the mixture, and comparing the fabG polypeptide and/or polynucleotide activity of the mixture to a standard. Fusion proteins, such as those made from Fc portion and fabG polypeptide, as hereinbefore described, can also be used for high-throughput screening assays to identify 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)). The polynucleotides, polypeptides and antibodies that bind to and/or interact with a polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of m-RNA and/or polypeptide in cells. For example, an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide using monoclonal and 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 invention also provides a method of screening compounds to identify those which enhance (agonist) or block (antagonist) the action of fabG polypeptides or polynucleotides, particularly those compounds that are bacteristatic and/or bactericidal. The method of screening may involve high-throughput techniques. 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, comprising fabG polypeptide and a labeled substrate or ligand of such polypeptide is incubated in the absence or the presence of a candidate molecule that may be a fabG agonist or antagonist- The ability of the candidate molecule to agonize or antagonize the fabG polypeptide is reflected in decreased binding of the labeled ligand or decreased production of product from such substrate. Molecules that bind gratuitously, i.e., without inducing the effects of fabG polypeptide are most likely to be good antagonists. Molecules that bind well and, as the case may be, increase the rate of product production from substrate, increase signal transduction. or increase 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 using a reporter system. Reporter systems that may be useful in this regard include but are not limited to colorimetric, labeled substrate converted into product, a reporter gene that is responsive to changes in fabG polynucleotide or polypeptide activity, and binding assays known in the art.

Polypeptides of the invention may be used to identify membrane bound or soluble receptors, if any. for such polypeptide, through standard receptor binding techniques known in the art. These techniques include, but are not limited to, ligand binding and crosslinking assays in which the polypeptide is labeled with a radioactive isotope (for instance, ^ I), 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 bodily materials) Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy These screemng methods may also be used to identify agonists and antagonists of the polypeptide which compete with the bmding of the polypeptide to its receptor(s), if any Standard methods for conducting such assays are well understood m the art The fluorescence polarization value for a fluorescently-tagged molecule depends on the rotational correlation time or tumbling rate Protein complexes, such as formed by fabG po peptide associating with another fabG polypeptide or other polypeptide, labeled to comprise a fluorescentlv-labeled molecule will have higher polarization values than a fluorescently labeled monomeπc protein It is preferred that this method be used to charactenze small molecules that disrupt polypeptide complexes

Fluorescence energy transfer may also be used characterize small molecules that interfere with the fonnation of fabG polypeptide dimers tnmers, tetramers or higher order structures, or structures formed by fabG polypeptide bound to another polypeptide FabG polypeptide can be labeled with both a donor and acceptor fluorophore Upon mixing of the two labeled species and excitation of the donor fluorophore, fluorescence energy transfer can be detected by observmg 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 fabG polypeptide self-association as well as an association of fabG polypeptide and another polypeptide or small molecule fabG polypeptide can be coupled to a sensor chip at low site density such that covalently bound molecules will be monomeπc Solution protem can then passed over the fabG polypeptide -coated surface and specific bmding can be detected m real-time by monitoring the change in resonance angle caused by a change m local refractive index This techmque can be used to characterize the effect of small molecules on kinetic rates and equihbnum bindmg constants for tabG polypeptide self-association as well as an association of fabG polypeptide and another polypeptide or small molecule

A scintillation proximity assay may be used to characterize the interaction between an association of fabG polypeptide with another fabG polypeptide or a different polypeptide fabG polypeptide can be coupled to a scmtillation-filled bead Addition of radio-labeled fabG polypeptide results m binding where the radioactive source molecule is in close proximity to the scintillation fluid Thus signal is emitted upon fabG polypeptide binding and compounds that prevent fabG polypeptide self-association or an association of fabG polypeptide and another polypeptide or small molecule will diminish signal ICS biosensors have been descnbed by AMBRI (Australian Membrane Biotechnology Research Institute) They couple the self-association of macromolecules to the closing of gramacidin-facilitated 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 combmatonal libranes

In 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 polypeptide and/or polynucleotide of the mvention compnsmg contacting a polypeptide and/or polynucleotide of the mvention with a compound to be screened under conditions to permit bmdmg to or other mteraction between the compound and the polypeptide and/or polynucleotide to assess the bmdmg to or other mteraction with the compound such bmdmg 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 and/or pohnucleotide with the compound and determining whether the compound binds to or otherwise interacts with and activates or inhibits an activity or expression of the polypeptide and/or polynucleotide by detecting the presence or absence of a signal generated from the bmdmg or mteraction of the compound with the polypeptide and/or polynucleotide

Another example of an assay for fabG agonists is a competitive assay that combines fabG and a potential agonist with fabG-binding molecules, recombinant fabG bmdmg molecules, natural substrates or ligands or substrate or ligand mimetics under appropnate conditions for a competitive inhibition assay fabG can be labeled, such as by radioactivity or a colonmetnc compound, such that the number of fabG molecules bound to a bindmg molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist

Potential antagonists include, among others, small organic molecules, peptides, polypeptides and antibodies that bind to a polynucleotide and/or polypeptide of the invention and thereby inhibit or extinguish its activity or expression Potential antagonists also may be small organic molecules, a peptide, a poh peptide such as a closely related protem or antibody that bmds the same sites on a bmdmg molecule, such as a bindmg molecule without inducing fabG-induced activities, thereby preventmg the action or expression of fabG polypeptides and/or polynucleotides by excluding fabG polypeptides and/or polynucleotides from bmding

Potential antagonists include a small molecule that bmds to and occupies the bmdmg site of the polypeptide thereby preventing bmdmg to cellular bmdmg molecules, such that normal biological activity is prevented Examples of small molecules include but are not limited to small organic molecules, peptides or peptide-like molecules Other potential antagonists mclude antisense molecules (see Okano, J Neurochem 56 560 (1991), OUGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION CRC Press Boca Raton. FL (1988), for a descnption of these molecules) Preferred potential antagonists mclude compounds related to and vanants of fabG Other examples of potential polypeptide antagonists mclude antibodies or, m some cases, oligonucleotides or proteins which are closely related to the ligands, substrates, receptors, enzymes, etc , as the case may be. of the polypeptide, e g . a fragment of the ligands, substrates, receptors, enzymes, etc , or small molecules which bmd to the polypeptide of the present mvention 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 fabG polypeptide These functional mimetics may be used for, among other things, antagonizing the activity of fabG polypeptide or as a antigen or lmmunogen in a manner descnbed elsewhere herem Functional mimetics of the polypeptides of the mvention mclude but are not limited to truncated polypeptides For example, prefened functional mimetics mclude, a polypeptide compnsmg the polypeptide sequence set forth in SEQ ID NO 2 lackmg 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 encodmg each of these functional mimetics may be used as expression cassettes to express each mimetic polypeptide It is prefened drat these cassettes compnse 5' and 3' restnction sites to allow for a convement 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 herem

Thus in another aspect, the present invention relates to a screening kit for identifying agonists, antagonists, ligands, 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 polypeptide and/or a polynucleotide of the present invention, (b) a recombinant cell expressing a polypeptide and/or polynucleotide of the present invention, (c) a cell membrane expressing a polypeptide and/or polynucleotide of the present invention, or (d) antibody to a polypeptide and/or polynucleotide of the present mvention, which pol peptide is preferably that of SEQ ID NO 2, and which polynucleotide is preferably that of SEQ ID NO 1 It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component

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

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

If the expression and/or activity of the polypeptide and/or polynucleotide is m excess, several approaches are available One approach compnses administering to an individual m need thereof an inhibitor compound (antagonist) as herem descnbed, optionally in combination with a pharmaceutically acceptable earner in an amount effective to inhibit the function and/or expression of the polypeptide and/or polynucleotide, such as, for example, by blocking the bmdmg of 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 bmdmg the ligand. substrate, enzymes, receptors, etc in competition with endogenous polypeptide and/or polynucleotide may be administered Typical examples of such competitors include fragments of the fabG polypeptide and/or polypeptide

In a further aspect, the present invention relates to geneticalh engineered soluble fusion proteins comprising a polypeptide of the present invention, or a fragment thereof, and vanous portions of the constant regions of heavy or light chains of immunoglobuhns of various subclasses (IgG, IgM. IgA IgE) Preferred as an lmmunoglobulin is the constant part of the heavy chain of human IgG, particularly IgG 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 by genetic engineering, and to the use thereof for drug screemng, diagnosis and therapy A further aspect of the invention also relates to polynucleotides encodmg such fusion proteins Examples of fusion protein technology can be found m International Patent Application Nos W094/29458 and W094/22914

In still another approach, expression of the gene encoding endogenous fabG polypeptide can be inhibited using expression blocking techniques This blockmg may be targeted agamst 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 Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL ( 1988))- Alternatively, oligonucleotides which form triple helices with the gene can be supplied (see, for example, Lee et al, Nucleic Acids Res (1979) 6:3073; Cooney et al, Science (1988) 241 :456; Dervan et al. Science (1991) 251 : 1360). These oligomers can be administered per se or the relevant oligomers can be expressed in vivo.

Each of the polynucleotide sequences provided herein may be used in the discovery and development of antibacterial compounds. The encoded protein, upon expression, can be used as a target for the screening of antibacterial drugs. Additionally, the polynucleotide sequences encoding the amino terminal regions of the encoded protein or Shine-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the coding sequence of interest. The invention also provides the use of the polypeptide, polynucleotide, agonist or antagonist of the invention to interfere with the initial physical interaction between a pathogen or pathogens and a eukaryotic. preferably mammalian, host responsible for sequelae of infection- In particular, the molecules of the invention may be used: in the prevention of adhesion of bacteria, in particular gram positive and/or gram negative bacteria, to eukaryotic, preferably mammalian, extracellular matrix proteins on in-dwelling devices or to extracellular matrix proteins in wounds; to block bacterial adhesion between eukaryotic, preferably mammalian, extracellular matrix proteins and bacterial fabG proteins that mediate tissue damage and/or; to block the normal progression of pathogenesis in infections initiated other than by the implantation of in-dwelling devices or by other surgical techniques. In accordance with yet another aspect of the invention, there are provided fabG agonists and antagonists, preferably bacteristatic or bactericidal agonists and antagonists.

The antagonists and agonists of the invention may be employed, for instance, to prevent, inhibit and/or treat diseases.

Helwobacter pylori (herein "H. pylori") bacteria infect the stomachs of over one-third of the world's population causing stomach cancer, ulcers, and gastritis (International Agency for Research on Cancer ( 1994) Schistosomes. Liver Flukes and Helicobacter Pylori (International Agency for Research on Cancer, Lyon, France, http://www.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 fabG polypeptides and/or polynucleotides) found using screens provided by the invention, or known in the art, particularly narrow-spectrum antibiotics, should be useful in the treatment of H. pylori infection. Such treatment should decrease the advent of H. /τy/or/-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 invention, products, compositions and methods for assessing fabG expression, treating disease, assaying genetic variation, and administering a fabG polypeptide and/or polynucleotide to an organism to raise an immunological response against a bacteria, especially a Pseudomonas aeruginosa bacteria.

Another aspect of the invention relates to a method for inducing an immunological response in an individual, particularly a mammal which comprises inoculating the individual with fabG polynucleotide and/or 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 Pseudomonas aeruginosa infection. Also provided are methods whereby such immunological response slows bacterial replication. Yet another aspect of the invention relates to a method of inducing immunological response in an individual which comprises delivering to such individual a nucleic acid vector, sequence or ribozyme to direct expression of fabG polynucleotide and/or polypeptide, or a fragment or a variant thereof, for expressing fabG polynucleotide and/or polypeptide, or a fragment or a variant thereof in vivo in order to induce an immunological response, such as, to produce antibody and or T cell immune response, including, for example, cytokine- producing T cells or cytotoxic T cells, to protect said individual, preferably a human, from disease, whether that disease is already established within the individual or not. One example of administering the gene is by accelerating it mto the desired cells as a coating on particles or otherwise. Such nucleic acid vector may comprise DNA, RNA, a ribozyme, a modified nucleic acid, a DNA/RNA hybrid, a DNA-protein complex or an RNA-protein complex.

A further aspect of the invention relates to an immunological composition that when introduced into an individual, preferably a human, capable of having induced within it an immunological response, induces an immunological response in such individual to a fabG polynucleotide and/or polypeptide encoded therefrom, wherein the composition comprises a recombinant fabG polynucleotide and/or polypeptide encoded therefrom and/or comprises DNA and/or RNA which encodes and expresses an antigen of said fabG polynucleotide, polypeptide encoded therefrom, or other polypeptide of the invention The immunological response may be used therapeutically or prophylactically and may take the form of antibody immunity and/or cellular immunity, such as cellular immunity arising from CTL or CD4+ T cells

A fabG polypeptide or a fragment thereof may be fused with co-protein or chemical moiety which may or may not by itself produce antibodies, but which is capable of stabihzmg the first protem and producing a fused or modified protem which will have antigemc and/or lmmunogenic properties, and preferably protective properties Thus fused recombinant protem, preferably further compnses an antigemc co-protein, such as lipoprotein D from Hemophilus mfluenzae, Glutathione-S-transferase (GST) or beta-galactosidase or any other relatively large co-protem which solubihzes the protem and facilitates production and purification thereof Moreover, the co-protein may act as an adjuvant m the sense of providing a generalized stimulation of the immune system of the organism receivmg the protein The co-protein may be attached to either the ammo- or carboxy-teπrunus of the first protem

Provided by this invention are compositions, particularly vaccine compositions, and methods comprising the polypeptides and/or polynucleotides of the invention and immunostimulatory DNA sequences such as those described in Sato, Y et al Science 273 352 (1996)

Also provided by this invention are methods using the described polynucleotide or particular fragments thereof, which have been shown to encode non-variable regions of bactenal cell surface proteins, in polynucleotide constructs used m such genetic immunization expenments m animal models of infection with Pseudomonas aeruginosa Such experiments will be particularly useful for identifying protein epitopes able to provoke a prophylactic or therapeutic immune response It is believed that this approach will allow for the subsequent preparation of monoclonal antibodies of particular value, 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 Pseudomonas aeruginosa infection, in mammals, particularly humans A polypeptide of the invention may 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 tor 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 compnses an lmmunogenic recombinant polypeptide and/or polynucleotide of the mvention together with a suitable earner, such as a pharmaceutically acceptable earner Since the polypeptides and polynucleotides may be broken down in the stomach, each is preferably administered parenterally, mcludmg, for example, administration that is subcutaneous, intramuscular, intravenous, or mtradermal Formulations suitable for parenteral administration mclude aqueous and non-aqueous stenle injection solutions which may contain anti-oxidants, buffers, bactenstatic compounds and solutes which render the formulation lsotonic with the bodily fluid, preferably the blood, of the mdividual, and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents The formulations may be presented in umt-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored m a freeze-dπed condition requiring only the addition of the stenle liquid carrier immediately prior to use The vaccine formulation may also include adjuvant systems for enhancing the immunogemcity of the formulation, such as oil-in water systems and other systems known m the art The dosage will depend on the specific activity of the vaccme and can be readily determined by routine experimentation

While the mvention has been described with reference to certain fabG polypeptides and polynucleotides, it is to be understood that this covers fragments of the naturally occurring polypeptides and polynucleotides, 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 mvention there are provided compositions compnsmg a fabG polynucleotide and/or a fabG polypeptide for admmistration to a cell or to a multicellular organism

The invention also relates to compositions compnsmg a polynucleotide and/or a polypeptides discussed herein or then- agonists or antagonists The polypeptides and polynucleotides of the mvention may be employed in combination with a non-sterile or stenle earner or earners for use with cells, tissues or organisms, such as a pharmaceutical earner suitable for administration to an mdividual Such compositions compnse, for instance, a media additive or a therapeutically effective amount of a polypeptide and/or polynucleotide of the mvention and a pharmaceutically acceptable earner or excipient Such earners may include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof The formulation should suit the mode of administration The my ention further relates to diagnostic and pharmaceutical packs and kits compnsmg one or more containers filled with one or more of the ingredients of the aforementioned compositions of the mvention

Polypeptides polynucleotides and other compounds of the invention may be employed alone or m conjunction with other compounds, such as therapeutic compounds The pharmaceutical compositions may be administered in any effective, convenient manner including, for instance, administration by topical, oral, anal, vaginal, intravenous, intrapentoneal, intramuscular, subcutaneous, lntranasal or intrade mal 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 isotonic. 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 in ointments and creams Such topical formulations may also contain compatible co entional carriers, for example cream or ointment bases, and ethanol or oleyl alcohol for lotions. Such earners may constitute from about 1% to about 98% by weight of the formulation; more usually they w ill constitute up to about 80% by weight of the formulation

In a further aspect, the present mvention provides for pharmaceutical compositions comprising a therapeutically effective amount of a polypeptide and/or polynucleotide, such as the soluble form of a polypeptide and or polynucleotide of the present invention, agonist or antagonist peptide or small molecule compound, in combination with a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention. 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 administration, for instance by a systemic or an oral route Prefened forms of systemic administration include injection, typically by intravenous injection. Other injection routes, such as subcutaneous, intramuscular, or intrapentoneal, can be used Alternative means for svstemic administration mclude transmucosal and transdermal administration using penetraπts such as bile salts or fusidic acids or other detergents. In addition, if a polypeptide or other compounds of the present invention can be formulated in an enteric or an encapsulated formulation, oral administration may also be possible Administration of these compounds may also be topical and/or localized, in 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 phy sician in any event will determine the actual dosage which will be most suitable for an individual and will vary with the age. weight and response of the particular individual. The above dosages are exemplary of the average case There can. of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention ln-dwelhng devices include surgical implants, prosthetic devices and catheters, i.e., devices that are introduced to the body of an individual and remain in position for an extended time. Such devices include, for example, artificial joints, heart valves, pacemakers, vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinary catheters, continuous ambulatory peritoneal 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 contmued after surgery during the in-body time of the device. In addition, the composition could also be used to broaden perioperative cover for any surgical technique to prevent bacterial wound infections, especially Pseudomonas aeruginosa 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 infection 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 this invention may be used generally as a wound treatment agent to prevent adhesion of bacteria to matrix proteins exposed in wound tissue and for prophylactic use m dental treatment as an alternative to, or in conjunction with, antibiotic prophylaxis

Alternatively, the composition of the invention may be used to bathe an indwelling device immediately before insertion The active agent will preferably be present at a concentration of 1 μg/ml to 1 Omg/ml for bathing of wounds or indwelling devices v accine composition is conveniently in injectable form Conventional adjuvants may be employed to enhance the immune response. A suitable unit dose for vaccination is 0.5-5 microgram/kg of antigen, and such dose is preferably administered 1-3 times and with an interval of 1- 3 weeks With the indicated dose range, no adverse toxicological effects will be observed with the compounds of the invention which would preclude their administration to suitable individuals. Sequence Databases, Sequences in a Tangible Medium, and Algorithms

Polynucleotide and polypeptide sequences form a valuable information resource with which to determine their 2- and 3 -dimensional structures as well as to identify further sequences of similar homology. These approaches are most easily facilitated by storing the sequence in a computer readable medium and then using the stored data in a known macromolecular structure program or to search a sequence database usmg well known searching tools, such as GCC

T he poly nucleotide and poly peptide sequences of the invention are particularly useful as components in databases useful for search analyses as well as in sequence analysis algorithms. As used in this section entitled "Sequence Databases. Sequences in a Tangible Medium, and Algorithms," and in claims related to this section, the terms "polynucleotide of the invention" and "polynucleotide sequence of the invention" mean any detectable chemical or physical characteristic of a polynucleotide of the invention that is or may be reduced to or stored in a tangible medium, preferably a computer readable fonn For example, chromatographic scan data or peak data, photographic data or scan data therefrom, called bases, and mass spectrographic data As used this section entitled Databases and Algorithms and in claims related thereto, the terms "polypeptide of the invention" and "polypeptide sequence of the mvention" mean any detectable chemical or physical charactenstic of a polypeptide of the invention that is or may be reduced to or stored m a tangible medium, preferably a computer readable form For example, chromatographic scan data or peak data, photographic data or scan data therefrom, and mass spectrographic data.

The invention provides a computer readable medium having stored thereon polypeptide sequences of the invention and or polynucleotide sequences of the invention For example, a computer readable medium is provided compnsmg and havmg stored thereon a member selected from the group consisting of a polynucleotide compnsmg the sequence of a polynucleotide of the invention; a polypeptide comprising the sequence of a polypeptide sequence of the mvention, a set of polynucleotide sequences wherein at least one of the sequences comprises the sequence of a polynucleotide sequence of the invention, a set of polypeptide sequences wherein at least one of the sequences comprises the sequence of a polypeptide sequence of the mvention; a data set representmg a polynucleotide sequence compnsmg the sequence of polynucleotide sequence of the invention; a data set representing a polynucleotide sequence encoding a polypeptide sequence comprising the sequence of a polypeptide sequence of the invention, a polynucleotide compnsmg the sequence of a polynucleotide sequence of the invention: a polypeptide comprising the sequence of a polypeptide sequence of the invention, a set of polynucleotide sequences wherem at least one of the sequences comprises the sequence of a polynucleotide sequence of the invention, a set of polypeptide sequences wherein at least one of said sequences comprises the sequence of a polypeptide sequence of the invention; a data set representmg a polynucleotide sequence comprising the sequence of a polynucleotide sequence of the invention, a data set representing a polynucleotide sequence encoding a polypeptide sequence comprising the sequence of a polypeptide sequence of the invention- The computer readable medium can be any composition of matter used to store information or data, including, for example, commercially available floppy disks, tapes, chips, hard drives, compact disks, and video disks. Also provided by the invention are methods for the analysis of character sequences or strings, particularly genetic sequences or encoded genetic sequences. Prefened methods of sequence analysis include, for example, methods of sequence homology analysis, such as identity and similarity analysis, RNA structure analysis, sequence assembly, cladistic analysis, sequence motif analysis, open reading frame determination, nucleic acid base calling, nucleic acid base trimming, and sequencing chromatogram peak analysis.

A computer based method is provided for performing homology identification. This method comprises the steps of providing a first polynucleotide sequence comprising the sequence a polynucleotide of the invention in a computer readable medium; and comparing said first polynucleotide sequence to at least one second polynucleotide or polypeptide sequence to identify homology.

A computer based method is also provided for performing homology identification, said method comprising the steps of: providing a first polypeptide sequence comprising the sequence of a polypeptide of the invention -in a computer readable medium; and comparing said first polypeptide sequence to at least one second polynucleotide or polypeptide sequence to identify homology. A computer based method is still further provided for polynucleotide assembly, said method comprising the steps of: providing a first polynucleotide sequence comprising the sequence of a polynucleotide of the invention in a computer readable medium; and screening for at least one overlapping region between said first polynucleotide sequence and at least one second polynucleotide or polypeptide sequence- A computer based method is still further provided for polynucleotide assembly, said method comprising the steps of: providing a first polypeptide sequence comprising a polypeptide of the invention m a computer readable medium; and screening for at least one overlapping region between said first polypeptide sequence and at least one second polynucleotide or polypeptide sequence.

In another prefened embodiment of the invention there is provided a computer readable medium having stored thereon a member selected from the group consisting of: a polynucleotide comprising the sequence of SEQ ID NO: l ; a polypeptide comprising the sequence of SEQ ID NO:2; a set of polynucleotide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO: 1 : a set of polypeptide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO 2 a data set representing a polynucleotide sequence compnsmg the sequence of SEQ ID NO 1 a data set representing a polynucleotide sequence encoding a polypeptide sequence compnsmg the sequence of SEQ ID NO 2 a polynucleotide compnsmg the sequence of SEQ ID NO 1, a polypeptide comprising the sequence of SEQ ID NO 2, a set of polynucleotide sequences wherein at least one of said sequences compnses the sequence of SEQ ID NO 1, a set of polypeptide sequences wherem at least one of said sequences compnses the sequence of SEQ ID NO 2, a data set representing a polynucleotide sequence comprising the sequence of SEQ ID NO 1 a data set representing a polvnucleotide sequence encoding a polypeptide sequence compnsing the sequence of SEQ ID NO 2 A further prefened embodiment of the invention provides a computer based method for performing homology identification, said method comprising the steps of providing a polynucleotide sequence compnsmg the sequence of SEQ ID NO 1 m a computer readable medium, and comparing said polynucleotide sequence to at least one polynucleotide or polypeptide sequence to identify homology

A still further preferred embodiment of the invention provides a computer based method for performing homology identification, said method compnsmg the steps of providing a polypeptide sequence comprising the sequence of SEQ ID NO 2 a computer readable medium, and comparing said polypeptide sequence to at least one polynucleotide or polypeptide sequence to identify homology

A further embodiment of the mvention provides a computer based method for polynucleotide assembly, said method compnsmg the steps of providing a first polynucleotide sequence compnsmg the sequence of SEQ ID NO 1 in a computer readable medium, and screemng for at least one overlapping region between said first polynucleotide sequence and a second polynucleotide sequence

A further embodiment of the mvention provides a computer based method for performing homology identification, said method compnsmg the steps of providing a polynucleotide sequence compnsmg the sequence of SEQ ID NO 1 in a computer readable medium, and comparing said polynucleotide sequence to at least one polynucleotide or polypeptide sequence to identify homology

All publications and references, including but not limited to patents and patent applications, cited in this specification are herem incorporated by reference in their entirety as if each mdividual 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 by reference herein m its entirety in the manner descnbed above for publications and references GLOSSARY

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

"Antιbody(ιes)" as used herem includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin expression library.

"Antigenically equivalent denvatιve(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(ies)" means an antibody compnsmg at least two antigen binding domains, each domain directed agamst a different epitope

"Bodily mateπal(s) means any matenal denved from an mdividual or from an organism infecting, infesting or inhabiting an mdividual, mcludmg but not 1-ιmited to, cells, tissues and waste, such as, bone, blood, serum, cerebrospmal fluid, semen, saliva, muscle, cartilage, organ tissue, skin. unne. stool or autopsy mateπals.

"Dιsease(s)" means any disease caused by or related to infection 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 cerebrospinal fluid. "Fusion protein(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 immunoglobulin molecules together with another human protem or part thereof. In many cases, employing an immunoglobulin Fc region as a part of a fusion protem is advantageous for use in therapy and diagnosis resultmg in, for example, improved pharmacok etic 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 the art. is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determined by comparing the sequences. In the art. "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology. Lesk, A M . ed , Oxford University Press, New York, 1988, Biocomputing Informatics and Genome Projects, Smith, D W , ed , Academic Press, New York, 1993, Computer Analysis of Sequence Data, Part I, Griffin, A M , and Gnffin, H G , eds , Humana Press, New Jersey, 1994, Sequence Analysis in Molecular Biology, v on Hemje, G , Academic Press, 1987, and Sequence Analysis Primer Gnbskov, M and Devereux, .! eds , M Stockton Press. New York, 1991, and Canllo, H , and Lipman, D , S-IAM J Applied Math , 48 1073 (1988) Methods to determine identity are designed to give the largest match between the sequences tested Moreover, methods to determine identity are codified in publicly available computer programs Computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package (Devereux, J . et al , Nucleic Acids Research 12(1) 387 (1984)), BLASTP, BLASTN. and FASTA (Altschul, S F et al N Molec Biol 215 403-410 (1990) The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S , et al , NCBI NLM NIH Bethesda, MD 20894 Altschul, S . et al , J Mol Biol 215 403-410 (1990) The well known Smith Waterman algorithm may also be used to determine identity Parameters for polypeptide sequence comparison include the following Algorithm

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

Companson matrix BLOSSUM62 from Hentikoff and Hentikoff, Proc Natl Acad Sci USA 89 10915-10919 (1992) Gap Penalty 12 Gap Length Penalty 4

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

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

Companson 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, wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO 1 or may mclude up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion substitution, mcludmg transition and transversion, or insertion and wherein said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, mterspersed either individually among the nucleotides m 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 by the mteger defining the percent identity divided by 100 and then subtracting that product from said total number of nucleotides m SEQ ID NO 1, or

n_n < x_n (^χ _n • y)>

wherein n_n is the number of nucleotide alterations, x_n is the total number of nucleotides m SEQ ID NO 1 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 herein 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 encodmg the polypeptide of SEQ ID NO 2 may create nonsense, missense or frameshift mutations m this codmg sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations

By way of example, a polynucleotide sequence of the present mvention may be identical to the reference sequence of SEQ ID NO 1, that is it may be 100% identical, or it may include up to a certain integer number of nucleic acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity Such alterations are selected from the group consisting of at least one nucleic acid deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleic acids in the reference sequence or in one or more contiguous groups within the reference sequence The number of nucleic acid alterations for a given percent identity is determined by multiplying the total number of nucleic acids in SEQ ID NO 1 by the mteger defining the percent identity divided by 100 and then subtracting that product from said total number of nucleic acids in SEQ ID NO: 1, or:

ⁿn ≤ ^xn " (^xn * y)>

wherein n_n is the number of nucleic acid alterations, x_n is the total number of nucleic acids in SEQ ID NO: 1, y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., • 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. (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, wherein said polypeptide sequence may be identical to the reference sequence of SEQ ID NO:2 or may include up to a certain integer number of amino acid alterations as compared to the reference sequence, wherein 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 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in SEQ ID NO:2, or:

n_a < x_a (^χ _a • y)>

wherein n_a is the number of amino acid alterations, x_a is the total number of amino acids in SEQ ID NO:2, 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.

By way of example, a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ID NO:2, that is it may be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity Such alterations are selected from the group consistmg of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherem said alterations may occur at the amino- or carboxy-termmal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the ammo acids in the reference sequence or in one or more contiguous groups withm the reference sequence The number of ammo acid alterations for a given % identity is determined by multiplying the total number of ammo acids in SEQ ID NO 2 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, or

ⁿa ≤ ^xa ^" (^χa ^# )»

wherein n_a is the number of ammo acid alterations. x_a is the total number of amino acids in SEQ ID NO 2 y is, for instance 0 70 for 70%, 0 80 for 80%, 0 85 for 85% etc , 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, polynucleotide, 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 mteraction 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 pnor art

"Indιvιdual(s)" means a multicellular eukaryote, mcludmg, but not limited to a meta-zoan, 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 m a living orgamsm is not "isolated," but the same polynucleotide or polypeptide separated from the coexistmg matenals of its natural state is "isolated" as the term is employed herein Moreover, a polynucleotide or polypeptide that is mtroduced mto an orgamsm by transformation, genetic manipulation or by any other recombinant method is "isolated" even if it is still present m said orgamsm which orgamsm may be living or non-living

"Organιsm(s)" means a (1) prokaryote, mcludmg but not limited to, a member of the genus Streptococcus, Staphylococcus, Bordetella, Corynebactenum, Mycobactenum, Neissena, Haemophilus, Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia, Fanasella, Pasturella, Moraxella, Acinetobacter, Erysipelothnx, Branhamella, Actinobacillus, Streptobacillus, Listena, Calymmatobacterium Brucella Bacillus, Clostndium, Treponema, Eschenchia, Salmonella, Kleώsiella, Vibrio, Proteus Erwinia, Borrelia, Leptospira, Spirillum, Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borrelia and Mycoplasma, and further mcludmg, but not limited to. a member of the species or group. Group A Streptococcus Group B Streptococcus, Group C Streptococcus, Group D Streptococcus, Group G Streptococcus Streptococcus pneumoniae Streptococcus pyogenes Streptococcus agalactiae, Streptococcus faecalis, Streptococcus faecium Streptococcus durans Neissena gonorrheae, Neissena meningitidts Staphylococcus aureus, Staphylococcus epidermidis, Corynebactenum dψthenae, Gardnerella vagtnalis, Mycobactenum tuberculosis, Mycobactenum bovis, Mycobactenum ulcerans, Mycobactenum leprae, Actmomyctes israelu, Listena monocytogenes, Bordetella pertusis, Bordatella parapertusis, Bordetella bronchiseptica, Eschenchia cob, Shigella dysentenae, Haemophilus lnβuenzae, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonella typhi, Citrobacter freundn, Proteus mirabihs Proteus vulgaris, Yersinia pestis, Kleibsiella pneumoniae Serratia marcessens, Serratia liquefaciens Vibrio cholera Shigella dysenteni Shigella flexnen, Pseudomonas aeruginosa, Franscisella tularensis Brucella abortis, Bacillus anthracis, Bacillus cereus, Clostndium perfnngens, Clostndium tetan , Clostndium botuhnum, Treponema palbdum, Rickettsia nckettsu and Chlamydia trachomitis, (u) an archaeon, mcludmg but not limited to Archaebacter , 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 cenviseae, Kluveromyces lactis, or Candida albicans

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

"Polypeptide(s)" refers to any peptide or protem compnsmg two or more ammo acids jomed to each other by peptide bonds or modified peptide bonds "Polypeptide(s)" refers to both short chains, commonly refened to as peptides, ohgopeptides and oligomers and to longer chains generally refened to as proteins Polypeptides may contain ammo acids other than the 20 gene encoded ammo acids "Polypeptide(s)" mclude those modified either by natural processes, such as processmg and other post-translational modifications, but also by chemical modification techniques Such modifications are well descnbed m basic texts and m more detailed monographs, as well as m a volummous research hterature, 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 m the same or varying degree at several sites m a given polypeptide Also, a given polypeptide may contain many types of modifications Modifications can occur anywhere in a polypeptide, mcludmg the peptide backbone, the ammo acid side-chains, and the ammo or carboxyl termini Modifications mclude, for example, acetylation, acylation ADP-nbosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide denvative, covalent attachment of a hpid or hpid denvative, covalent attachment of phosphotidylmositol, cross-linking, cycbzation, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, GPI anchor formation, hydroxylation, lodination, methylation, mynstoylation, oxidation, proteolytic processmg, phosphorylatioi---, prenylation, racemizatio-α, glycosylation, hpid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP- nbosylation., selenoylation, sulfation, transfer-RNA mediated addition of ammo acids to proteins, such as arginylatioα and ubiquitmation See. for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed , T E Creighton- W H Freeman and Company, New York (1993) and Wold, F , Posttranslational Protem Modifications Perspectives and Prospects, pgs 1-12 m POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B C Johnson, Ed , Academic Press, New York (1983), Seifter et al . Meth Enzymol 182 626-646 (1990) and Rattan et al Protein Synthesis Posttranslational Modifications and Aging. Ann N Y Acad Sci 663 48-62 (1992) Polypeptides may be branched or cyclic, with or without branching Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well

"Recombinant expression system(s)" refers to expression systems or portions thereof or polynucleotides of the mvention introduced or transformed into 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 compnsmg at least one polynucleotide of the invention

Vaπant(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 in nucleotide sequence from another, reference polynucleotide Changes m the nucleotide sequence of the vanant may or may not alter the am o acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result m ammo acid substitutions, additions, deletions, fusion protems and truncations in the polypeptide encoded by the reference sequence, as discussed below A typical variant of a polypeptide differs m ammo acid sequence from another, reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the vanant are closely similar overall and, in many regions, identical A vanant and reference polypeptide may differ m ammo acid sequence by one or more substitutions, additions, deletions m any combination A substituted or inserted ammo acid residue may or may not be one encoded by the genetic code The present mvention also mcludes mclude vanants of e^ch of the polypeptides of the mvention, that is polypeptides that vary from the referents by conservative ammo acid substitutions whereby a residue is substituted by another with like charactenstics Typical such substitutions are among Ala, Val, Leu and lie, among Ser and Thr, among the acidic residues Asp and Glu, among Asn and Gin, and among the basic residues Lys and Arg, or aromatic residues Phe and Tyr Particularly prefened are vanants m which several, 5-10, 1-5, 1-3, 1-2 or 1 ammo acids are substituted, deleted, or added m any combination A variant of a polynucleotide or polypeptide may be a naturally occurring such as an allehc variant, or it may be a variant that is not known to occur naturally Non- naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques by direct synthesis, and by other recombinant methods known to skilled artisans EXAMPLES

The examples below are earned out usmg standard techniques, which 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 having a DNA sequence given m Table 1 [SEQ ID NO 1] was obtained from a library of clones of chromosomal DNA of Pseudomonas aeruginosa m E cob The sequencmg data from two or more clones containing overlapping Pseudomonas aeruginosa DNAs was used to construct the contiguous DNA sequence in SEQ ID NO 1 Libranes may be prepared by routine methods for example Methods 1 and 2 below

Total cellular DNA is isolated from Pseudomonas aeruginosa strain 4 accordmg to standard procedures and size-fractionated by either of two methods

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

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combination of restnction 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 accordmg to standard procedures EcoRI linkers are ligated to the DNA and the fragments then ligated mto 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 FabG Characterization

We have used chemical mutagenesis to isolate temperature-sensitive (ts) mutants m an attempt to identify essential P aeruginosa gene products Over 100 mutants, which show ts growth on complex medium at 44oC, have been isolated A genomic library containing 5 to 6 kb DNA fragments of P aeruginosa was constructed to complement these ts mutants Nucleotide sequence analysis of plasmids complementing the ts mutants revealed many known essential genes as well as genes with unknown functions. One of the ts mutants, ts-67, was shown to contain a C->T transition mutation at nucleotide position 394 in Table 1 [SEQ ID NO: l], which caused an amino acid substitution resulting in the change of arginine at position 135 in Table 1 [SEQ ID NO: 2] to cysteine in the FabG ORF. Furthermore, a revertant (i.e., restore growth at 44oC) of the fabG ts mutant was isolated and found to have the original mutation (C to T) reverted back to wild type sequence (T to C). Therefore, these results demonstrate that the fabG gene is essential for cell growth and can be used as an antimicrobial target.

Claims

What is claimed is:

1 An isolated polypeptide selected from the group consisting of

(0 an isolated polypeptide comprising an amino acid having at least

(a) 70% identity,

(b) 80% identity.

(c) 90% identity, or

(d) 95% identity to the ammo acid sequence of SEQ ID NO 2 over the entire length of SEQ ID NO 2, (n) an isolated polypeptide compnsing the ammo acid sequence of SEQ ID NO 2, (in) an isolated polypeptide which is the ammo acid sequence of SEQ ID NO 2, and (iv) a polypeptide which is encoded by a recombmant polynucleotide compnsmg the polynucleotide sequence of SEQ ID NO 1

An isolated polynucleotide selected from the group consisting of

(0 an isolated polynucleotide compnsmg a polynucleotide sequence encodmg a polypeptide that has at least

(a) 70% identity

(b) 80%> ιdentιtv

(c) 90% identity, or

(d) 95% identity, to the ammo acid sequence of SEQ ID NO 2, over the entire length of SEQ ID NO 2, (u) an isolated polynucleotide compnsmg a polynucleotide sequence that has at least

(a) 70%) identity

(b) 80% identity,

(c) 90% identity , or

(d) 95% identity, over its entire length to a polynucleotide sequence encodmg the polypeptide of SEQ ID

NO 2,

(in) an isolated polynucleotide compnsmg a nucleotide sequence which has at least

(a) 70% identity.

(b) 80% identity

(c) 90% identity, or (d) 95% identity, to that of SEQ ID NO 1 over the entire length of SEQ ID NO 1 , ) an isolated polynucleotide compnsmg a nucleotide sequence encodmg the polypeptide of SEQ

ID NO 2

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

(vi) an isolated polynucleotide obtainable by screemng an appropnate library under strmgent hy bπdization conditions with a probe havmg the sequence of SEQ ID NO 1 or a fragment thereof, (vn) an isolated polynucleotide encodmg a mature polypeptide expiessed by the fabG gene contained m the Pseudomonas aentgmosa and

(v in) a poh nucleotide sequence complementarv to said isolated polynucleotide of (l) (n) (in)

3 An antibody antige c to or lmmunospecific for the polypeptide of claim 1

4 A method for the treatment of an individual

(0 in need of enhanced activity or expression of the polypeptide of claim 1 comprising the step of

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

(b) pro\ iding to the individual an isolated polynucleotide compnsmg a polynucleotide sequence encodmg said polypeptide in a form so as to effect production of said poh peptide actn ιty in vivo or

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

(a) administering to the individual a thei apeutically effective amount of an antagonist to said polypeptide or

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

(c) administering to the individual a therapeutically effective amount of a polypeptide that competes with said poly peptide for its ligand, substrate or receptor 5 A process for diagnosing or prognosing a disease or a susceptibility to a disease in an individual related to expression or activity of the polypeptide of claim 1 in an individual comprising the step of

(a) detennmmg the presence or absence of a mutation in the nucleotide sequence encoding said polypeptide in the genome of said individual, or

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

6 A method for screening to identify' compounds that activate or that inhibit the function of the polypeptide of claim 1 which compnses a method selected from the group consisting of

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

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

(c) testing whethei the candidate compound results in a signal generated by activation or inhibition of the poh peptide using detection systems appropriate to the cells or cell membranes bearing the polypeptide.

(d) mixing a candidate compound with a solution containing a polypeptide of claim 1 , to fonn 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 poly peptide and said polypeptide in cells, using for instance, an ELISA assay, oi

(f) ( 1 ) contacting a composition compnsmg the polypeptide with the compound to be screened under conditions to penmt mteraction between the compound and the polypeptide to assess d e interaction of a compound, such mteraction bemg associated with a second component capable of prov iding a detectable signal in response to the interaction of the polypeptide with the compound and

(2) determining whether the compound interacts with and activates or inhibits an activity of the polypeptide by detecting the presence or absence of a signal generated from the mteraction of the compound witii the polypeptide

7 An agonist or an antagonist of the activity or expression polypeptide of claim 1 8 An expression system compnsmg a polynucleotide capable of producing a polypeptide of claim 1 when said expression system is present m a compatible host cell

9 \ host cell comprising the expression sv stem of claim 8 or a membrane thereof expressing a poh peptide selected trom the group consisting of

(I ) an isolated poly peptide comprising an amino acid sequence selected from the group having at least

(α) 70% identity ,

(b) 80% identity,

(c) 90% identity or

(d) 95% identity to the amino acid sequence of SEQ ID NO 2 over the entire length of SEQ ID NO 2, (n) an isolated polypeptide comprising the ammo acid sequence of SEQ ID NO 2. (in) an isolated polypeptide which is the amino acid sequence of SEQ ID NO 2, and (iv) a polypeptide which is encoded by a recombinant polynucleotide comprising the polynucleotide sequence of SEQ ID NO 1

I n loi producing α poly peptide selected trom the group consisting of

(0 an isolated polypeptide comprising an ammo acid sequence selected from the group having at least

(a) 70% identity,

(b) 80% identity

(c) 90% identity . or

(d) 95% identity to the amino acid sequence of SEQ ID NO 2 over the entire length of SEQ ID NO 2 (n) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO 2. (in) an isolated polypeptide which is the ammo acid sequence of SEQ ID NO 2, and (iv ) a polypeptide which is encoded by a recombinant polynucleotide comprising the poh nucleotide sequence of SEQ ID NO 1 comprising the step of cultuπng a host cell of claim 9 under conditions sufficient for the production ol said poly peptide 1 1 A process for producing a host cell comprising the expression system of claim 8 or a membrane thereof expressing a polypeptide selected from the group consisting of

(I) an isolated poly peptide comprising an amino acid sequence selected from the group having at least

(a) 70% identity,

(b) 80% identity.

(c) 90% identity or

(d) 95% identity to the ammo acid sequence of SEQ ID NO 2 over the entire length of SEQ ID NO 2, (u) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO 2. (in) an isolated poly peptide which is the ammo acid sequence of SEQ ID NO 2, and (i ) α poly peptide w hich is encoded by a recombinant polynucleotide comprising the poh nuc υtidi- --^quαic of S EQ ID NO 1 said process comprising the step of transforming or transfecting a cell with an expression system compnsmg a polynucleotide capable of producing said polypeptide of (l), (n), (in) or (iv) when said expression system is present in a compatible host cell such the host cell, under appropriate culture conditions, produces said polypeptide of (l), (n), (m) or (iv)

12 A host cell produced by the process of claim 1 1 oi a membrane thereof expressing a polypeptide selected from the group consisting of

(I) an isolated polypeptide comprising an amino acid sequence selected from the group having at least

(a) 70% identity

(b) 80% identity

(c) 90% identity or

(d) 9S^» , . identity to the amino acid sequence of SEQ ID NO 2 over the entire length of SEQ ID NO 2 (n) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO 2, (in) an isolated polypeptide which is the amino acid sequence of SEQ ID NO 2, and (iv) a polypeptide which is encoded by a recombinant polynucleotide comprising the pol^ynucleotide sequence of SEQ ID NO 1 13 A computer readable medium having stored thereon a member selected from the group consisting of a poh nucleotide comprising the sequence of SEQ ID NO 1. a polypeptide comprising the sequence of SEQ ID NO 2, a set of polynucleotide sequences wherein at least one of said sequences comprises the sequence ot SEQ ID NO 1 a set of poly peptide sequences wherein at least one of said sequences comprises the sequence ofSEQ ID NO 2. a data set representing a polynucleotide sequence comprising the sequence of SEQ ID NO 1 , a data set representing a polynucleotide sequence encoding a polypeptide sequence comprising the sequence of SEQ ID NO 2, a polynucleotide comprising the sequence of SEQ ID NO 1 , a polypeptide comprising the sequence of SEQ ID NO 2. a set of polynucleotide sequences wherem at least one of said sequences comprises the sequence of SEQ ID NO 1. a set of polypeptide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO 2 a data set representmg a polynucleotide sequence compnsing the sequence of SEQ ID NO 1. a data set representing a polynucleotide sequence encoding a polypeptide sequence comprising the sequence of SEQ ID NO 2

14 A computer based method for performing homology identification, said method comprising the steps ol prov iding α polv nucleotide sequence comprising the sequence of SEQ ID NO 1 in a computer readable medium, and comparing said polynucleotide sequence to at least one polynucleotide or poh peptide sequence to identify homology

15 A further embodiment of the invention provides a computer based method for polynucleotide assembly, said method comprising the steps of providing a first polynucleotide sequence compnsing the sequence of SEQ ID NO 1 in a computer readable medium, and screening for at least one overlapping region between said first polynucleotide sequence and a second polynucleotide sequence

16 A poly nucleotide consisting of a polynucleotide of die fonnula

X-(R₁)_m-(R₂)-(R₃)_n-Y wherein, at the 5' end of the molecule, X is hydrogen, a metal or a modified nucleotide residue, or together w ith Y defines a cov alent bond and at the 3' end of the molecule. Y is hydrogen, a metal, or a modified nucleotide residue or together with X defines the covalent bond each occurrence of R_j and R3 is independently any nucleic acid residue or modified nucleic acid residue, m is an integer between 1 and 3000 or zero n is an integer between 1 and 3000 01 zero and R₂ is a nucleic acid sequence or modified nucleic acid sequence set forth 111 SEQ ID NO 1