WO2001019979A1 - Murc gene and enzyme of pseudomonas aeruginosa - Google Patents

Abstract

This invention provides isolated polynucleotides that encode the MurC protein of Pseudomonas aeruginosa. Purified and isolated MurC recombinant proteins are also provided. Nucleic acid sequences which encode functionally active MurC proteins are described. Assays for the identification of modulators of the expression of murC and inhibitors of the activity of MurC, are also provided.

Description

TITLE OF THE INVENTION

MURC GENE AND ENZYME OF PSEUDOMONAS AERUGINOSA

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S Provisional Application No.

60/154,073, filed September 14, 1999, the contents of which are incorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY-SPONSORED R&D Not applicable.

REFERENCE TO MICROFICHE APPENDIX

Not applicable.

FIELD OF THE INVENTION

This invention relates to the genes and enzymes involved in cell wall synthesis in bacteria, and particularly to the inhibition of such enzymes.

BACKGROUND OF THE INVENTION The emegence of mluti-drug resistant bacteπa has led to an increased demand for new antibiotics with new modes of action. The biosynthetic pathway of the bacterial cell wall contains several attractive targets Some of the enzymes in that pathway are proven targets for antibiotics such as β-lactams and glycopeptides antibiotics. The bacterial cell wall is a polymer — a single molecule composed of peptidoglycan — that defines the boundary and shape of the cell. Assembled by crosshnking glycan chains with short peptide bπdges (Rogers, H. J., H. R. Perkins, and J. B. Ward, 1980, Biosynthesis of peptidoglycan. p 239-297. In Microbial cell walls and membranes Chapman & Hall Ltd. London), the completed structure is strong enough to maintain cell integπty against an osmotic pressure differential of over four atmospheres, but also flexible enough to allow the cell to move, grow and divide

The construction of the peptidoglycan begins in the cytoplasm with an activated sugar molecule, UDP-N-acetylglucosamine. After two reactions (catalyzed by MurA and MurB) that result in the placement of a lactyl group on the 3-OH of the glucosamme moiety, a seπes of ATP-dependent amino acid ligases (MurC, -D, -E, and -F) catalyze the stepwise synthesis of the pentapeptide sidecha using the newly synthesized lactyl carboxylate as the first acceptor site. After attachment of the sugar pentapeptide to a pid earner in the plasma membrane, another glucosamme unit is added to the 4-OH of the muramic acid moiety. The completed monomeπc building block is moved across the membrane into the peπplas where the penicillin-binding proteins enzymatically add it into the growing cell wall (Lugtenberg, E. J. J., 1972, Studies on Escherichia coh enzymes involved in the synthesis of Undine Diphosphate-N-Acetyl-Muramyl-pentapeptide. J. Bacteπol. 110:26-34; Mengin- Lecreulx, D., B. Flouret, and J. van Heijenoort, 1982, Cytoplasmic steps of peptidoglycan synthesis in Escherichia coli. J. Bacteπol. 151: 1109-1117).

Among the potential enzyme targets involved in cell wall biosynthesis is MurC, UDP-N-acetylmuramoyl hgase. This enzyme catalyses the ATP-dependent addition of L-alanine to UDP-N-acetylmuramoyl to form the precursor UDP-N- acetylmuramoyl-L-alanine. This step is essential for cell wall formation in both Gram (-ve) and Gram (+ve) bacteπa. Thus, inhibitors of this enzyme are likely broad spectrum antibiotics.

SUMMARY OF THE INVENTION Polynucleotides and polypeptides of Pseudomonas aeruginosa MurC, an enzyme involved in bacteπal cell wall biosynthesis are provided. The recombmant MurC enzyme is catalytically active in ATP-dependent D-glutamate addition reactions. The enzyme is used in in vitro assays to screen for antibacteπal compounds that target cell wall biosynthesis. The invention includes the polynucleotides, proteins encoded by the polynucleotides, and host cells expressing the recombmant enzyme, probes and primers, and the use of these molecules in assays.

An aspect of this invention is a polynucleotide having a sequence encoding a Pseudomonas aeruginosa MurC protein, or a complementary sequence. In a particular embodiment the encoded protein has a sequence corresponding to SEQ ID NO:2. In other embodiments, the encoded protein can be a naturally occurring mutant or polymorphic form of the protein. In preferred embodiments the polynucleotide can be DNA, RNA or a mixture of both, and can be single or double stranded. In particular embodiments, the polynucelotide is compπsed of natural, non-natural or modified nucleotides. In some embodiments, the mternucleotide linkages are linkages that occur in nature. In other embodiments, the mternucleotide linkages can be non- natural linkages or a mixture of natural and non-natural linkages. In a most preferred embodiment, the polynucleotide has a sequence shown in SEQ ED NO:l.

An aspect of this invention is a polynucleotide having a sequence of at least about 25 contiguous nucleotides that is specific for a naturally occurring polynucleotide encoding a Pseudomonas aeruginosa MurC protein. In particular preferred embodiments, the polynucleotides of this aspect are useful as probes for the specific detection of the presence of a polynucleotide encoding a Pseudomonas aeruginosa MurC protein. In other particular embodiments, the polynucleotides of this aspect are useful as pπmers for use in nucleic acid amplification based assays for the specific detection of the presence of a polynucleotide encoding a Pseudomonas aeruginosa MurC protein. In preferred embodiments, the polynucleotides of this aspect can have additional components including, but not limited to, compounds, isotopes, proteins or sequences for the detection of the probe or pπmer.

An aspect of this invention is an expression vector including a polynucleotide encoding a Pseudomonas aeruginosa MurC protein, or a complementary sequence, and regulatory regions. In a particular embodiment the encoded protein has a sequence corresponding to SEQ ID NO:2. In particular embodiments, the vector can have any of a vaπety of regulatory regions known and used m the art as appropπate for the types of host cells the vector can be used in. In a most preferred embodiment, the vector has regulatory regions appropπate for the expression of the encoded protein in gram-negative prokaryotic host cells. In other embodiments, the vector has regulatory regions appropπate for expression of the encoded protein in gram-positive host cells, yeasts, cyanobacteπa or actmomycetes. In some preferred embodiments the regulatory regions provide for inducible expression while in other preferred embodiments the regulatory regions provide for constitutive expression. Finally, according to this aspect, the expression vector can be deπved from a plasmid, phage, virus or a combination thereof.

An aspect of this invention is host cell compπsing an expression vector including a polynucleotide encoding a Pseudomonas aeruginosa MurC protein, or a complementary sequence, and regulatory regions. In a particular embodiment the encoded protein has a sequence corresponding to SEQ ID NO:2. In preferred embodiments, the host cell is a yeast, gram-positive bacteπum, cyanobacteπum or actmomycete. In a most preferred embodiment, the host cell is a gram-negative bactenum. An aspect of this invention is a process for expressing a MurC protein of P. aeruginosa in a host cell. In this aspect a host cell is transformed or transfected with an expression vector including a polynucleotide encoding a Pseudomonas aeruginosa MurC protein, or a complementary sequence. According to this aspect, the host cell is cultured under conditions conducive to the expression of the encoded MurC protein In particular embodiments the expression is inducible or constitutive. In a particular embodiment the encoded protein has a sequence corresponding to SEQ ID NO:2.

An aspect of this invention is a puπfied polypeptide having an amino acid sequence of SEQ ID NO:2 or the sequence of a naturally occurring mutant or polymorphic form of the protein.

An aspect of this invention is a method of determining whether a candidate compound can inhibit the activity of a P. aeruginosa MurC polypeptide. According to this aspect a polynucleotide encoding the polypeptide is used to construct an expression vector appropπate for a particular host cell. The host cell is transformed or transfected with the expression vector and cultured under conditions conducive to the expression of the MurC polypeptide. The cell is contacted with the candidate. Finally, one measures the activity of the MurC polypeptide in the presence of the candidate. If the activity is lower relative to the activity of the protein in the absence of the candidate, then the candidate is a inhibitor of the MurC polypeptide. In preferred embodiments, the polynucleotide encodes a protein having an amino acid sequence of SEQ ID NO:2 or a naturally occurring mutant of polymorphic form thereof. In other preferred embodiments, the polynucleotide has the sequence of SEQ XT) NO: 1. In particular embodiments, the relative activity of MurC is determined by compaπng the activity of the MurC in a host cell. In some embodiments, the host cell is disrupted and the candidate is contacted to the released cytosol. In other embodiments, the cells can be disrupted contacting with the candidate and before determining the activity of the MurC protein. Finally, according to this aspect the relative activity can determined by compaπson to a previously measured or expected activity value for the MurC activity in the host under the conditions. However, in preferred embodiments, the relative activity is determined by measuπng the activity of the MurC in a control cell that was not contacted with a candidate compound. In particular embodiments, the host cell is a pseudomonad and the protein inhibited is the MurC produced by the pseudomonad. An aspect of this invention is a compound that is an inhibitor of a P. aeruginosa MurC protein an assay described herein. In preferred embodiments, the compound is an inhibitor of a P. aeruginosa MurC protein produced by a host cell compπsing an expression vector of this invention. In most preferred embodiments, the compound is also an inhibitor of MurC protein produced by a pathogenic strain P. aeruginosa and also inhibits the growth of said pseudomonad.

An aspect of this invention is a pharmaceutical preparation that includes an inhibitor of P. aeruginosa MurC and a pharmaceutically acceptable earner. An aspect of this invention is a method of treatment compπsing admmistenng a inhibitor of the P. aeruginosa MurC to a patient The treatment can be prophylactic or therapeutic. In preferred embodiments, the appropπate dosage for a particular patient is determined by a physician.

By "about" it is meant within 10% to 20% greater or lesser than particularly stated.

As used herein an "inhibitor" is a compound that interacts with and inhibits or prevents a polypeptide of MurC from catalyzing the ATP-dependent addition of L-alamne to UDP-N-acetylmuramoyl precursor

As used herein a "modulator" is a compound that interacts with an aspect of cellular biochemistry to effect an increase or decrease in the amount of a polypeptide of MurC present in, at the surface or in the peπplasm of a cell, or m the surrounding serum or media. The change in amount of the MurC polypeptide can be mediated by the effect of a modulator on the expression of the protein, e g., the transcnption, translation, post-translational processing, translocation or folding of the protein, or by affecting a component(s) of cellular biochemistry that directly or indirectly participates in the expression of the protein. Alternatively, a modulator can act by accelerating or decelerating the turnover of the protein either by direct interaction with the protein or by interacting with another component(s) of cellular biochemistry which directly or indirectly effects the change. All of the references cited herein are incorporated by reference in their entirety as background mateπal

BRIEF DESCRIPTION OF THE DR AW INGS

FIGS 1 A & IB. Nucleotide sequence (SEQ ID NO: 1) and the predicted amino acid sequence (SEQ ID NO.2) of P. aeruginosa murC. The amino acid sequence (SEQ ID NO.2) is presented in three-letter code below the nucleotide sequence (nucleotides 59 to 1520 of SEQ ID NO: 1).

FIG 2. Production of recombmant P. aeruginosa MurC. Lane 1, Molecular weight markers; Lane2, IPTG- duced lysate of cells (BL21(DE3)/pLysS) containing the control vector pET-15b; Lane 3, uninduced cell lysate containing the control vector pET-15b; lane 4, column-punfied MurC; Lane 5 IPTG-mduced lysate of cells expressing MurC; Lane 6, uninduced lysate of cells containing murC.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides polynucleotides and polypeptides of a cell wall biosynthesis gene from Pseudomonas aeruginosa, referred to herein as MurC. The polynucleotides and polypeptides are used to further provide expression vectors, host cells compnsing the vectors, probes and pπmers, antibodies against the MurC protein and polypeptides thereof, assays for the presence or expression of MurC and assays for the identification of modulators and inhibitors of MurC.

Bactenal MurC, UDP-N-acetylmuramyl:L-alanme hgase, a cytoplasmic peptidoglycan biosynthetic enzyme, catalyzes the ATP-dependent addition of L-alanine to the UDP-N-acetylmuramyl precursor, generating the UDP-N- acetylmuramoyl-L-alanme

The murC gene was cloned from Pseudomonas aeruginosa. Sequence analysis of the P. aeruginosa murC gene revealed an open reading frame of 487 amino acids. The deduced ammo acid sequence of P. aeruginosa MurC is homologous to MurC from Escherichia coli, Haemophύus influenza, Bacillus subtilis and S. aureus . Recombmant MurC protein from P. aeruginosa was over-produced as His-tagged fusion protein in Escherichia coli host cells and the enzyme was punfied to apparent homogeneity. The recombmant enzyme catalyzed the ATP-dependent addition of L-alanine to the UDP-N-acetylmuramyl precursor.

Nucleic acids encoding murC from Pseudomonas aeruginosa are useful in the expression and production of the P. aeruginosa MurC protein. The nucleic acids are also useful in providing probes for detecting the presence of P. aeruginosa. Polynucleotides

Polynucleotides useful in the present invention include those descnbed herein and those that one of skill in the art will be able to denve therefrom following the teachings of this specification. A preferred aspect of the present invention is an isolated nucleic acid encoding a MurC protein of Pseudomonas aeruginosa. A preferred embodiment is a nucleic acid having the sequence disclosed in FIG. 1, SEQ ID NO:l and disclosed as follows:

CTCCATGGCA GACCAGGCAC GCAGCCTGGC GAAACCCGAG GCTACCCGGA

CGGTGGTCGA TGCCTGCCTG GAGGTGGCCC GTGGTTAAAG AACCGAATGG

CGTCACCCGG ACCATGCGCC GTATCCGCCG CATCCATTTC GTCGGCATCG

GCGGCGCCGG TATGTGCGGG ATCGCCGAAG TGCTGCTGAA CCTCGGCTAC

GAGGTATCCG GCTCGGACCT CAAGGCCTCG GCGGTGACCG AGCGCCTGGA

GAAGTTCGGC GCGCAGATCT TCATCGGCCA CCAGGCGGAA AACGCCGACG

GCGCCGACGT GCTGGTGGTG TCCAGTGCCA TCAACCGGGC CAACCCGGAA

GTGGCATCGG CCCTGGAACG GCGGATTCCG GTGGTGCCGC GTGCGGAGAT

GCTCGCCGAG CTGATGCGCT ACCGGCACGG CATCGCGGTA GCCGGCACCC

ACGGCAAGAC CACCACTACC AGCCTGATCG CCTCGGTGTT CGCCGCCGGC

GGCCTGGACC CGACCTTCGT CATCGGCGGC CGGCTGAACG CCGCCGGGAC

CAACGCCCAG CTCGGCGCCA GCCGCTACCT GGTGGCCGAG GCCGACGAGA

GCGACGCCAG CTTCCTGCAC CTGCAACCGA TGGTCGCGGT GGTCACCAAT

ATCGACGCCG ACCACATGGC GACCTACGGC GGCGACTTCA ACAAGCTGAA GAAGACCTTC GTCGAGTTCC TCCACAACCT GCCGTTCTAC GGACTGGCGG

TGATGTGCGT GGATGATCCG GTTGTGCGTG AGATCCTCCC GCAGATCGCC

CGCCCGACCG TGACCTACGG CCTCAGCGAA GACGCCGACG TGCGCGCGAT

CAACATCCGC CAGGAAGGCA TGCGCACCTG GTTCACCGTG TTGCGCCCGG

AGCGCGAGCC GCTGGACGTC TCGGTGAACA TGCCCGGCCT GCACAACGTG

CTGAATTCCC TGGCGACCAT CGTCATCGCT ACCGACGAGG GCATCTCCGA

CGAAGCCATC GTCCAGGGGC TGTCCGGCTT CCAGGGCGTA GGCCGGCGCT

TCCAGGTCTA CGGCGAGCTG CAGGTCGAGG GTGGCAGCGT GATGCTGGTG

GACGATTACG GCCACCATCC GCGCGAAGTC GCCGCGGTGA TCAAGGCGAT

CCGTGGCGGT TGGCCGGAGC GTCGCCTGGT GATGGTCTAC CAGCCGCATC

GCTATACCCG TACCCGCGAC CTGTACGAAG ACTTCGTGCA GGTGCTGGGC

GAAGCCAACG TGCTGCTGTT GATGGAGGTC TATCCGGCCG GCGAAGAGCC

GATCCCGGGA GCCGACAGCC GCCAGCTGTG CCACAGCATC CGCCAGCGCG

GCCAGCTTGA CCCGATCTAC TTCGAGCGCG ACGCCGACCT GGCGCCGCTG

GTCAAGCCGC TGCTGCGCGC TGGCGACATC CTGCTTTGCC AGGGCGCTGG

CGATGTCGGC GGCCTGGCCC CGCAACTGAT CAAGAACCCG CTGTTCGCCG

GCAAGGGAGG GAAGGGCGCA TGAACCTTTG CCTCGATAGC CTGCTGAACG (SEQ ID NO : 1 ) The translation initiation and termination codons are underlined

The isolated nucleic acid molecule of the present invention can include a nbonucleic or deoxyπbonucleic acid molecule, which can be single (coding or noncodmg strand) or double stranded, as well as synthetic nucleic acid, such as a synthesized, single stranded polynucleotide

The present invention also relates to recombmant vectors and recombmant hosts, both prokaryotic and eukaryotic, which contain the substantially punfied nucleic acid molecules disclosed throughout this specification

As used herein a "polynucleotide" is a nucleic acid of more than one nucleotide A polynucleotide can be made up of multiple polynucleotide units that are referred to by descnption of the unit For example, a polynucleotide can compπse within its bounds a polynucleotιde(s) having a coding sequence(s), a polynucleotιde(s) that is a regulatory regιon(s) and/or other polynucleotide units commonly used in the art An "expression vector" is a polynucleotide having regulatory regions operably linked to a coding region such that, when in a host cell, the regulatory regions can direct the expression of the coding sequence The use of expression vectors is well known in the art Expression vectors can be used in a vanety of host cells and, therefore, the regulatory regions are preferably chosen as appropnate for the particular host cell

A "regulatory region" is a polynucleotide that can promote or enhance the initiation or termination of transcnption or translation of a coding sequence A regulatory region includes a sequence that is recognized by the RNA polymerase, πbosome, or associated transcπption or translation initiation or termination factors of a host cell Regulatory regions that direct the initiation of transcπption or translation can direct constitutive or inducible expression of a coding sequence

Polynucleotides of this invention contain full length or partial length sequences of the MurC gene sequences disclosed herein Polynucleotides of this invention can be single or double stranded If single stranded, the polynucleotides can be a coding, "sense," strand or a complementary, "antisense," strand Antisense strands can be useful as modulators of the gene by interacting with RNA encoding the MurC protein Antisense strands are preferably less than full length strands having sequences unique or specific for RNA encoding the protein

The polynucleotides can include deoxyπbonucleotides, nbonucleotides or mixtures of both The polynucleotides can be produced by cells, in cell-free biochemical reactions or through chemical synthesis Non-natural or modified nucleotides, including osine, methyl-cytos e, deaza-guanosine, etc., can be present. Natural phosphodiester mternucleotide linkages can be appropnate. However, polynucleotides can have non-natural linkages between the nucleotides. Non-natural linkages are well known in the art and include, without limitation, methylphosphonates, phosphorothioates, phosphorodithionates, phosphoroamidites and phosphate ester linkages Dephospho-lmkages are also known, as bπdges between nucleotides. Examples of these include siloxane, carbonate, carboxymethyl ester, acetamidate, carbamate, and thioether bndges "Plastic DNA," having, for example, N-vinyl, methacryloxyethyl, methacrylamide or ethyleneimme mternucleotide linkages, can be used "Peptide Nucleic Acid" (PNA) is also useful and resists degradation by nucleases. These linkages can be mixed m a polynucleotide.

As used herein, "punfied" and "isolated" are utilized interchangeably to stand for the proposition that the polynucleotide, protein and polypeptide, or respective fragments thereof in question have been removed from the in vivo environment so that they exist in a form or punty not found in nature. Purified or isolated nucleic acid molecules can be manipulated by the skilled artisan, such as but not limited to sequencing, restnction digestion, site-directed mutagenesis, and subcloning into expression vectors for a nucleic acid fragment as well as obtaining the wholly or partially punfied protein or protein fragment so as to afford the opportunity to generate polyclonal antibodies, monoclonal antibodies, or perform ammo acid sequencing or peptide digestion Therefore, the nucleic acids claimed herein can be present in whole cells or in cell lysates or in a partially or substantially punfied form It is preferred that the molecule be present at a concentration at least about five-fold to ten-fold higher than that found in nature. A polynucleotide is considered substantially pure if it is obtained punfied from cellular components by standard methods at a concentration of at least about 100-fold higher than that found in nature. A polynucleotide is considered essentially pure if it is obtained at a concentration of at least about 1000-fold higher than that found in nature We most prefer polynucleotides that have been punfied to homogeneity, that is, at least 10,000 - 100,000 fold. A chemically synthesized nucleic acid sequence is considered to be substantially punfied when punfied from its chemical precursors by the standards stated above. Included in the present invention are assays that employ further novel polynucleotides that hybndize to P. aeruginosa murf sequences under stnngent conditions. By way of example, and not limitation, a procedure using conditions of high stnngency is as follows: Prehybπdization of filters containing DNA is earned out for 2 hr. to overnight at 65°C in buffer composed of 6X SSC, 5X Denhardt's solution, and 100 μg/ml denatured salmon sperm DNA. Filters are hybndized for 12 to 48 hrs at 65°C in prehybndization mixture containing 100 μg/ml denatured salmon sperm DNA and 5-20 X 106 cpm of 32p_ι_abeled probe. Washing of filters is done at 37°C for 1 hr in a solution containing 2X SSC, 0.1% SDS. This is followed by a wash in 0.1X SSC, 0.1% SDS at 50°C for 45 m . before autoradiography.

Other procedures using conditions of high stnngency would include either a hybndization step earned out in 5XSSC, 5X Denhardt's solution, 50% formamide at 42°C for 12 to 48 hours or a washing step earned out in 0.2X SSPE, 0.2% SDS at 65°C for 30 to 60 minutes. Reagents mentioned in the foregoing procedures for carrying out high stnngency hybndization are well known in the art. Details of the composition of these reagents can be found in, e.g., Sambrook, et al, 1989, Molecular Cloning: A Laboratory Manual, second edition, Cold Spπng Harbor Laboratory Press. In addition to the foregoing, other conditions of high stnngency which may be used are well known in the art.

Polypeptides

A preferred aspect of the present invention is a substantially punfied form of the MurC protein from Pseudomonas aeruginosa. A preferred embodiment is a protein that has the amino acid sequence which is shown in FIG.1, in SEQ ID NO:2 and disclosed as follows:

MetProAlaTrpArgTrpProValValLysGluProAsnGlyValThrArgThrMetArg ArglleArgArglleHisPheValGlylleGlyGlyAlaGlyMetCysGlylleAlaGlu ValLeu euAsn euGlyTyrGluValSerGlySerAspLeuLysAlaSerAlaValThr GluArgLeuGlu ysPheGlyAlaGlnllePhelleGlyHisGlnAlaGluAsnAlaAsp GlyAlaAspValLeuValValSerSerAlalleAsnArgAlaAsnProGluValAlaSer AlaLeuGluArgArglleProValValProArgAlaGluMetLeuAlaGl LeuMetArg TyrArgHisGlylleAlaValAlaGlyThrHisGlyLysThrThrThrThrSerLeuIle AlaSerValPheAlaAlaGlyGlyLeuAspProThrPheVallleGlyGlyArgLeuAsn AlaAlaGlyThrAsnAlaGlnLeuGlyAlaSerArgTyr euValAlaGluAlaAspGlu SerAspAlaSerPheLeuHisLeuGlnProMetValAlaValValThrAsnlleAspAla AspHisMetAlaThrTyrGlyGlyAspPheAsnLys euLysLysThrPheValGluPhe euHisAsnLeuProPheTyrGlyLeuAlaValMetCysValAspAspProValValArg GlulleLeuProGlnlleAlaArgProThrValThrTyrGlyLeuSerGluAspAlaAsp ValArgAlalleAsnlleArgGlnGluGly etArgThrTrpPheThrValLeuArgPro GluArgGluPro euAspValSerValAsnMetProGlyLeuHisAsnValLeuAsnSer LeuAlaThrlleVallleAlaThrAspGluGlylleSerAspGluAlalleValGlnGly LeuSerGlyPheGlnGlyValGlyArgArgPheGlnValTyrGlyGluLeuGlnValGlu GlyGlySerValMetLeuValAspAspTyrGlyHisHisProArgGluValAlaAlaVal IleLysAlalleArgGlyGlyTrpProGluArgArgLeu alMetValTyrGlnProHis ArgTyrThrArgThrArgAspLeuTyrGluAspPheValGlnVal euGlyGluAlaAsn ValLeuLeuLeuMetGluValTyrProAlaGlyGluGluProIleProGlyAlaAspSer ArgGlnLeuCysHisSerlleArgGlnArgGlyGlnLeuAspProIleTyrPheGluArg AspAlaAspLeuAlaProLeuValLysProLeuLeuArgAlaGlyAspIleLeuLeuCys GlnGlyAlaGlyAspValGlyGly euAlaProGlnLeuIleLysAsnPro euPheAla GlyLysGlyGly ysGlyAla (SEQ ID NO : 2 )

The present invention also relates to biologically active fragments and mutant or polymorphic forms of MurC polypeptide sequence as set forth as SEQ ID NO: 2, including but not limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy-terminal truncations such that these mutations provide for proteins or protein fragments of diagnostic, therapeutic or prophylactic use and would be useful for screening for modulators, and/or inhibitors of MurC function.

Using the disclosure of polynucleotide and polypeptide sequences provided herein to isolate polynucleotides encoding naturally occumng forms of

MurC, one of skill in the art can determine whether such naturally occumng forms are mutant or polymorphic forms of MurC by sequence companson. One can further determine whether the encoded protein, or fragments of any MurC protein, is biologically active by routine testing of the protein of fragment in a in vitro or in vivo assay for the biological activity of the MurC protein. For example, one can express N-termmal or C-terminal truncations, or internal additions or deletions, in host cells and test for their ability to catalyze the ATP-dependent addition of L-alanine to the UDP-N-acetylmuramyl precursor.

It is known that there is a substantial amount of redundancy in the vanous codons which code for specific ammo acids Therefore, this invention is also directed to those DNA sequences that encode RNA compπsing alternative codons which code for the eventual translation of the identical amino acid

Therefore, the present invention discloses codon redundancy which can result in different DNA molecules encoding an identical protein For purposes of this specification, a sequence beaπng one or more replaced codons will be defined as a degenerate vanation Also included within the scope of this invention are mutations either in the DNA sequence or the translated protein which do not substantially alter the ultimate physical properties of the expressed protein. For example, substitution of vahne for leucine, arginine for lysme, or asparag e for glutam e may not cause a change in functionality of the polypeptide. However, any given change can be examined for any effect on biological function by simply assaying for the ability to catalyze the ATP-dependent addition of L-alanme to an alanyl residue of the UDP-N- acetylmuramyl precursor as compared to an unaltered MurC protein

It is known that DNA sequences coding for a peptide can be altered so as to code for a peptide having properties that are different than those of the naturally occumng peptide. Methods of altenng the DNA sequences include but are not limited to site directed mutagenesis. Examples of altered properties include but are not limited to changes in the affinity of an enzyme for a substrate.

As used herein, a "biologically active equivalent" or "functional deπvative" of a wild-type MurC possesses a biological activity that is substantially similar to the biological activity of a wild type MurC. The term "functional denvative" is intended to include the "fragments," "mutants," "vaπants," "degenerate vaπants," "analogs," "orthologues," and "homologues" and "chemical denvatives" of a wild type MurC protein that can catalyze the ATP-dependent addition of L-alanine to the UDP-N-acetylmuramyl precursor.

The term "fragment" refers to any polypeptide subset of wild-type MurC. The term "mutant" is meant to refer to a molecule that may be substantially similar to the wild-type form but possesses distinguishing biological charactenstics Such altered characteristics include but are in no way limited to altered substrate binding, altered substrate affinity and altered sensitivity to chemical compounds affecting biological activity of the MurC or MurC functional denvative. The term "vaπant" refers to a molecule substantially similar in structure and function to either the entire wild-type protein or to a fragment thereof A molecule is "substantially similar" to a wild-type MurC-hke protein if both molecules have substantially similar structures or if both molecules possess similar biological activity. Therefore, if the two molecules possess substantially similar activity, they are considered to be variants even if the exact structure of one of the molecules is not found in the other or even if the two ammo acid sequences are not identical. The term "analog" refers to a molecule substantially similar in function to either the full-length MurC protein or to a biologically active fragment thereof.

As used herein in reference to a MurC gene or encoded protein, a "polymorphic" MurC is a MurC that is naturally found in the population of Pseudomonads at large. A polymorphic form of MurC can be encoded by a different nucleotide sequence from the particular murC gene disclosed herein as SEQ ID NO: l. However, because of silent mutations, a polymorphic murC gene can encode the same or different ammo acid sequence as that disclosed herein. Further, some polymorphic forms MurC will exhibit biological charactenstics that distinguish the form from wild-type MurC activity, in which case the polymorphic form is also a mutant

A protein or fragment thereof is considered punfied or isolated when it is obtained at least partially free from it's natural environment in a composition or puπty not found in nature. It is preferred that the molecule be present at a concentration at least about five-fold to ten-fold higher than that found in nature. A protein or fragment thereof is considered substantially pure if it is obtained at a concentration of at least about 100-fold higher than that found in nature. A protein or fragment thereof is considered essentially pure if it is obtained at a concentration of at least about 1000-fold higher than that found in nature We most prefer proteins that have been punfied to homogeneity, that is, at least 10,000 -100,000 fold.

Probes and Pnmers Polynucleotide probes compπsing full length or partial sequences of

SEQ ID NO: 1 can be used to determine whether a cell or sample contains P. aeruginosa MurC DNA or RNA. The effect of modulators that effect the transcnption of the murC gene can be studied via the use of these probes. A preferred probe is a single stranded antisense probe having at least the full length of the coding sequence of MurC. It is also preferred to use probes that have less than the full length sequence, and contain sequences specific for P. aeruginosa murC DNA or RNA The identification of a sequence(s) for use as a specific probe is well known in the art and involves choosing a sequence(s) that is unique to the target sequence, or is specific thereto It is preferred that polynucleotides that are probes have at least about 25 nucleotides, more preferably about 30 to 35 nucleotides The longer probes are believed to be more specific for P aeruginosa murC gene(s) and RNAs and can be used under more stπngent hybndization conditions Longer probes can be used but can be more difficult to prepare synthetically, or can result in lower yields from a synthesis. Examples of sequences that are useful as probes or pnmers for P. aeruginosa murC gene(s) are Pπmer A (sense)

5' TTCATATGCCTGCCTGGAGGTG 3' (SEQ ID NO.3) and Pnmer B (antisense) 5' TTGGATCCTCATGCGCCCTTCCCTCCCTTG 3'(SEQ ID NO:4) These pnmers are nucleotides 55-76 (A) and the complement of nucleotides 1442-1464 (B) respectively, of SEQ ID NO: l. Restnction sites, underlined, for Ndel and BamHI are added to the 5' ends of the pnmers to allow cloning between the Ndel and BamHI sites of the expression vector pET-15b. However, one skilled in the art will recognize that these are only a few of the useful probe or pπmer sequences that can be denved from SEQ ID NO: 1.

Polynucleotides havmg sequences that are unique or specific for P. aeruginosa murC can be used as pnmers m amplification reaction assays. These assays can be used in tissue typing as descnbed herein. Additionally, amplification reactions employing pnmers denved from P. aeruginosa murC sequences can be used to obtain amplified P. aeruginosa murC DNA using the murC DNA of the cells as an initial template. The murC DNA so obtained can be a mutant or polymorphic form of P. aeruginosa murC that differs from SEQ ID NO: 1 by one or more nucleotides of the MurC open reading frame or sequences flanking the ORF. The differences can be associated with a non-defective naturally occumng form or with a defective form of MurC. Thus, polynucleotides of this invention can be used in identification of vanous polymorphic P. aeruginosa murC genes or the detection of an organism having a P aeruginosa murC gene Many types of amplification reactions are known in the art and include, without limitation, Polymerase Chain Reaction, Reverse Transcnptase Polymerase Cham Reaction, Strand Displacement Amplification and Self-Sustained Sequence Reaction. Any of these or like reactions can be used with pnmers denved from SEQ ID NO: 1.

Expression of MurC

A vaπety of expression vectors can be used to express recombmant MurC in host cells. Expression vectors are defined herein as nucleic acid sequences that include regulatory sequences for the transcnption of cloned DNA and the translation of their mRNAs in an appropπate host Such vectors can be used to express a bacteπal gene in a vanety of hosts such as bactena, bluegreen algae, plant cells, insect cells and animal cells. Specifically designed vectors allow the shuttling of genes between hosts such as bactena-yeast or bacteπa-animal cells. An appropπately constructed expression vector should contain: an oπgm of replication for autonomous replication in host cells, selectable markers, a limited number of useful restnction enzyme sites, a potential for high copy number, and regulatory sequences. A promoter is defined as a regulatory sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. A strong promoter is one which causes mRNAs to be initiated at high frequency. Expression vectors can include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses

In particular, a vaπety of bacteπal expression vectors can be used to express recombmant MurC in bacteπal cells. Commercially available bactenal expression vectors which are suitable for recombmant MurC expression include, but are not limited to pQE (Qiagen), pETl la or pET15b (Novagen), lambda gtl 1 (Invitrogen), and pKK223-3 (Pharmacia).

Alternatively, one can express murC DNA in cell-free transcnption- translation systems, or murC RNA in cell-free translation systems. Cell-free synthesis of MurC can be in batch or continuous formats known in the art. One can also synthesize MurC chemically, although this method is not preferred.

A vaπety of host cells can be employed with expression vectors to synthesize MurC protein These can include E. coli, Bacillus, and Salmonella. Insect and yeast cells can also be appropnate. Following expression of MurC in a host cell, MurC polypeptides can be recovered. Several protein puπfication procedures are available and suitable for use. MurC protein and polypeptides can be punfied from cell lysates and extracts, or from culture medium, by vanous combinations of. or individual application of methods including ultrafiltration, acid extraction, alcohol precipitation, salt fractionation, ionic exchange chromatography, phosphocellulose chromatography, lecithin chromatography, affinity (e.g.. antibody or His-Ni) chromatography, size exclusion chromatography, hydroxylapatite adsorption chromatography and chromatography based on hydrophobic or hydrophilhc interactions. In some instances, protein denaturation and refolding steps can be employed High performance liquid chromatography (HPLC) and reversed phase HPLC can also be useful. Dialysis can be used to adjust the final buffer composition

The MurC protein itself is useful in assays to identify compounds that modulate the activity of the protein — including compounds that inhibit the activity of the protein. The MurC protein is also useful for the generation of antibodies against the protein, structural studies of the protein, and structure/function relationships of the protein.

Modulators and Inhibitors of MurC The present invention is also directed to methods for screening for compounds which modulate or inhibit a MurC protein. Compounds which modulate or inhibit MurC can be DNA, RNA, peptides, proteins, or non-proteinaceous organic or inorganic compounds or other types of molecules. Compounds that modulate the expression of DNA or RNA encoding MurC or are inhibitors of the biological function of MurC can be detected by a vanety of assays. The assay can be a simple "yes/no" assay to determine whether there is a change in expression or function. The assay can be made quantitative by companng the expression or function of a test sample with the levels of expression or function in a standard sample, that is, a control. A compound that is a modulator can be detected by measuπng the amount of the MurC produced in the presence of the compound An compound that is an inhibitor can be detected by measunng the specific activity of the MurC protein in the presence and absence of the compound.

The proteins, DNA molecules, RNA molecules and antibodies lend themselves to the formulation of kits suitable for the detection and anaysis of MurC Such a kit would compnse a compartmentalized earner suitable to hold in close confinement at least one container. The earner would further compnse reagents such as recombmant MurC or anti- MurC antibodies suitable for detecting MurC. The earner can also contain a means for detection such as labeled antigen or enzyme substrates or the like.

Pharmaceutical Compositions

Pharmaceutically useful compositions compπsing a modulator or inhibitor of MurC can be formulated according to known methods such as by the admixture of a pharmaceutically acceptable earner. Examples of such earners and methods of formulation can be found in Remington's Pharmaceutical Sciences To form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of the inhibitor.

Therapeutic, prophylactic or diagnostic compositions of the invention are administered to an individual in amounts sufficient to treat, prevent or diagnose disorders The effective amount can vary according to a vanety of factors such as the individual's condition, weight, sex and age. Other factors include the mode of administration The appropnate amount can be determined by a skilled physician

The pharmaceutical compositions can be provided to the individual by a vanety of routes such as subcutaneous, topical, oral and intramuscular. The term "chemical deπvative" descπbes a molecule that contains additional chemical moieties which are not normally a part of the base molecule. Such moieties can improve the solubility, half-life, absorption, etc. of the base molecule. Alternatively the moieties can attenuate undesirable side effects of the base molecule or decrease the toxicity of the base molecule. Examples of such moieties are descnbed in a vanety of texts, such as Remington's Pharmaceutical Sciences.

Compounds identified according to the methods disclosed herein can be used alone at appropπate dosages. Alternatively, co-admmi strati on or sequential administration of other agents can be desirable.

The present invention also provides a means to obtain suitable topical, oral, systemic and parenteral pharmaceutical formulations for use in the methods of treatment of the present invention. The compositions containing compounds identified according to this invention as the active ingredient can be administered in a wide vaπety of therapeutic dosage forms in conventional vehicles for administration. For example, the compounds can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection. Likewise, they can also be administered in intravenous (both bolus and infusion), intrapeπtoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts.

Advantageously, compounds of the present invention can be administered in a single daily dose, or the total daily dosage can be administered in divided doses of two, three or four times daily Furthermore, compounds for the present invention can be administered in mtranasal form via topical use of suitable mtranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.

For combination treatment with more than one active agent, where the active agents are in separate dosage formulations, the active agents can be administered concurrently, or they each can be administered at separately staggered times.

The dosage regimen utilizing the compounds of the present invention is selected m accordance with a vaπety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated, the route of administration; the renal, hepatic and cardiovascular function of the patient; and the particular compound thereof employed. A physician or veteπnanan of ordinary skill can readily determine and prescnbe the effective amount of the drug required to prevent, counter or arrest the progress of the condition. Optimal precision m achieving concentrations of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This involves a consideration of the distribution, equihbπum, and elimination of a drug.

The following examples are presented by the way of illustration and, because vanous other embodiments will be apparent to those in the art, the following is not to be construed as a limitation on the scope of the invention. For example, while particular preferred embodiments of the invention are presented herein, it is within the ability of persons of ordinary skill in the art to modify or substitute vectors, host cells, compositions, etc , or to modify or design protocols or assays, all of which may reach the same or equivalent performance or results as the embodiments shown herein.

EXAMPLE 1

General Matenals and Methods

All reagents were purchased from SIGMA CHEMICAL CO., St

Louis, MO, unless otherwise indicated. UDP-N-acetylmuramyl-L-alanine was synthesized and punfied by a method known in the art (Jm, H , Emanuele, J J.. Jr , Fairman, R., Robertson, J G., Hail, M E., Ho, H.-T , Falk, P. and Villafranca. J. J, 1996. Structural studies of Escherichia coli UDP-N-acetylmuramate. L-alanine hgase, Biochemistry 35: 14423-14431).

DNA manipulations reagents and techniques.

Restnction endonucleases and T4 hgase were obtained from GIBCO- BRL. Agarose gel electrophoresis and plasmid DNA preparations were performed according to published procedures (Sambrook, J., E. F. Fntsch, and T. Maniatis, 1989, Molecular cloning: a L, Laboratory Manual, 2nd ed. Cold Spπng Harbor, NY- Cold Spπng Harbor Laboratory). Recombmant plasmids containing P. aeruginosa murC were propagated in E. coli DH5a (GIBCO-BRL, Rockville, MD) pπor to protein expression m E. coli BL21(DE3)/plysS (NOVAGEN, Madison, WI). SDS-PAGE was performed with precast gels (NOVAGEN). DNA sequences were determined using an automated ABI PRISMTM DNA sequencer (PERKIN-ELMER ABI, Foster City, CA).

EXAMPLE 2

Cloning of Pseudomonas aeruginosa murC

Genomic DNA from P. aeruginosa (strain MB4439) was prepared from 100 ml late stationary phase culture in Brain Heart Infusion broth (DIFCO, Detroit, MI). Cells were washed with 0.2 M sodium acetate, suspended in 10 ml of TEG (100 mM Tπs, pH 7, containing 10 mM EDTA and 25% glucose) and lysed by incubation with 200 μg of N-acetylmuramidase (SIGMA) for lh at 37°C. Chromosomal DNA was punfied from the cell lysate using a QIAGEN (Santa Clanta, CA) genomic DNA preparation kit and following the manufacturers protocol. Bπefly, the cell lysate was treated with protease K at 50°C for 45 mm, loaded onto an equilibrated QIAGEN genomic tip, entered into the resin by centnfugation at 3000 rpm for 2 min. Following washing the genomic tip, the genomic DNA was eluted in distilled water and kept at 4°C. Approximately 50 ng genomic DNA was used as a template in PCR reactions to clone murC.

Two ohgonucleotide pnmers (GIBCO/BRL, Bethesda, MD) complementary to sequences at the 5' and the 3' ends of P. aeruginosa murC were used to clone this gene using KLENTAQ ADVANTAGETM polymerase (CLONTECH, Palo Alto, CA). The pnmer nucleotide sequences were as follows. 5'- TTCATATGCCTGCCTGGAGGTG -3' (SEQ ID NO.3) (a Ndel linker plus nucleotides of SEQ ID NO: 1) and

5'- TTGGATCCTCATGCGCCCTTCCCTCCCTTG -3' (SEQ ID NO:4) (a BamHI linker plus the complement of nucleotides of SEQ ID NO: 1). A PCR product representing P. aeruginosa murC was veπfied by nucleotide sequence, digested with Ndel and BamHI, and cloned between the Ndel and BamHI sites of pET-15b, creating plasmid pPaeMurC. This plasmid was used for expression of the murC gene in E. coli.

The plasmid pPaeMurC has been deposited with the Amencan Type Culture Collection on , 1999, under the terms of the Budapest Treaty for the

Deposit of Microorganisms and has been designated as ATCC . The deposited matenal is provided as a convenience and is not an indication that the deposited matenal is required to descnbe or practice the invention. The sequence of the polynucleotide of the deposit, and the encoded ammo acid sequence, are incorporated herein by reference and are controlling in the event of a conflict with any descπption of the sequences provided in this specification or the associated drawings. A license may be required to make, use, sell or offer to sell the polynucleotide of the deposit or a protein of the ammo acid sequence encoded by the polynucleotide. No such license is granted herein.

EXAMPLE 3

Sequence analysis of Pseudomonas aeruginosa murC

The nucleotide sequence of murC, determined in both oπentations, and the deduced amino acid sequence of the MurC protein is depicted in FIGS. 1A-1B. Sequence companson using the BLAST algoπthm (Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) "Basic local alignment search tool." J. Mol. Biol. 215:403-410) against the GenBank database showed that, to varying degrees, the cloned region is homologous (76% similar, 59% identical) to murC gene from E. coli (Ikeda, M., Wachi, M., Jung, H. K., Ishino, F. and Matsuhashi, M. 1990. Nucleotide sequence involving murG and murC in the mra gene cluster of Escherichia coli. Nucleic Acids Res 18-4014). EXAMPLE 4

Overexpression, purification and enzymatic activity of Pseudomonas aeruginosa MurC

murC was cloned into the expression vector pET-15b (NOVAGEN) as descnbed above to create plasmid pPaeMurC. The pET-15b vector incorporates the 6xHιstιdme-tag into the protein construct to allow rapid puπfication of MurC by affinity chromatography. The pET (Plasmids for Expression by T7 RNA polymerase) plasmids are denved from pBR322 and designed for protein over-production in E coli. The vector pET-15b contains the ampicilhn resistance gene, ColEl oπgin of replication in addition to T7 phage promoter and terminator. The T7 promoter is recognized by the phage T7 RNA polymerase but not by the E. coli RNA polymerase. A host E coli strain such as BL21(DE3)pLysS is engineered to contain integrated copies of T7 RNA polymerase under the control of lacUV5 that is inducible by IPTG. Production of a recombmant protein in the E. coli strain BL21(DE3)pLysS occurs after expression of T7RNA polymerase is induced.

The pPaeMurC plasmid was introduced into the host strain BL21 DE3/pLysS (NOVAGEN) for expression of His-tagged MurC. Colonies were grown at 37°C in 100 ml of LB broth containing 100 mg/ml ampicillm and 32 μg/ml chloramphenicol When cultures reached a cell density of A600⁼0.5, cells were pelleted and then resuspended in M9ZB medium (NOVAGEN) containing 1 mM IPTG. Cells were induced for 3 h at 30°C, pelleted at 3000g, and frozen at -80°C.

Cultures containing either the recombmant plasmid pPaeMurC or the control plasmid vector, pET-15b were grown at 30°C and induced with IPTG. Cells transformed with pPaeMurC contained an inducible protein of approximately 54.7 kDa, corresponding to the expected size of P. aeruginosa MurC protein as shown by SDS-PAGE. (FIG 2.) There were no comparable detectable protein bands after induction of cells transformed with the control plasmid vector, pET-15b

Punfication of recombmant MurC enzyme The cell pellet from 100 ml of induced culture prepared as descnbed above was resuspended in 10 ml BT buffer (50 mM bis-tns-propane, pH 8.0, containing 100 mM potassium chloπde and 1% glycerol) at 4°C. Cells were lysed either by freeze-thaw or by French Press. After centnfugation, the supernatant was

99 - mixed with 15 ml of freshly prepared TALON (CLONTECH) resin and incubated for 30 mm at room temp The resin was washed twice by centnfugation with 25 ml of BT buffer at room temperature Finally, the res was loaded into a column and washed with 20 ml of BT, pH 7 0, containing 5 mM imidazole Protein was eluted with 20 ml of BT buffer pH 8 0, containing 100 mM imidazole Fractions (0 5 ml) were collected and analyzed by SDS-Gel electrophoresis (FIG 2) This resulted in a partially punfied preparation of P aeruginosa MurC protein that could be used in activity assays The protein may be punfied further, if desired, using methods known in the art

Assay for activity of MurC enzyme

The ATP-dependent MurC activity was assayed by monitonng the formation of product ADP using the pyruvate kinase and lactate dehydrogenase coupled enzyme assay The reaction was monitored spectrophotometncally Typically, the assay contained 100 mM BIS-TRIS-propane, pH 8 0,

200 μM NADH, 1 mM ATP, 20 mM PEP, 5 mM MgCl2, 1 mM DTT, 350 μM UDP-

N-acetyl-muramyl, 1 mM L-alanine, 33 units/ml of pyruvate kinase and 1660 units/ml of lactate dehydrogenase in a final volume of 200 or 400 μl The mixture was incubated at 25°C for 5 mm and the reaction initiated by the addition of 1-10 μg of MurC These conditions are one example of an assay useful for evaluating the activity of MurC Other assays can be used, or amounts of buffers, substrate and enzyme can be changed, as desired, to alter the rate of production of ADP

ADP formation was monitored by the decrease in absorbance at 340 nm as a function of time using a MOLECULAR DEVICES SPECTRAMAXPLUSTM microtiterplate spectrophotomer (for 200 μl assays) or a HEWLETT-PACKARD HP8452A spectrophotometer equipped with a circulating water bath (for 400 μl assays) Rates were calculated from the linear portions of the progress curves using the extinction coefficient for NADH, e = 6220 cnr* M^~l One unit of MurC activity is equal to 1 μmol of ADP formed per mm at 25°C MurC activity co-eluted with a -51 kDa protein Table 1

Specific activities of recombmant MurC from E. coli and P. aeruginosa.

Mur Ligase P. aeruginosa E. coli μmol x mιn-1 x mg-1 μmol x miir- x mg-1

MurC 0.3 0.066

EXAMPLE 5

Screening for inhibitors of MurC

One assay for the measurement of the activity of MurC is provided in Example 4. That assay, and other assays for MurC activity can be adapted for screening assays to detect inhibitors of MurC. For example, for inhibition assays, inhibitors in DMSO are added at the desired concentration to the assay mixture. In a separate, control reaction, only DMSO is added to the assay mixture. The reactions are initiated by the addition of enzyme (MurC). Rates are calculated as descnbed above. Relative activities are calculated from the equation L

relative activity = rate with inhibitor/rate without inhibitor. (1)

Inhibition constant (IC50) values are determined from a range of inhibitor concentrations and calculated from equation 2. relative

1/(1 + [I]/IC5θ) (2)

One can use computer software to assist in the analysis, e.g., SIGMA PLOTTM (JANDEL SCIENTIFIC, San Rafeal, CA).

We prefer inhibitors of MurC that result in relative activities of the MurC enzyme of at least less than 75%, more preferably, 25-50% or 10-25% We most prefer inhibitors resulting in relative activities of less than 20%, particularly less than 10% of the activity of MurC in the absence of the inhibitor

We also prefer inhibitors that effectively lower the relative activity of MurC when the inhibitor is present at a very low concentration EXAMPLE 8

Therapy using inhibitors of MurC

A patient presenting with an indication of infection with a microorganism susceptible to inhibitors of MurC, e.g , gram positive and negative bacteπa, including P. aeruginosa, can be treated by administration of inhibitors of MurC Physicians skilled in the art are familiar with administenng therapeutically effective amounts of inhibitors or modulators of microbial enzymes. Such skilled persons can readily determine an appropnate dosing scheme to achieve a desired therapeutic effect.

Therapy can also be prophylactic For example, a patient at nsk for developing a bacteπal infection, including infection with P. aeruginosa, can be treated by administration of inhibitors of MurC. Physicians skilled in the art are familiar with administenng therapeutically effective amounts of inhibitors or modulators of microbial enzymes. Such skilled persons can readily determine an appropπate dosing scheme to achieve a desired therapeutic effect.

Claims

WHAT IS CLAIMED:

1. A punfied and isolated polynucleotide selected from the group consisting of:

(a) a polynucleotide encoding a polypeptide having an amino acid sequence of SEQ ID NO: 2.

(b) a polynucleotide which is complementary to the polynucleotide of (a),

(c) a polynucleotide representing a naturally occu ng mutant or polymorphic form of (a), and (d) a polynucleotide that hybπdizes with a polynucleotide of (a),

(b), or (c) under stnngent conditions, and

(e) a polynucleotide compπsing at least 25 nucleotides of the polynucleotide of (a), (b) or (c), said 25 nucleotides being specific for murC gene of

Pseudomonas aeruginosa.

2. The polynucleotide of claim 1 wherein the polynucleotide compnses nucleotides selected from the group consisting of natural, non-natural and modified nucleotides.

3. The polynucleotide of claim 1 wherein the mternucleotide linkages are selected from the group consisting of natural and non-natural linkages.

4. The polynucleotide of claim 1 compπsing the nucleotide sequence of SEQ ID NO: 1.

5. A polynucleotide that is an expression vector compnsing a polynucleotide of claim 1.

6. A host cell compπsing the expression vector of claim 5.

7. A process for expressing a MurC protein of Pseudomonas aeruginosa in a recombmant host cell, compπsing:

(a) transforming a suitable host cell with an expression vector of claim 5; and, (b) cultunng the host cell of step (a) in conditions under which allow expression of said the MurC protein from said expression vector.

8. A punfied and isolated polypeptide having an amino acid sequence selected from the group consisting of

(a) a polypeptide having an ammo acid sequence of SEQ ID NO:2,

(b) a polypeptide that is a naturally occumng mutant or polymorphic form of (a).

9. A method of determining whether a candidate compound is an inhibitor of a Pseudomonas aeruginosa MurC polypeptide compnsing:

(a) providing at least one host cell harbonng an expression vector that includes a polynucleotide selected from the group consisting of:

(l) a polynucleotide encoding a polypeptide having an ammo acid sequence of SEQ ID NO: 2.

(n) a polynucleotide which is complementary to the polynucleotide of (l),

(in) a polynucleotide representing a naturally occumng mutant or polymorphic form of (I), and

(b) contacting at least one of said cells with the candidate to permit the interaction of the candidate with the MurC polypeptide, and

(c) determining whether the candidate is an inhibitor of the MurC polypeptide by ascertaining the relative activity of the polypeptide in the presence of the candidate.

10 The method of claim 9 wherein the polynucleotide has the nucleotide sequence of SEQ ID NO: 1.

11 The method of claim 9 wherein in step (c) the relative activity is determined by companng a measurement of MurC polypeptide activity of at least one cell before step (b) to a measurement of MurC polypeptide activity of at least one cell after step (b).

12 A compound that is an inhibitor of a polypeptide having an amino acid sequence selected from the group consisting of (a) a polypeptide having an ammo acid sequence of SEQ ID NO.2, (b) a polypeptide that is a naturally occumng mutant or polymorphic form of (a).

13. A pharmaceutical composition compnsing a pharmaceutically acceptable earner and an inhibitor of a polypeptide havmg an amino acid sequence selected from the group consisting of

(a) a polypeptide having an ammo acid sequence of SEQ ID NO:2,

(b) a polypeptide that is a naturally occumng mutant or polymorphic form of (a).

14 A method of treatment of a patient in need of prophylactic or therapeutic treatment for a bacteπal infection compnsing administenng to the patient an effective amount of an inhibitor of a polypeptide having an amino acid sequence selected from the group consisting of (a) a polypeptide having an ammo acid sequence of SEQ ID NO:2,

(b) a polypeptide representing a naturally occurring mutant or polymorphic form of (a).

15. A method of determining whether a candidate compound is an inhibitor of a Pseudomonas aeruginosa MurC polypeptide compπsing:

(a) providing a sample that includes a MurC polypeptide selected from the group consisting of:

(I) a polypeptide having an amino acid sequence of SEQ ID NO: 2 (n) a polypeptide that is a functional denvative of the polypeptide of (l),

(in) a polypeptide representing a naturally occumng mutant or polymorphic form of (I), and

(b) contacting said sample with the candidate to permit the interaction of the candidate with the MurC polypeptide, and (c) determining whether the candidate is an inhibitor of the MurC polypeptide by ascertaining the relative activity of the MurC polypeptide in the presence of the candidate

16 The method of claim 15 wherein the polypeptide has the ammo acid sequence of SEQ ID NO:2

17. The method of claim 15 wherein in step (c) the relative activity is determined by companng a measurement of MurC polypeptide activity of the sample before step (b) to a measurement of MurC polypeptide activity of the sample after step (b)