This invention relates to newly identified polynucleotides and polypeptides, and their production and uses, as well as their variants, agonists and antagonists, and their uses. In particular, the invention relates to polynucleotides and polypeptides of a Staphylococcus aureus (S. aureus) DnaI related protein, as well as its variants. The invention also relates to a specific interaction between the S. aureus DnaI related protein or specific regions thereof, and a growth-inhibitory protein encoded by the S. aureus bacteriophage 77 genome. The phage open reading frame (ORF) product interacts with amino acids 150 313 of S. aureus DnaI polypeptide, and the invention relates to the use of this interaction target site as the basis of drug screening assays. Accordingly, the invention provides a method for the inhibition of bacterial growth, and the treatment of bacterial infection via the inhibition of DnaI.

DESCRIPTION OF THE INVENTION

The invention is based on the discovery of an essential gene and its encoded polypeptide in S. aureus and portions thereof useful in screening, diagnostics, and therapeutics. The invention also relates to S. aureus DnaI polypeptides and polynucleotides as described in greater detail below, and to a pair of polynucleotides encoding a pair of interacting polypeptides, and the pair of polypeptides themselves, or interacting domains thereof, where the pair includes a S. aureus DnaI polypeptide and a 77 ORF 104 polypeptide. Also, the invention relates to polynucleotides and polypeptides of a protein complex, thought to be involved in initiation of DNA replication, containing DnaI and DnaC related proteins, as well as their variants. In particular, the invention relates to polypeptides and polynucleotides of a DnaI of S. aureus, which is related by amino acid sequence homology to B. subtilis DnaI polypeptide. The invention relates especially to DnaI having the nucleotide and amino acid sequences disclosed as SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The sequences presented as SEQ ID NOs: 1 and 2 represent an exemplification of the invention, since those of ordinary skill will recognize that such sequences can be usefully employed in polynucleotides in general, including ribopolynucleotides.

We have used the methodology of two previous inventions (U.S. patent application Ser. No. 09/407,804, filed Sep. 28, 1999, and U.S. Provisional Patent Application 60/110,992 filed Dec. 3, 1998) to identify and characterize an essential polynucleotide and polypeptide sequence from S. aureus. Thus, the present invention provides polynucleotide and polypeptide sequences isolated from S. aureus that can be used in a drug screening assay to identify compounds with anti-microbial activity. The polynucleotide and polypeptide sequences can be isolated using a method similar to those described herein, or using another method. In addition, such polynucleotide and polypeptide sequences can be chemically synthesized.

How to Identify a S. aureus dnaI Sequence:

Using methodology described in detail in Example 1, a S. aureus polypeptide that specifically bound the P77 phage ORF 104 protein was isolated. The sequence of a tryptic peptide of the S. aureus polypeptide, GHVPENVTDNDR (SEQ ID NO: 19), was used to BLAST search the S. aureus nucleotide sequence in the University of Oklahoma S. aureus genomic database at http://www.genome.ou.edu/staph.html.

The University of Oklahoma's Advanced Center for Genome Technology web site. One sequence contig of 4850 nucleotides in length (Contig 981), when converted into amino acid sequence, contained within it the similar amino acid sequence GHVPELYVDNNR (SEQ ID NO: 11; FIG. 5). This tentative identification of the candidate protein was then confirmed upon in silico tryptic digestion of the open reading frame found in the contig (FIG. 5). The obtained PSD/CID spectra for tryptic peptides with monoisotopic MH+ masses of 1351.8, 1412.7, and 1617.8 Da were similar to the predicted PSD/CID fragmentation patterns of the tryptic peptides with monoisotopic MH+ masses of 1351.8 and 1617.8 Da found in the contig's +3 open reading frame (FIG. 5).

Comparison of the ORF of the S. aureus contig that encodes a tryptic peptide similar to that identified in the S. aureus phage 77 ORF 104 binding studies with all other sequences in the public domain databases revealed that the ORF is related to the DnaI protein from Bacillus subtilis (Table 1, SEQ ID NOs: 14 15) a protein implicated in chromosome replication. No other significant similarity was found with any other protein in publicly accessible databases. The degree of relatedness of the identified ORF to the B. subtilis DnaI protein shows 41% identity and 63% similarity (Table 1, SEQ ID NOs: 14 15).

Many genes of B. subtilis involved in DNA replication have been identified through the isolation of thermosensitive mutants. One of these, dnaI2, affected an unknown step of chromosome replication at the restrictive temperature (Karamata, D. and Gross, J. D. (1970) Mol. Gene. Genet. 108, 277 287). The gene was mapped around 250.degree. on the B subtilis chromosome and resides immediately downstream of the dnaB gene on the B. subtilis chromosome (Bruand, C. and Ehrlich, S. D. (1995) Microbiology 141, 1199 1200). The dnaI2 mutation has been characterized and resides within the dnaI gene and consists of a G to A substitution at nucleotide position 922 (FIG. 1; SEQ ID NO: 1) resulting in a glycine to glutamate change at position 307 (FIG. 1; SEQ ID NO: 2) (Bruand, C. and Ehrlich, S. D. (1995) Microbiology 141, 1199 1200).

DnaC has been genetically identified to be the major component DNA helicase of chromosome replication (Sakamoto, Y., Nakai, S., Moriya, S., Yoshikawa, H., and Ogasawara, N. (1995) Microbiology 141, 641 644) and is thought to unwind duplex DNA progressively and allow for binding of the DNA polymerase III holoenzyme necessary for priming and DNA synthesis. One possible function of DnaI is as a helicase loader, being responsible for transferring DnaC helicase to the oriC. The product of the dnaC and dnaI genes are required for chromosome replication and are all essential for DNA replication in B. subtilis (Ceglowski, P., Lurz, R., Alonso, J. C. J. (1993) Mol. Biol. 236, 1324 1340).

Databases were searched for S. aureus genes which may be related to the B. subtilis dnaC gene. Utilizing the B. subtilis amino acid sequence for DnaC (Accession Number P37469), a BLAST search was performed of the Staphylococcus database at The Institute of Genomic Research (TIGR) web site and revealed the presence of an ORF within the S. aureus genome encoding a related protein. The nucleotide sequence and corresponding protein sequence are presented in FIG. 6B (SEQ ID NO: 7) and FIG. 6D (SEQ ID NO: 9), respectively.

Identification of the Surface of Interaction on DnaI

This invention relates, in part, to a specific interaction between a growth-inhibitory protein encoded by the Staphylococcus aureus bacteriophage 77 genome and an essential S. aureus protein. This interaction forms the basis for drug screening assays. More specifically, the invention relates to the interacting regions of the protein encoded by the S. aureus bacteriophage 77 and the S. aureus DnaI proteins, forming the basis for screening assays. The invention provides a method for the identification of DnaI polypeptide fragments that are involved in said interaction between DnaI and ORF 104 from bacteriophage 77. Several approaches and techniques known to those skilled in the art can be used to identify and to characterize fragments of the DnaI interacting with 77 ORF 104. These fragments may include, for example, truncation polypeptides having a portion of an amino acid sequence of the proteins, or variants thereof, such as a continuous series of residues that includes an amino- and/or carboxyl-terminal amino acid sequence for DnaI.

A) Affinity Chromatography

Partial proteolysis of proteins in solution is one method to delineate the domain boundaries in multi-domain proteins. By subjecting proteins to limited digestion, the most accessible cleavage sites are preferentially hydrolyzed. These cleavage sites preferentially reside in less structured regions which include loops and highly mobile areas typical of the joining amino acids between highly structured domains. For this analysis, a purified recombinant DnaI polypeptide (including a fragment of DnaI either purified from a previous protease digestion or expressed from a recombinant nucleic acid vector as a fragment) can be subjected to partial proteolysis. The proteolysis can be performed with low concentrations of proteases, including, but not limited to trypsin, chymotrypsin, endoproteinase Glu-C, and Asp-N with a DnaI polypetide in solution, resulting in the generation of defined proteolytic products as observed by SDS-PAGE. An acceptable concentration and reaction time is defined by the near complete conversion of the full-length protein to stable proteolytic products. The partial proteolytic fragments are then subjected to affinity chromatography with immobilized 77 ORF 104 to determine the region of the DnaI polypeptide containing the 77 ORF 104 binding site. Interacting domains are identified by mass spectrometry to determine the masses of both the intact fragment and the series of fragments from a tryptic digest to identify the amino acid residues contained within the partial proteolytic fragment. Using both sets of data, the amino acid sequence of the partial proteolytic fragment can be precisely determined.

B) Yeast Two-Hybrid analysis

The interaction between 77 ORF 104 and portions of the DnaI polypeptide can also be assessed in vivo using the yeast two hybrid system. To do this, bacteriophage 77 ORF 104 is fused to the DNA binding domain of the yeast transcriptional transactivator Gal4, and different portions of the DnaI polypeptide are fused to the carboxyl terminus of the Gal4 activation domain. The two plasmids bearing such constructs can be introduced sequentially, or in combination, into a yeast cell line, for example AH109 (Clontech Laboratories), previously engineered to contain chromosomally-integrated copies of E. coli lacZ and the selectable HIS3 and ADE2 genes. The lacZ, HIS, and ADE2 reporter genes, each driven by a promoter containing Gal4 binding sites, are used for measuring protein--protein interactions. If the two recombinant proteins interact within the yeast cell, the resulting protein:protein complex activates transcription from promoters containing Gal4 binding sites. Expression of HIS3, and ADE2 genes is manifested by relief of histidine and adenine auxotrophy. As described in the examples below, full length DnaI, as well as DnaI fragments, was found to interact with bacteriophage 77 ORF 104 fusion polypeptides using this system.

Further elucidation of the bacteriophage 77 ORF 104 interacting domain of DnaI can be carried out by first subjecting the full length DnaI polypeptide to deletional mutagenesis, the methods of which are known to those of skill in the art. The mutated DnaI polypeptides can then be subjected to yeast two hybrid analysis as described above, to further narrow those amino acid sequences or polypeptide fragments, for example, those within SEQ ID NO: 16, that are required for the binding of DnaI to bacteriophage 77 ORF 104.

S. aureus DnaI Polypeptides

In one aspect of the invention there are provided polypeptides of S. aureus referred to herein as "DnaI" and "DnaI polypeptides" as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same.

Among the particularly preferred embodiments of the invention are variants of S. aureus DnaI polypeptides encoded by naturally occurring alleles of the dnaI gene. The present invention provides for an isolated polypeptide which comprises or consists of: (a) an amino acid sequence which has at least 50% identity, preferably at least 80% identity, more preferably at least 90%, yet more preferably at least 95%, 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 or b) an amino acid sequence that has at least 70% similarity, at least 80% similarity, at least 90% similarity, at least 95% similarity, at least 97 99% similarity or even 100% similarity over the entire length of SEQ ID NO: 2.

The polypeptides of the invention include a polypeptide of FIG. 1 (SEQ ID NO: 2) (in particular the mature polypeptide) as well as polypeptides and fragments, particularly those which have the biological activity of DnaI, and also those which have at least 50% identity over 20, 40, 50 or more amino acids to a polypeptide of SEQ ID NO: 2 or the relevant portion, preferably at least 60%, 70%, or 80% identity, more preferably at least 90% identity to a polypeptide of SEQ ID NO: 2 and more preferably at least 90% identity to a polypeptide of SEQ ID NO: 2 and still more preferably at least 95% identity to a polypeptide of SEQ ID NO: 2 and yet still more preferably at least 99% identity to a polypeptide of SEQ ID NO: 2.

The polypeptides of the invention also include a polypeptide or protein fragment that has at least 60%, 70%, 80% or 90% similarity, 95% similarity or even 97 99% similarity over 10, 20, 25, 30 or more amino acids to a polypeptide of SEQ ID NO: 2. It is preferred that a polypeptide of the invention has at least 60% similarity to a polypeptide of SEQ ID NO: 2 over at least 20 amino acids.

It is most preferred that a polypeptide of the invention is derived from S. aureus, however, it may 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.

Fragments of DnaI also are included in the invention. These fragments may include, for example, truncation polypeptides having a portion of an amino acid sequence of FIG. 1 (SEQ ID NO: 2), or variants thereof, such as a continuous series of residues that includes an amino- and/or carboxyl-terminal amino acid sequence. Degradation forms of the polypeptides of the invention produced by or in a host cell, particularly S. aureus, are also preferred. Further preferred are fragments characterized by structural or functional attributes such as fragments that comprise alpha-helix and alpha-helix-forming regions, beta-sheet and beta-sheet-forming 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 antigenic index regions. Fragments of DnaI may be expressed as fusion proteins with other proteins or protein fragments.

Preferred fragments also include an isolated polypeptide comprising an amino acid sequence having at least 20, 30, 40, 50, or 100 contiguous amino acids from the amino acid sequence of SEQ ID NO: 2.

Also preferred are biologically "active" fragments which are those fragments that mediate activities of S. aureus DnaI, 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 domains that confer a function essential for viability of S. aureus.

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.

S. aureus Polynucleotides

It is an object of the invention to provide polynucleotides that encode DnaI polypeptides, particularly polynucleotides that encode the polypeptide herein designated S. aureus DnaI.

In one aspect of the invention a polynucleotide is provided that comprises a region encoding a S. aureus DnaI polypeptide, the polynucleotide comprising a sequence set out in SEQ ID NO: 1. Such a polynucleotide encodes a full length DnaI gene, or a variant thereof. It is contemplated that this full-length gene is essential to the growth and/or survival of an organism which possesses it, such as S. aureus.

As a further aspect of the invention there are provided isolated nucleic acid molecules encoding and/or expressing a fragment of a full-length DnaI polypeptide, particularly a S. aureus DnaI polypeptide or a variant thereof. Further embodiments of the invention include biologically, diagnostically, prophylactically, clinically or therapeutically useful polynucleotides and polypeptides, and variants thereof, and compositions comprising the same.

A polynucleotide of the invention is obtained using S. aureus cells as starting material, the nucleotide sequence information disclosed in SEQ ID NO: 1, and standard cloning and screening methods, such as those for cloning and sequencing chromosomal DNA fragments from bacteria. For example, to obtain a polynucleotide sequence of the invention, such as the polynucleotide sequence disclosed as in SEQ ID NO: 1, a library of clones of chromosomal DNA of S. aureus in E. coli or another 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 be distinguished using stringent hybridization conditions. As herein used, the terms "stringent conditions" and "stringent hybridization conditions" mean hybridization occurring only if there is at least 95% and preferably at least 97% identity between the sequences. A specific example of stringent hybridization conditions is of a overnight incubation of a hybridization support (e.g., a nylon or nitrocellulose membrane at 42.degree. C. in a solution comprising: 1.times.10.sup.6 cpm/ml labeled probe, 50% formamide, 5.times.SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5.times. Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml of denatured, sheared salmon sperm DNA, followed by washing the hybridization support in 0.1.times.SSC at about 65.degree. C. Hybridization and wash conditions are well known to those skilled in the art and are exemplified in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), particularly Chapter 11 therein. Solution hybridization may also be used with the polynucleotide sequences provided by the invention. By sequencing the individual clones thus identified by hybridization, it is possible to confirm the identity of the clone.

Alternatively, an amplification process can be utilized to isolate the polynucleotide. In this approach, the sequence disclosed as SEQ ID NO: 1 is targeted by two oligonucleotides, one identical to a sequence on the coding DNA strand at or upstream of the ATG initiation codon and the other which anneals to the opposite strand at or downstream of the stop codon. Priming from these oligonucleotides in a polymerase chain reaction yields a full length gene coding sequence. Such suitable techniques are described by Maniatis, T., Fritsch, E. F. and Sambrook, MOLECULAR CLONING: A LABORATORY MANUAL, 2.sup.nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

In a further aspect, the present invention provides for an isolated polynucleotide comprising or consisting of: (a) a polynucleotide sequence which has at least 60% identity, preferably at least 70% identity, more preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95%, most preferably at least 97 99% or exact identity, to that of SEQ ID NO: 1 over the entire length of SEQ ID NO: 1; (b) a polynucleotide sequence encoding a polypeptide which has at least 50% identity, preferably at least 60% identity, more preferably at least 70% identity, more preferably at least 80% identity, more preferably at least 90%, yet more preferably at least 95%, most preferably at least 97 99% or exact identity to SEQ ID NO:2 over the entire length of SEQ ID NO:2; or the complement of a sequence of (a) or (b) above.

The invention provides a polynucleotide sequence identical over its entire length to the coding sequence of SEQ ID NO: 1. Also provided by the invention is a coding sequence for a mature polypeptide or a fragment thereof (Including, for example, a fragment encoding a polypeptide of SEQ ID NO: 16), by itself as well as a coding sequence for a mature polypeptide or a fragment in reading frame with another coding sequence, such as a sequence encoding a leader or secretory sequence, a pre-, or pro-, or prepro-protein sequence. The polynucleotide of the invention may also contain at least one non-coding sequence, including for example, but not limited to at least one non-coding 5' and 3' sequence, such as the transcribed but non-translated sequences, termination signals (such as rho-dependent and rho-independent termination signals), ribosome binding sites, Kozak sequences, sequences that stabilize or destabilize mRNAs, introns, and polyadenylation signals. The polynucleotide sequence may also comprise additional coding sequence encoding additional amino acids. For example, a marker sequence that facilitates purification of the fused polypeptide can be encoded. In certain embodiments of the invention, the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc. Natl. Acad. Sci. 86: 821 824 (1989), or an HA peptide tag (Wilson et al., Cell 37: 767 (1984), both of which may be useful in purifying polypeptide sequences fused to them. Polynucleotides of the invention also include, but are not limited to, polynucleotides comprising a structural gene and its naturally associated sequences that control gene expression.

It is most preferred that a polynucleotide of the invention is derived from Staphylococcus aureus, however, it may also 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.

Further preferred embodiments are polynucleotides encoding S. aureus dnaI variants that have the amino acid sequence of S. aureus DnaI polypeptide of SEQ ID NO: 2 in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, modified, deleted and/or added, in any combination. Especially preferred among these polynucleotides are those encoding silent nucleotide alterations that do not alter the coding sequence or activities of S. aureus DnaI polypeptides they encode.

Preferred embodiments are polynucleotides encoding polypeptides that retain substantially the same biological function or activity as the mature polypeptide encoded by a DNA of SEQ ID NO: 1.

In accordance with certain preferred embodiments of this invention there are provided polynucleotides that hybridize, particularly under stringent conditions, to S. aureus dnaI polynucleotide sequences, such as those polynucleotides in FIG. 1.

The polynucleotides of the invention are useful as hybridization probes for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding genes that have a high degree of sequence identity to the dnaI gene. Such probes generally will comprise at least 15 to about 100 residues or base pairs, although such probes will preferably have about 20 to 50 nucleotide residues or base pairs. Particularly preferred probes are about 20 to about 30 nucleotide residues or base pairs in length.

A coding region of a related dnaI gene from a bacterial species other than S. aureus may be isolated by screening a library using a DNA sequence provided in SEQ ID NO: 1 to synthesize an oligonucleotide probe. A labeled oligonucleotide having a sequence complementary to that of a gene of the invention is then used to screen a library of cDNA, genomic DNA or mRNA to determine to which member(s) of the library the probe hybridizes.

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

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

The polynucleotides of the invention that are oligonucleotides derived from a sequence of SEQ ID NO: 1 are useful for the design of PCR primers in reactions to determine whether or not the polynucleotides identified herein in whole or in part are transcribed in bacteria in infected tissue. That is, the polynucleotides of the invention are useful for diagnosis of infection with a bacterial strain carrying those sequences. It is recognized that such sequences 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. 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 facilitate manipulation of a protein for assay or production, among other things. As generally is the case in vivo, the additional amino acids may be processed away from the mature protein by cellular enzymes.

A precursor protein, having 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.

A polynucleotide of the invention thus 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 preproprotein, which is a precursor to a proprotein, having a leader sequence and one or more prosequences, which generally are removed during processing steps that produce active and mature forms of the polypeptide.

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 nucleotide that when taken in combination with adjacent nucleotide positions, read in the correct reading frame, would have the effect of generating a premature termination codon in such reading frame.

For each and every polynucleotide of the invention there is also provided a polynucleotide complementary to it.

Vectors, Host Cells, and Expression Systems

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

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

For recombinant production of DnaI polypeptides of the invention, host cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the invention. Representative examples of appropriate hosts include bacterial cells (Gram positive and Gram negative), fungal cells, insect cells, animal cells and plant cells. Polynucleotides are introduced to bacteria by standard chemical treatment protocols, such as the induction of competence to take up DNA by treatment with calcium chloride (Sambrook et al., supra). Introduction of polynucleotides into fungal (e.g., yeast) host cells is effected, if desired, by standard chemical methods, such as lithium acetate-mediated transformation.

A great variety of expression systems are useful to produce DnaI polypeptides of the invention. Such vectors include among others, chromosomal-, episomal- and virus-derived vectors. For example, vectors derived from bacterial plasmids, from bacteriophages, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses, and from vectors derived from combinations thereof, are useful in the invention.

DnaI polypeptides of the invention are recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid or urea extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. Well known techniques for refolding may be employed to regenerate an active conformation when the DnaI polypeptide is denatured during isolation and/or purification.

Diagnostic, Prognostic, Serotyping, and Mutation Assays

This invention is also related to the use of dnaI polynucleotides and polypeptides of the invention for use as diagnostic reagents. Detection of S. aureus dnaI 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 S. aureus dnaI 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 dnaI polynucleotide sequences. Perfectly or significantly matched sequences can be distinguished from imperfectly or more significantly mismatched duplexes by DNase or RNase 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 mobility 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, (1985) Science 230, 1242. Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase, V1 and S1 protection assay or a chemical cleavage method. See, for example, Cotton et al., (1985) Proc. Natl. Acad. Sci., USA 85, 4397 4401.

In another embodiment, an array of oligonucleotide probes comprising dnaI 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., (1996) Science 274, 610).

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: 1, 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 dnaI polynucleotides of the present invention as diagnostic reagents. Detection of a mutated form of a polynucleotide of the invention, preferably, SEQ ID NO: 1, 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 dnaI nucleotide sequences of the present invention are also valuable for organism chromosome identification. The sequence is specifically targeted to, and can hybridize with, a particular location on an organism's chromosome, particularly to a S. aureus chromosome. The mapping of relevant sequences to chromosomes according to the present invention may be an important step in correlating 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-line in 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 (coinheritance 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 possessing a different, second different phenotype can also be determined. If a mutation is observed in some or all organisms possessing the first phenotype but not in any organisms possessing the second phenotype, then the mutation is likely to be the causative agent of the first phenotype.

Polypeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtained from a putatively infected and/or infected individual's bodily materials. Particularly DNA or polynucleotides, from any of these sources may be used directly for detection or may be amplified enzymatically using PCR or other amplification technique with oligonucleotide amplification primers derived from the polynucleotide sequence of S. aureus dna 1. RNA, particularly mRNA, or RNA reverse transcribed to cDNA, is also useful for diagnostics. Following amplification of a S aureus dnaI-related polynucleotide from a sample, characterization of the species and strain of infecting or resident organism is made by an analysis of the amplified polynucleotide relative to one or more reference polynucleotides or sequences relative to a standard from a related organism (i.e. a known strain of S. aureus).

Point mutations can be identified by hybridizing amplified DNA to known dnaI polynucleotide sequences and by detecting differences in melting temperatures or renaturation kinetics. Perfectly or significantly matched sequences can be distinguished from imperfectly or more significantly mismatched duplexes by RNase protection or S1 nuclease mapping. (See, for example, Cotton et al., (1988) Proc. Natl. Acad. Sci. USA 85:4397 4401). Polynucleotide sequence differences may also be detected by alterations in the electrophoretic mobility 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, (1985) Science 230, 1242. Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase, V1 and S1 protection assay or a chemical cleavage method. (Cotton et al., 1988 Supra).

In another embodiment, an array of oligonucleotide probes comprising dnaI 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., (1996) Science 274, 610).

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: 1, 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. Such a kit will be of use in diagnosing a disease or susceptibility to a disease, among other uses.

The invention further provides a process for diagnosing bacterial infections such as those caused by S. aureus, the process comprising determining from a sample derived from an individual, such as a bodily material, an increased level of expression of a polynucleotide having a sequence disclosed in SEQ ID NO: 1 relative to a sample taken from a non-diseased individual. Increased or decreased expression of a dnaI polynucleotide can be measured using any one of the methods well known in the art for the quantitation of polynucleotides, such as, for example, PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods, and spectrometry.

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

Gridding and Polynucleotide Subtraction of S. aureus Genomic Sequences

The dnaI polynucleotides of the invention may 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 comprising a different gene, and further comprising a polynucleotide or polynucleotides of the invention, may be used for probing, such as hybridization or nucleic acid amplification, using a probe obtained or derived from a bodily sample, to determine the presence a particular polynucleotide sequence or related sequence in an individual.

Antibodies Specific for S. aureus Peptides or Polypeptides

The DnaI polypeptides and polynucleotides of the invention or variants thereof, or cells expressing them are useful as immunogens to produce antibodies immunospecific for such polypeptides or polynucleotides, respectively.

In certain preferred embodiments of the invention there are provided antibodies against S. aureus DnaI polypeptides or polynucleotides encoding them. Antibodies against DnaI-polypeptide or dnaI-polynucleotide are useful for treatment of infections, particularly bacterial infections.

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

Techniques for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies to polypeptides or polynucleotides of this invention. Also, transgenic mice, or other mammals, are useful to express humanized antibodies immunospecific to the polypeptides or polynucleotides of the invention.

When antibodies are administered therapeutically, the antibody or variant thereof is preferably modified to make it less immunogenic in the individual. For example, if the individual is human the antibody is most preferably "humanized," where the complimentarily determining region or regions of the hybridoma-derived antibody has been transplanted into a human monoclonal antibody, for example as described in Jones et al. (1986), Nature 321, 522 525 or Tempest et al., (1991) Biotechnology 9, 266 273.

Alternatively, phage display technology is useful to select antibody genes with binding activities towards a DnaI polypeptide of the invention. In one approach, antibody fragments specific for S. aureus DnaI are selected from an immune library of antibody genes expressed as fusions with coat protein of filamentous phage. Alternatively, naive libraries are screened by phage display techniques to identify genes encoding antibodies specific for DnaI or from naive libraries (McCafferty, et al., (1990), Nature 348, 552 554; Marks, et al., (1992) Biotechnology 10, 779 783; a recent reference is de Haard et al. (1999) J Biol Chem 274: 18218 18230). The ability to recover, for various targets, antibodies with subnanomolar affinities obviates the need for immunization. The affinity of these antibodies can also be improved by, for example, chain shuffling (Clackson et al., (1991) Nature 352: 628).

The above-described antibodies may be employed to isolate or to identify clones expressing the polypeptides or polynucleotides of the invention, for example to purify the polypeptides or polynucleotides by immunoaffinity chromatography.

A variant polypeptide or polynucleotide of the invention, such as an antigenically or immunologically equivalent derivative or a fusion protein of the polypeptide is also useful as an antigen to immunize a mouse or other animal such as a rat or chicken. A fused protein provides stability to the polypeptide acting as a carrier, or acts as an adjuvant or both. Alternatively, the antigen is associated, for example by conjugation, with an immunogenic carrier protein, such as bovine serum albumin, keyhole limpet haemocyanin or tetanus toxoid. Alternatively, when antibodies are to be administered therapeutically, alternatively a multiple antigenic polypeptide comprising multiple copies of the polypeptide, or an antigenically or immunologically equivalent polypeptide thereof may be sufficiently antigenic to improve immunogenicity so as to obviate the use of a carrier.

In accordance with an aspect of the invention, there is provided the use of a dnaI polynucleotide of the invention for therapeutic or prophylactic purposes, in particular genetic immunization. The use of a dnaI polynucleotide of the invention in genetic immunization preferably employs a suitable delivery method such as direct injection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet (1992) 1: 363, Manthorpe et al., Hum. Gene Ther. (1983) 4: 419), delivery of DNA complexed with specific protein carriers (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 al., DNA Cell Biol (1993) 12: 791) or in vivo infection using cloned retroviral vectors (Seeger et al., PNAS USA (1984) 81: 5849).

Antagonists and Agonists: Assays and Molecules

The invention is based in part on the discovery that DnaI is a target for the bacteria phage 77ORF104 inhibitory factor. Applicants have recognized the utility of the interaction in the development of antibacterial agents. Specifically, the inventors have recognized that 1) DnaI is a critical target for bacterial inhibition; 2) 77ORF104 or derivatives or functional mimetics thereof are useful for inhibiting bacterial growth; and 3) the interaction between dnaI and of S. aureus and 77ORF104 may be used as a target for the screening and rational design of drugs or antibacterial agents. In addition to methods of directly inhibiting DnaI activity, methods of inhibiting DnaI expression are also attractive for antibacterial activity.

In several embodiments of the invention, 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 invention comprising: contacting a polypeptide and/or polynucleotide of the invention with a compound to be screened under conditions to permit binding to or other interaction between the compound and the polypeptide and/or polynucleotide to assess the binding to or other interaction with the compound, such binding or interaction preferably being associated with a second component capable of providing a detectable signal in response to the binding or interaction of the polypeptide and/or polynucleotide with the compound; and determining whether the compound binds to or otherwise interacts with and activates or inhibits an activity or expression of the polypeptide and/or polynucleotide by detecting the presence or absence of a signal generated from the binding or interaction of the compound with the polypeptide and/or polynucleotide.

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 polypeptide such as a closely related protein or antibody that binds the same sites on a binding molecule, such as a binding molecule, without inducing dnaI-induced activities, thereby preventing the action or expression of S. aureus DnaI polypeptides and/or polynucleotides by excluding S. aureus DnaI polypeptides and/or polynucleotides from binding.

Potential antagonists also include a small molecule that binds to and occupies the binding site of the polypeptide thereby preventing binding to cellular binding molecules, such that normal biological activity is prevented. Examples of small molecules include but are not limited to small organic molecules, peptides or peptide-like molecules. Other potential antagonists include antisense molecules (see Okano, (1991) J. Neurochem. 56, 560; see also OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for a description of these molecules). Preferred potential antagonists include compounds related to and variants of 77ORF104 and of DnaI. Other examples of potential polypeptide antagonists include antibodies or, in 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 bind to the polypeptide of the present invention but do not elicit a response, so that the activity of the polypeptide is prevented.

Compounds may be identified from a variety of sources, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures. These substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics. See, e.g., Coligan et al., Current Protocols in Immunology 1(2): Chapter 5 (1991). Peptide modulators can also be selected by screening large random libraries of all possible peptides of a certain length.

Compounds could also be derived from the polypeptide sequence of 77ORF104 itself. Peptide fragments representing small overlapping fragments or peptides spanning the entire amino acid sequence of the protein can be used to perform extensive screens. Fragments of 77ORF104 can be produced by proteolytic digestion of the full-length protein as described above. Alternatively, suitable 77ORF104-derived peptide or polypeptide fragments representative of the complete sequence of the protein can be chemically synthesized. For instance, in the multi-pin approach, peptides are simultaneously synthesized by the assembly of small quantities of peptides on plastic pins derivatized with an ester linker based on glycolate and 4-(hydroxymethyl) benzoate (Maeji et al. (1991) Pept Res, 4:142 6).

Certain of the polypeptides of the invention are biomimetics, functional mimetics of the natural S. aureus DnaI polypeptide. These functional mimetics are useful for, among other things, antagonizing the activity of S. aureus DnaI polypeptide or as an antigen or immunogen in a manner described above. Functional mimetics of the polypeptides of the invention include but are not limited to truncated polypeptides. For example, preferred functional mimetics include a polypeptide comprising the polypeptide sequence set forth in SEQ ID NO: 2 lacking 20, 30, 40, 50, 60, 70 or 80 amino- or carboxy-terminal amino acid residues, including fusion proteins comprising one or more of these truncated sequences. Polynucleotides encoding each of these functional mimetics may be used as expression cassettes to express each mimetic polypeptide. It is preferred that these cassettes comprise 5' and 3' restriction sites to allow for a convenient means to ligate the cassettes together when desired. It is further preferred that these cassettes comprise gene expression signals known in the art or described elsewhere herein.

Screening Assays According to the Invention

It is desirable to devise screening methods to identify compounds which stimulate or which inhibit the function of the DnaI polypeptide or polynucleotide of the invention. Accordingly, the present invention provides for a method of screening compounds to identify those that modulate the function of a polypeptide or polynucleotide of the invention. In general, antagonists may be employed for therapeutic and prophylactic purposes. It is contemplated that an agonist of DnaI may be useful, for example, to enhance the growth rate of bacteria in a sample being cultured for diagnostic or other purposes.

Screening methods generally fall into two broad categories: those that assay binding of candidate compounds; and those that assay a functional aspect of the target.

a) Binding Assays

There are a number of methods of examining binding of a candidate compound to a protein target such as DnaI. Screening methods that measure the binding of a candidate compound to the DnaI polypeptide or polynucleotide, or to cells or supports bearing the polypeptide or a fusion protein comprising the polypeptide, by means of a label directly or indirectly associated with the candidate compound, are useful in the invention.

The screening method may involve competition for binding of a labeled competitor such as 77ORF104 or a fragment that is competent to bind DnaI.

i) Phage Display

Phage display is a powerful assay to measure protein:protein interaction. In this scheme, proteins or peptides are expressed as fusions with coat proteins or tail proteins of filamentous bacteriophage. A comprehensive monograph on this subject is Phage Display of Peptides and Proteins. A Laboratory Manual edited by Kay et al. (1996) Academic Press. For phages in the Ff family that include M13 and fd, gene III protein and gene VIII protein are the most commonly-used partners for fusion with foreign protein or peptides. Phagemids are vectors containing origins of replication both for plasmids and for bacteriophage. Phagemids encoding fusions to the gene III or gene VIII can be rescued from their bacterial hosts with helper phage, resulting in the display of the foreign sequences on the coat or at the tip of the recombinant phage.

In the simplest assay, purified recombinant DnaI protein, or a fragment of DnaI, for example comprising the amino acid sequence of SEQ ID NO: 16, could be immobilized in the wells of a microtitre plate and incubated with phages displaying 77ORF104 in fusion with the gene III protein. Washing steps are performed to remove unbound phages and bound phages are detected with monoclonal antibodies directed against phage coat protein (gene VIII protein). Color development by means of an enzyme-linked secondary antibody allows quantitative detection of bound fusion protein. Screening for inhibitors is performed by the incubation of the compound with the immobilized target before the addition of phages. The presence of an inhibitor will specifically reduce the signal in a dose-dependent manner relative to controls without inhibitor.

ii) Surface Plasmon Resonance

Another powerful assay to screen for inhibitors of a for protein: protein interaction is surface plasmon resonance. Surface plasmon resonance is a quantitative method that measures binding between two (or more) molecules by the change in mass near the sensor surface caused by the binding of one protein or other biomolecule from the aqueous phase to a second protein or biomolecule immobilized on the sensor. This change in mass is measured as resonance units versus time after injection or removal of the second protein or biomolecule and is measured using a Biacore Biosensor (Biacore AB). DnaI could be immobilized on a sensor chip (for example, research grade CM5 chip; Biacore AB) using a covalent linkage method (e.g. amine coupling in 10 mM sodium acetate [pH 4.5]). A blank surface is prepared by activating and inactivating a sensor chip without protein immobilization. The binding of 77ORF104 to DnaI, or a fragment of DnaI, for example comprising the amino acid sequence of SEQ ID NO: 16, is measured by injecting purified 77ORF104 over the chip surface. Measurements are performed at room temperature. Conditions used for the assay (i.e., those permitting binding) are as follows: 25 mM HEPES-KOH (pH 7.6), 150 mM sodium chloride, 15% glycerol, 1 mM dithiothreitol, and 0.001% Tween 20 with a flow rate of 10 ul/min. Preincubation of the sensor chip with candidate inhibitors will predictably decrease the interaction between 77ORF104 and DnaI. A decrease in 77ORF104 binding is indicative of competitive binding by the candidate compound.

iii) Fluorescence Resonance Energy Transfer (FRET)

Another method of measuring inhibition of binding of two proteins uses fluorescence resonance energy transfer (FRET; de Angelis, 1999, Physiological Genomics). FRET is a quantum mechanical phenomenon that occurs between a fluorescence donor (D) and a fluorescence acceptor (A) in close proximity (usually <100 A of separation.) if the emission spectrum of D overlaps with the excitation spectrum of A. Variants of the green fluorescent protein (GFP) from the jellyfish Aequorea Victoria are fused to a polypeptide or protein and serve as D-A pairs in a FRET scheme to measure protein--protein interaction. Cyan (CFP: D) and yellow (YFP: A) fluorescence proteins are linked with DnaI polypeptide, or a fragment of DnaI, for example comprising the amino acid sequence of SEQ ID NO: 16, and 77ORF104 protein respectively. Under optimal proximity, interaction between DnaI, or a fragment of DnaI, for example comprising the amino acid sequence of SEQ ID NO: 16 and 77ORF104 causes a decrease in intensity of CFP concomitant with an increase in YFP fluorescence.

The addition of a candidate modulator to the mixture of appropriately labeled DnaI and 77ORF104 protein, will result in an inhibition of energy transfer evidenced by, for example, a decease in YFP fluorescence at a given concentration of 77ORF104 relative to a sample without the candidate inhibitor.

iv) Fluorescence Polarization

In addition to the surface plasmon resonance and FRET methods, fluorescence polarization measurement is useful to quantitate protein--protein binding. The fluorescence polarization value for a fluorescently-tagged molecule depends on the rotational correlation time or tumbling rate. Protein complexes, such as those formed by S. aureus DnaI polypeptide; or a fragment of DnaI, for example comprising the amino acid sequence of SEQ ID NO: 16 associating with a fluorescently labeled polypeptide (e.g., 77ORF104 or a binding fragment thereof), have higher polarization values than a fluorescently labeled monomeric protein. Inclusion of a candidate inhibitor of the DnaI interaction results in a decrease in fluorescence polarization relative to a mixture without the candidate inhibitor if the candidate inhibitor disrupts or inhibits the interaction of DnaI with its polypeptide binding partner. It is preferred that this method be used to characterize small molecules that disrupt the formation of polypeptide or protein complexes.

v) Scintillation Proximity Assay

A scintillation proximity assay may be used to characterize the interaction between a S. aureus DnaI polypeptide, or a fragment of DnaI polypeptide, for example comprising the amino acid sequence of SEQ ID NO: 16 and another polypeptide. For the assay, S. aureus DnaI polypeptide can be covalently coupled to beads. Addition of radio-labeled 77ORF104 results in binding where the radioactive source molecule is in close proximity to the scintillation fluid. Thus, signal is emitted upon 77ORF104 polypeptide binding, and compounds that prevent association between S. aureus DnaI polypeptide and 77ORF104 diminish the scintillation signal.

vi) Bio Sensor Assay

ICS biosensors have been described by AMBRI (Australian Membrane Biotechnology Research Institute; http//www.ambri.com.au/). In this technology, the self-association of macromolecules such as DnaI, or a fragment of DnaI, for example comprising the amino acid sequence of SEQ ID NO: 16, and bacteriophage 77 ORF 104, is coupled 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 order of magnitude of admittance change and is ideally suited for large scale, high through-put screening of small molecule combinatorial libraries.

It is important to note that in assays of protein--protein interaction, it is possible that a modulator of the interaction need not necessarily interact directly with the domain(s) of the proteins that physically interact. It is also possible that a modulator will interact at a location removed from the site of protein--protein interaction and cause, for example, a conformational change in the DnaI polypeptide. Modulators (inhibitors or agonists) that act in this manner are of interest since the change they induce may modify the activity of the DnaI polypeptide.

b. Assays of DnaI Functional Activity.

i) Assay for DNA Replication, .sup.3H-thymidine Incorporation

To measure the effect of 77ORF104 expression on S. aureus DNA replication, the level of radiolabeled thymidine incorporation into DNA is measured in the presence or in the absence of sodium arsenite (5 uM). Samples (0.5 ml) are withdrawn from the cultures at appropriate time intervals and mixed to 4.5 ul of labeling solution (0.2 uCi/ml of .sup.3H-thymidine (73 Ci/mmol, NEN Life Science Products, Inc) and 70 pmol of cold thymidine). After 15 min of reaction, incorporation is stopped by adding solution containing 0.2% NaN.sub.3 and 1 mM cold thymidine. Samples are precipitated with 10% w/v trichloroacetic acid and filtered through glass fiber filters (GF-C, Whatman). The results are expressed as .sup.3H-thymidine counts incorporated normalized to OD culture.

The assay is performed in the presence of varying concentrations of candidate inhibitors in place of 77 ORF104 to screen for inhibitors. At least a 10-fold reduction in 3H-thymidine incorporation in the presence of 77 ORF104 or other inhibitor indicates a reduction in DnaI activity.

ii) Plasmid Replication

The plasmid pC194 replicates in S. aureus by rolling circle mechanism. The single stranded origin, sso of the pC194 is involved in the synthesis of the lagging DNA strand. The plasmid pADG6406 is a derivative of pC194 lacking sso. The absence of sso leads the accumulation of plasmid single-stranded DNA. The single-stranded (ss) initiation site, ssiA, is located on the lagging strand of pAM 1 and is a site for primosome assembly. SsiA was inserted into plasmid pADG6404. S aureus harboring plasmids are grown to mid-log phase and their total DNA is extracted and analyzed by Southern hybridization, using .sup.32P-labeled plasmid DNA as probe. The presence of pADG6406 with ssiA is associated to a decrease in the ratio of ss to double stranded (ds) DNA compared to that of the plasmid without ssiA. This system is used to measure the effect of 77ORF104 or a candidate inhibitor polypeptideexpression on ss DNA synthesis. In an assay, a plasmid containing 77ORF104 or a candidate inhibitor polypeptide coding sequence under an arsenite inducible promotor is introduced into a S aureus strain harboring pADG6406. The ratio of ss to ds DNA of pADG6406 is measured in the presence or in the absence of sodium arsenite (5 uM). An increase in the ratio of ss to ds DNA (10% or more) indicates an effect of the candidate modulator. 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 invention; which polypeptide 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 site(s), binding site(s) or motif(s) of an agonist, antagonist or inhibitor; (c) synthesizing candidate compounds that are predicted to bind to or react with the deduced binding site(s), reactive site(s), and/or motif(s); and (d) testing whether the candidate compounds are indeed 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.

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 that is 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 DnaI 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 dnaI antagonists, preferably bacteriostatic or bacteriocidal antagonists.

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

Compositions, Kits and Administration

In a further aspect of the invention there are provided compositions comprising a dnaI polynucleotide and/or a S. aureus DnaI polypeptide for administration to a cell or to a multicellular organism.

The present invention 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, 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 pharmaceutical compositions may be administered in any effective, convenient manner including, for instance, administration by topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes among others.

In therapy or as a prophylactic, the active agent may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion, preferably 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 conventional carriers, for example cream or ointment bases, and ethanol or oleyl alcohol for lotions. Such carriers may constitute from about 1% to about 98% by weight of the formulation; more usually they will constitute up to about 80% by weight of the formulation. Alternative means for systemic administration include transmucosal and transdermal administration using penetrants 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 physician 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.

As used herein, the term "in-dwelling device" refers to 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, but are not limited to, 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 continued 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 S. aureus 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. 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 in dental treatment as an alternative to, or in conjunction with, antibiotic prophylaxis.

Alternatively, the composition of the invention may be used to bathe an indwelling device immediately before insertion. The active agent will preferably be present at a concentration of 1 mg/ml to 10 mg/ml for bathing of wounds or indwelling devices.

A vaccine 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 using well known searching tools, such as GCC.

The polynucleotide and polypeptide 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 form. For example, chromatographic scan data or peak data, photographic data or scan data therefrom, called bases, and mass spectrographic data. As used in this section entitled Databases and Algorithms and in claims related thereto, the terms "polypeptide of the invention" and "polypeptide sequence of the invention" mean any detectable chemical or physical characteristic of a polypeptide of the invention that is or may be reduced to or stored in 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. 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.

In a preferred 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: 1 or SEQ ID NO: 17; a polypeptide comprising the sequence of SEQ ID NO: 2 or SEQ ID NO: 16; a set of polynucleotide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO: 1 or SEQ ID NO: 17: a set of polypeptide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO: 2 or SEQ ID NO: 16; a data set representing a polynucleotide sequence comprising the sequence of SEQ ID NO: 1 or SEQ ID NO: 17; a data set representing a polynucleotide sequence encoding a polypeptide sequence comprising the sequence of SEQ ID NO: 2 or SEQ ID NO: 16; a polynucleotide comprising the sequence of SEQ ID NO: 1 or SEQ ID NO: 17; a polypeptide comprising the sequence of SEQ ID NO: 2 or SEQ ID NO: 16; a set of polynucleotide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO: 1 or SEQ ID NO: 17; a set of polypeptide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO: 2 or SEQ ID NO: 16; a data set representing a polynucleotide sequence comprising the sequence of SEQ ID NO: 1 or SEQ ID NO: 17; a data set representing a polynucleotide sequence encoding a polypeptide sequence comprising the sequence of SEQ ID NO: 2 or SEQ ID NO: 16.
 

Claim 1 of 10 Claims

1. A method for inhibiting bacterial growth, comprising contacting bacteria in vitro with an amount of an inhibitor effective to reduce a DnaI activity of a polypeptide comprising the amino acid sequence of SEQ ID NO: 16, wherein said inhibitor inhibits bacterial growth.
 

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