JP2003517833A - Compositions and methods related to Staphylococcus aureus essential genes and proteins encoded by them - Google Patents

Abstract

  (57) [Summary] The present invention relates to newly discovered polynucleotides and polypeptides, as well as variants thereof, their production and use, agents and antagonists and their uses. In particular, it relates to a polynucleotide or polypeptide of Staphylococcus aureus (S. aureus) DnaI-related protein, or a mutant protein thereof. In addition, the present invention relates to a method for inhibiting the growth of a S. aureus Dnal-related protein or a specific portion thereof from a growth-inhibitory protein encoded by a S. aureus bacteriophage 77 chromosomal gene. Associated with specific interactions. The phage protein interacts with amino acids 313-150 of the S. aureus Dnal polypeptide, and the present invention involves using the target site of this interaction as the basis for a drug screening test I do. In addition, the present invention relates to polynucleotides and polypeptides of a protein complex comprising S. aureus Dnal and DnaC related proteins, as well as variants thereof.

Description

Detailed Description of the Invention

[0001] INVENTION FIELD The present invention relates to bacteria and bacteriophage genetic.

BACKGROUND Staphylococcus of the present invention (Staphylococcus aureus) is a medically important microorganisms, are known to cause the disease of various types of human beings. Staphylococcus aureus (S. aureus) is a Gram-positive bacterium that is also found in the skin of healthy human hosts. This bacterium causes many bacteremias, and the skin, lungs, urinary tract or contaminated endovascular device (medical device) may be the entry route (Steinberg et al., (1996) Clin.
Infect. Dis. 23: 255-259; Roder et al., (1999) Arch. Intem. Med. 159:
462469). In addition, this bacterium causes fatal endocarditis and damages the heart, and its exotoxin also causes death through “toxic shock”. (Frimodt-Moller et al., (1997) Clin. Microbiol. Infect. 3: 297-305;
20 Sanabria et al., (1990) Arch. Intem. Med. 150: 1305-1309).

Of the 19 Staphylococcus species listed in Bergey's Manual (1992), only Staphylococcus aureus and S. epidermidis are among humans. Have an important relationship with. They are in the normal human microflora and are found in the nasal route, skin and mucous membranes. When pathogenic to humans, Staphylococcus aureus can cause many purulent (pus-forming) infections as well as food poisoning, endocarditis, and toxic shock syndrome .

Staphylococcus aureus causes superficial skin disorders such as swelling, stye and stye. It also causes pneumonia, osteomyelitis and endocarditis, as well as more serious infections including mastitis, phlebitis, meningitis and urinary tract infections. In addition, Staphylococcus aureus is also a major cause of nosocomial infections via surgical wounds, medical devices inserted or implanted in the body. Lastly, Staphylococcus aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by releasing superantigens into the blood. Staphylococcus aureus (
S. aureus) also secretes two types of toxins with superantigenic activity.
The two are 1) enterotoxins with six antigen types (SE-A, B, C, D, E and G) and 2) toxic shock syndrome toxin (TSST-1).

[0005] Staphylococcus aureus (S. aureus) was previously treated effectively with the penicillin derivative methicillin, but is now resistant to this antibiotic (MRSA-methicillin-resistant Staphylococcus aureus (S. .aureus)) is increasing (Harbath et al., (1998) Arch. Intern. Med. 158: 182-189.). For example, the mortality rate of endocarditis due to Staphylococcus aureus is
It varies between 26-45%, indicating that treatment with a combination of beta-lactams and aminoglucosidases is becoming ineffective in the complete cure of the disease (Roder et al.
, (1999) Arch. Intern. Med. 159: 462-469).

However, MRSA infections remain sensitive to treatment with vancomycin, the drug of last resort. Infections caused by MRSA are increasing in children and adults and are isolated in 97% of large university-related education hospitals in the United States. Since 1996, three cases have been reported for vancomycin-resistant Staphylococcus aureus. The emergence of this new strain suggests the emergence of particularly dangerous mutant strains, such as highly pathogenic microbial pathogens against which known antibiotics do not work. The emergence of resistance to vancomycin has the potential, as a result, to render S. aureus infections an untreatable (and thus fatal) infection.

Staphylococcus aureus (resistant to most standard antibiotics
It is no longer uncommon for S. aureus strains to be isolated and therefore there is an unresolved medical need and demand for new antimicrobial agents, vaccines, drug screening and diagnostics against this organism. .

SUMMARY OF THE INVENTION [0008] The present invention relates to DnaI and DnaI-related proteins, and more particularly to recombinants of Staphylococcus aureus DnaI polypeptides and dnaI polynucleotides and their production methods. It is a thing. The invention further pertains to a set of interacting proteins that are growth inhibitory (or inhibitor) bacteriophage 77ORF104 gene products that interact with S. aureus DnaI polypeptides.

[0009] The interaction sites of the S. aureus DnaI-related protein and the protein encoded by the S. aureus bacteriophage 77ORF104 form the basis of screening tests.
The invention further relates to polynucleotides and polypeptides of protein complexes that are believed to be involved in the initiation of DNA replication containing DnaI as a subunit, and further include DnaC and related proteins, as well as variants thereof. There is also.

In another aspect, the invention relates to the use of these polypeptides and polynucleotides and may include treatment of diseases by other microorganisms. In a further aspect, the present invention provides a method for separating an active substance or an antagonist using the substance provided by the present invention, a treatment of a microbial infection or a condition associated with such an infection using the separated active substance or antagonist. Related to the law. Furthermore, the present invention also relates to diagnostic tests for detecting diseases associated with microbial infections and associated symptoms due to infections, such as tests for detecting the expression or activity of DnaI.

The invention includes a method of identifying a compound active against a polypeptide comprising the amino acid sequence of SEQ ID NO: 16. The method comprises contacting the candidate compound with the polypeptide and detecting binding of the candidate compound to the polypeptide, wherein detecting binding indicates that the compound is active against the polypeptide. Show. As a specific example, this detecting step includes the step of measuring the binding between the candidate compound and the polypeptide, and in this method, the candidate compound is directly or indirectly labeled with a detectable substance.

In another embodiment, the step of detecting comprises measurement by phage display. In another embodiment, the step of detecting includes measuring by surface plasmon resonance. In another embodiment, the detecting step includes measurement by the FRET method. In another embodiment, the detecting step comprises fluorescence polarization measurement. In another embodiment, the detection step is scintillation proximity
Includes assay measurements.

In another embodiment, the process of detection includes biosensor measurement. In another embodiment, the active compound is selected from the group consisting of small molecules, peptidomimetics, bacteriophage inhibitory protein fragments and derivatives. In another embodiment, the active compound is a peptide synthesized or purified by a recombinant expression system, or an artificially synthesized peptide.

The present invention comprises a method of identifying a compound active against a polypeptide comprising the amino acid of SEQ ID NO: 16, the method comprising the steps of first and second in the presence or absence of a candidate compound. The process involves examining the interference of two polypeptides. Then, the binding of the first and second polypeptides to each other is detected, and the binding of the first and second polypeptides is suppressed in the presence of the candidate compound, as compared with the case where there is no candidate compound. , It is confirmed that the candidate compound is an active compound for the polypeptide containing the amino acid sequence of SEQ ID NO: 16.

In one embodiment, the first or second polypeptide is directly or indirectly detectably labeled. In another embodiment, the detecting step comprises measurement by phage display. In another embodiment, the step of detecting comprises measuring by surface plasmon resonance. In another embodiment, the step of detecting comprises measuring by FRET. In another embodiment, the detecting step includes fluorescence polarization measurement. As another example, the step of detection is a scintillation proximity assay.
Including measurement by sei.

In another embodiment, the process of detection comprises measurement with a biosensor. The invention further includes agents and antagonists for the activity of the DnaI polypeptide or the gene encoding the polypeptide. The invention further includes a method for identifying a compound active against a DnaI polypeptide, which method comprises contacting a cell expressing a polypeptide comprising SEQ ID NO: 16 with a candidate compound, This includes the process of detecting intracellular DnaI activity. In this way, intracellular DnaI compared to no contact with candidate compounds
A decrease in activity indicates inhibition of DnaI activity.

The present invention further includes methods for synthesizing antimicrobial compounds. What is this method a)
Whether the candidate compound is active against a polypeptide comprising the amino acid sequence of SEQ ID NO: 16 or a gene encoding the polypeptide, b) to a bacterium which naturally produces the polypeptide comprising the amino acid sequence of SEQ ID NO: 16 When administered to an infected organism, it refers to the process of synthesizing or purifying a candidate compound in an amount sufficient to exert a therapeutic effect. In one embodiment, the candidate compound is selected from the group consisting of small molecules, peptidomimetics and fragments of bacteriophage inhibitory proteins or derivatives thereof. As one specific example, the candidate compound is synthesized by a recombinant expression system,
It is a purified or artificially synthesized peptide.

The present invention further includes methods of inhibiting bacteria by contacting the bacteria with a polypeptide comprising the amino acid sequence of SEQ ID NO: 16 or a compound active against the gene encoding this polypeptide. In one embodiment, this contacting process takes place in a test tube. In another embodiment, the contacting process takes place within the body of the animal. In other embodiments, the compound is selected from the group consisting of small molecules, peptidomimetic compounds and fragments of bacteriophage inhibitory proteins or derivatives thereof.

In another embodiment, the compound is a peptide synthesized, purified, or artificially synthesized by a recombinant expression system. The invention further includes a method of treating a bacterial infection in an animal afflicted with an infection. This involves administering to the animal a therapeutically effective amount of a compound which is active against the polypeptide comprising the amino acid sequence of SEQ ID NO: 16 or the gene encoding the polypeptide. This animal is preferably a mammal, but it is not always necessary, and it is more expected to be a human. In one specific example, the compound is selected from the group consisting of small molecules, peptidomimetic compounds and bacteriophage inhibitor proteins.

The present invention further encompasses prophylaxis to prevent bacterial infections. This involves contacting the indwelling device with a compound active against the polypeptide comprising the amino acid sequence of SEQ ID NO: 16 prior to implantation in the body of a mammal. Such contact is sufficient to prevent Staphylococcus aureus infection at the site of implantation. The invention further comprises a prophylactic method for preventing the infection of animals by bacteria, the amino acid sequence of SEQ ID NO: 16 or a polypeptide thereof in an amount sufficient to prevent the bacteria from adhering to the tissue surface of a mammal. Administering to the animal a compound that is active against a polypeptide comprising a gene encoding the gene. The invention further provides Staphylococcus aureu.
s), a method of diagnosing an individual infected, which includes a method of determining the presence of a polypeptide comprising the amino acid sequence of SEQ ID NO: 16 in the individual.

As one specific example, this determining step involves reacting a biological sample obtained from an individual with an antibody specific to an antigenic determinant present in a polypeptide containing the amino acid sequence of SEQ ID NO: 16. including. The present invention further provides a method for confirming the presence of a nucleic acid sequence encoding a polypeptide containing the amino acid sequence of SEQ ID NO: 16 in an individual, Staphylococcus aur
Includes diagnostics for eus-infected individuals. As one specific example, this confirmation step includes the step of reacting a nucleic acid probe of the aforementioned individual with a nucleic acid probe having a length of at least 15 nucleotides which has been separated, purified or concentrated, and the probe is subjected to severe hybridization conditions. Sequence number:
1 or hybridizes with the complementary strand of such a probe.

The invention further includes isolated, purified or concentrated polynucleotides. This polynucleotide consists of a nucleotide sequence that has at least 55% identity to the sequence of SEQ ID NO: 1, or a sequence that is complementary to the aforementioned nucleotide sequence. The invention further includes a polynucleotide consisting of isolated, purified or concentrated polynucleotide 448-942 of SEQ ID NO: 1 (referred to herein as SEQ ID NO: 17). This sequence includes the complement of the nucleotide sequence that encodes the polypeptide of SEQ ID NO: 16. The present invention further includes a polynucleotide comprising the sequence of SEQ ID NO: 17 isolated, purified or concentrated. The invention further includes a polypeptide having at least 55% identity to the isolated, purified or concentrated amino acid sequence of SEQ ID NO: 16. The invention further includes a polypeptide of at least 50 amino acids in length having at least 50% identity to the isolated, purified or concentrated amino acid sequence of SEQ ID NO: 16.

The invention further includes a polypeptide having at least 70% similarity to the isolated, purified or concentrated amino acid sequence of SEQ ID NO: 16. The invention further comprises a purified, isolated or enriched polypeptide of at least 20 amino acids in length having at least 60% similarity to the amino acid sequence of SEQ ID NO: 16. The invention further includes a polypeptide comprising the isolated amino acid sequence of SEQ ID NO: 16. The invention further includes a polypeptide consisting of the amino acid sequence of SEQ ID NO: 16, which is isolated.

The present invention further includes isolated, purified or concentrated antibodies specific for the polypeptide comprising SEQ ID NO: 16. The invention further provides a bacteriophage 77ORF104 polypeptide, SEQ ID NO: 16.
A polypeptide comprising the amino acid sequence of SEQ ID NO: or a complex consisting of two polypeptides of the variant of SEQ ID NO: 16 which specifically binds to the phage 77ORF104 polypeptide. The invention further includes a nucleic acid sequence encoding bacteriophage 77ORF104, a component comprising the nucleic acid sequence of SEQ ID NO: 17, or a variant thereof, encoding a polypeptide that specifically binds to the phage 77ORF104 polypeptide. Further features and advantages of the invention will be more fully apparent from the following description of specific embodiments, its illustrations and claims.

[0025] DESCRIPTION OF THE INVENTION The present invention is screening, essential genes of diagnostic and therapeutically useful Staphylococcus aureus and (S.aureus) part, based on the discovery of a polypeptide encoded by the gene is there. The invention also relates to S. aureus DnaI polypeptides and polynucleotides as described in further detail below. Then, when the interacting pair comprises a Staphylococcus aureus DnaI polypeptide and a 77ORF104 polypeptide, a pair of polynucleotides encoding the pair of interacting polypeptides and the pair of polypeptides themselves, Alternatively, it is related to its interaction domain. The invention also relates to polynucleotides and polypeptides of protein complexes that are thought to be involved in the initiation of DNA replication, including DnaI and DnaC derivatives as well as their related proteins.

In particular, the present invention relates to Staphylococcus aureus DnaI polypeptides and polynucleotides, which are related to B. subtilis.
lis) Classified in the same family by amino acid sequences homologous to DnaI polypeptides. The invention is particularly concerned with DnaI having the nucleotide and amino acid sequences published as SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The sequences shown as SEQ ID NO: 1 and 2 symbolize the best example of the invention. This is because these common techniques show that such a sequence can be effectively used in all polynucleotides including ribopolynucleotides.

We have identified two previous inventions (in order to separate and characterize essential polynucleotide and polypeptide sequences from S. aureus).
The US patent application serial number 09 / 407.804, filed September 28, 1999, and the methodology of the US provisional patent application 60 / 110,992) was used. Accordingly, the invention provides polypeptides and polynucleotide sequences from S. aureus that can be used in drug screening methods to find compounds with antimicrobial activity.

Definitions The phrase "active" with reference to a particular intracellular target (eg, the product of a particular gene) means an intracellular pathway in which the target comprises a target, substance or compound that functions on that pathway. Means an important part of. Thus, in some cases, the substance or compound may act on a component upstream or downstream of the stated target, which element comprises the modulator of the pathway or the component itself. In general, antimicrobial factors are active on essential cell functions and often on the products of essential genes.

As used herein, the terms “inhibit”, “inhibit”, “inhibitory” and “inhibitor (factor)” all mean the action of diminishing biological activity or function. Such a decrease in activity or function may be associated with, for example, an intracellular component (eg, enzyme), or an intracellular process (eg, specific protein synthesis), or an overall process of the cell (eg, cell proliferation). There is.

As used herein, the term “DnaI polypeptide” refers to Staphylococcus aureus DnaI (SEQ ID NO: 2) or Staphylococcus aureus (S.
.aureus) means a polypeptide containing a DnaI active domain. As used herein, the term "S. aureus DnaI active domain" refers to a Staphylococcus aureus DnaI that retains the activity of S. aureus DnaI. A polypeptide fragment or portion. The term "DnaI polypeptide" refers to Staphylococcus aureus (S.
aureus) DnaI or a fusion with another non-DnaI polypeptide sequence is meant to include the S. aureus DnaI active domain.

“DnaI activity” is defined by one or more of the following definitions. A) Bacteriophage 77ORF104 or its DnaI in a bacterial DNA replication assay
Polypeptides having a Staphylococcus aureus DnaI sequence provided herein, such that the uptake is reduced by at least 10-fold compared to the uptake of tritium thymidine in the absence of a binding fragment, fragments thereof Alternatively, the activity of an analog, or a protein comprising a Staphylococcus aureus DnaI polypeptide that interacts directly with the bacteriophage 77ORF104 protein or a DnaI binding fragment thereof.

To test DnaI activity by tritium thymidine ( ³ H-thymidine) uptake, arsenite-inducible in the presence or absence of 5 μM sodium arsenite was used.
The uptake of radiolabeled thymidine into DNA in S. aureus cells expressing the ORF104 construct is measured. Sample (0.5 m
l) was collected from the culture solution at appropriate time intervals and treated with 4.5 μl of labeling solution (0.2 μCi / ml of tritium thymidine (73 Ci / mmol, NEN Life Science Products, Inc.) and 70 pm).
ol unlabeled thymidine). After 15 minutes reaction, 5 μl 0.2% NaN ₃ and 5 μl 30
Stop the uptake by adding μl / ml unlabeled thymidine. Specimens are precipitated with 10% (W / V) trichloroacetic acid and filtered through glass filters (GF-C, Whatman). This result is expressed as a count of incorporated tritium thymidine and is normalized by the absorbance of the culture solution.

B) A polypeptide having a Staphylococcus aureus DNA sequence provided herein, which is required to inhibit plasmid replication by the bacteriophage 77ORF104 protein in a plasmid replication test by at least 10%, Fragment or analog or Staphylococcus aureus D
Activity of proteins, including NAI polypeptides. The test is as follows. Plasmid pC194 replicates in S. aureus by the rolling cycle mechanism. The origin of single-stranded (replication) of pC194, sso, is involved in the synthesis of the lagging strand of DNA. pADG6406 plasmid is sso-deficient p
It is a C194-derived strain. Deficiency of sso causes accumulation of single-stranded plasmid DNA. The single-strand initiation site, ssia, is on the lagging strand of pAM1 and is the assembly site for replication primosomes. SsiA was inserted into plasmid pADG6406.

Staphylococcus aureus cells carrying the plasmid are grown to mid-logarithmic phase, total DNA is extracted and analyzed by Southern hybridization using ³² P-labeled plasmid DNA as a probe. The presence of pADG6406 with ssiA is associated with a reduced ratio of single-stranded plasmid DNA to double-stranded as compared to cells with the same plasmid without the ssiA insertion. This system is single-stranded D
Used to measure the effect of 77ORF104 expression on NA synthesis. A plasmid containing 77ORF104 under the control of an arsenite-inducible promoter is transformed into S. aureus carrying pADG6406. Double strand of pADG6406
The ratio of single-stranded DNA to DNA is measured in the presence and absence of arsenous acid. 77OR
An increase in the ratio of single-stranded DNA to double-stranded DNA of 10% or more in the presence of F104 suggests an effect on DnaI activity.

C) Staphylococcus aureus (S. aureus) A polypeptide having the Staphylococcus aureus sequence (S. aureus) sequence provided herein in the process of carrying the DnaC helicase to the replicative primosome, a fragment or analog thereof, or The activity of S. aureus DnaI polypeptide. The helicase assay described below is based on the SPP 1 phages G38P (DnaA), G39P (Dnal) and G40P.
In vitro assay using (DnaC) polypeptide (Ayora et al., 1999, J. Mol.
Biol. 288: 71-85). Helicase has the ability to unwind (unwind) the double strands of DNA and is polarized from the 5'end to the 3'end.

Staphylococcus aureus (S. aureus) for the unwinding activity of this helicase
aureus) To determine the role of DnaI, a substrate (preformed fork) annealed to the 3'single-stranded (ss) DNA ends was used with a defined amount of purified DnaC helicase.
Increased amount of either purified DnaI or DnaA, or preformed DnaA-D
Incubate with naI complex. The reaction mixture is subject to conditions that support helicase activity.

This reaction contains 50 mM sodium chloride, 1 mM ATP, 50 μg / ml BSA and 0.24 nM ³² P-labeled oligomer annealed to M13 ssDNA provided as a substrate. It is analyzed whether the DNA molecule in the reaction mixture has been converted into single-stranded (ss) DNA. 5 μl
DNA loading buffer containing stop solution (100 mM EDTA, 2% (w / v) SDS)
Sambrook (1989)) was added to stop the reaction, and then loaded on a 10% non-denaturing PAGE gel. Run the gel and let dry before autoradiography. M13 ssDNA
Evaluate the percentage of oligos released from.

D) Binding or interaction of a polypeptide consisting of the amino acid sequence of SEQ ID NO: 16 provided herein with the bacteriophage 77ORF104 protein, or a bacterium capable of binding with a polypeptide comprising the amino acid sequence of SEQ ID NO: 16 Phage 77 ORF1
04 Binding or interaction with some of the proteins. Binding or interaction of the polypeptide comprising the amino acid sequence of SEQ ID NO: 16 with the bacteriophage 77ORF104 protein binding portion is expected to occur between isolated polypeptides containing the essential sequences required for binding. Alternatively, each polypeptide sequence will be contained within a larger polypeptide.

Described below are a number of methods useful in this invention for measuring the binding of bacteriophage 77ORF104 protein to a polypeptide comprising the amino acid sequence of SEQ ID NO: 16. For example, the phage display method is a calorimetric ELISA (enzyme-li
measurement of protein-protein interactions using a naked immunosorbent assay),
It is a powerful quantitative method. Surface plasmon resonance is a quantitative method that measures the binding between two molecules by the mass change near the immobilized sensor caused by the binding of one protein from the aqueous phase to another protein immobilized on the sensor. It is available. In addition, a useful binding assay is Fluorescence Resonance Energy
Transfer (FRET) method.

In this method, when two fluorescent groups respectively bound to different molecules come close to each other, the excitation light of one fluorescent group causes an overlap with the excitation light of the other fluorescent group. Therefore, double fluorescence upon excitation of only one fluorescent group suggests binding. A further inspection method useful in this invention is fluorescence polarization. In this method, the quantifiable degree of polarization of a known molecule labeled with a fluorophore is altered by binding to another protein. The scintillation proximity assay also
It can be used to measure the binding of a polypeptide containing the amino acid sequence of SEQ ID NO: 16 or bacteriophage 77ORF104.

In this method, the emittance of the radioactive particle changes with the binding. further,
Binding can also be assessed by biosensor methods. This method is based on a change in selective permeability of the sensor induced by a change in ion channel upon binding. A further method available for measuring binding of a polypeptide comprising the amino acid sequence of bacteriophage 77ORF104 with SEQ ID NO: 16 is the yeast two hybrid method. In this method, binding of the two polypeptides, under the condition of the two fusion proteins expressed in yeast, allows expression of the reporter molecule, resulting in the production of a measurable or detectable signal.

The activity of the dnaI gene is defined as the expression of RNA encoding the S. aureus DnaI polypeptide according to the invention. As used herein, the term "polynucleotide encoding a polypeptide" or equivalent term refers to a polypeptide of the invention, particularly a bacterial polypeptide, or more specifically, the amino acid sequence defined in Figure 1, SEQ ID NO: 2. With a staphylococcus aureus (S. aureus) DnaI protein. The term refers to one contiguous or non-contiguous portion (e.g., an integrating phage, an integrating insert sequence, that encodes a polypeptide with additional moieties that include coding or non-coding sequences).
Integrating vector sequences, integrating transposon sequences, or polynucleotides interrupted by RNA editing or chromosomal DNA reconstruction.

As used herein, the term “dnaI gene” refers to Staphylococcus aureus (S.au
reus) It has the meaning of including a polynucleotide encoding a DnaI polypeptide. All additional nucleotide sequences necessary to direct transcription of the RNA encoding the S. aureus DnaI polypeptide, either intracellularly or in vitro, are termed “regulatory sequences”. This includes, but is not limited to, transcription promoters, enhancers, and transcription terminators.

As used herein, the term “ORF 104” or “phage 77ORF 104” or “77ORF10”
4 "comprises a polynucleotide having the sequence provided in Figure 4 (SEQ ID NO: 4), which sequence encodes a gene product known as the 77ORF104 gene product. As used herein, the term “polynucleotide” generally refers to any polynucleotide or polydeoxyribonucleotide, including unmodified RNA or DNA or modified RNA or DNA.

“Polynucleotide” includes, without limitation, single-stranded and double-stranded DNA, mixed DNA of single-stranded and double-stranded portions, or mixed single-stranded, double-stranded and triple-stranded portions. DNA
, Single- and double-stranded RNA, mixed RNA of single-stranded and double-stranded portions, possibly consisting of single-stranded, or more typically double- or triple-stranded portions, or single-stranded And a hybrid molecule that can be composed of mixed DNA and RNA of double-stranded portions.

Furthermore, the term “polynucleotide” as used herein refers to RNA or DNA or
It may also mean a triple-stranded portion composed of both RNA and DNA. The chains of such moieties may consist of the same or different molecules. This part consists of one or more of these molecules, but usually contains only a fraction of some. One of the molecules that makes up the triple helix is often an oligonucleotide.

As used herein, the term “polynucleotide” also refers to DNA or RNA that includes one or more altered base pairs as described above. Thus, DNA or RNA with backbone moieties modified for stability or for other reasons are also "polynucleotides" as meant herein. Furthermore, just two examples:
A DNA or RNA consisting of unique base pairs, such as inosine, or modified base pairs, such as tritylated base pairs, is also a polynucleotide within the meaning of this invention.

It is expected that a wide variety of modifications will be made to DNA or RNA to serve many beneficial purposes known in the art. As used herein, the term "polynucleotide" refers to chemical forms of DNA or RNA that are characteristic of viruses or cells, including, for example, simple or complex cells, as well as such chemical forms. , Including enzymatically or metabolically modified polynucleotides. "Polynucleotide" means
It also means short polynucleotides, often referred to as oligonucleotides.

The term “polypeptide” as used herein refers to any peptide or protein of two or more amino acids linked to each other by peptide bonds or modified peptide bonds. "Polypeptide" refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and longer chains, commonly referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. “Polypeptide” includes polypeptides modified by chemical modification techniques as well as by naturally occurring processes such as processing and other post-transcriptional modifications.

Such modifications are described in detail in basic textbooks and more detailed monographs, as well as in many research papers, and these methods are well known in the art. It may be advantageous for the same type of modification to be present in the same place, or several places, to varying degrees in a particular polypeptide. Furthermore, a particular polypeptide may contain many types of modifications. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxy termini.

Modifications include, for example, acetylation of flavins, acylation, ADP-ribosylation, amidation, covalent attachment, covalent attachment of a portion of heme, covalent attachment of nucleotides or nucleotide derivatives, of lipids or lipid derivatives. Covalent binding, phosphatidylinositol covalent binding, cross-linking, cyclization, disulfide bond formation, demethylation, pyroglutamate formation, formylation, gamma-carboxylation, GP
I anchor formation, hydroxylation, iodide, methylation, myristoleination, oxidation, proteolytic treatment, phosphorylation, prenylation, racemization, glycation, lipid addition, sulfation, gamma-carboxylation of glutamic acid residues, Includes addition of amino acids to proteins via transfer RNA such as hydroxylation, selenization, sulfation, arginylation and ubiquitination.

As a reference, PROTEINS-STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T
E. Creighton, WH Freeman and Company, New York (1993); Wold, F., Po
sttranslational Protein Modifications: Perspectives and Prospects, pgs.
1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, BC Johns
on, Ed., Academic Press, New York (1983); Seifter et al., Meth. Enzymol.
182: 626-646 (1990); and Rattan et al., Protein Synthesis: Posttranslati
See onal Modifications and Aging, Ann. NY Acad. Sci. 663: 48-62 (1992). Polypeptides may have a branched or cyclic structure with or without branching. Cyclic, branched or branched cyclic polypeptides result from natural processes after transcription, as well as from complete synthetic methods.

As used herein, the term “variant” is derived from a reference polynucleotide or polypeptide, respectively, but Dn as described herein.
A polynucleotide or polypeptide in which one or more of the biological activities of aI remains. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference nucleotide. Changes in the nucleotide sequence of the variant may or may not change the amino acid sequence of the polypeptide encoded by the referenced polynucleotide. Nucleotide changes may result in amino acid substitutions, insertions, deletions, truncations in the polypeptide encoded by the referenced sequence, or in the formation of fusion proteins, as described below.

In general, the differences are limited and the sequence of the referenced polypeptide and the sequence of the variant are very similar over their entire length and are in many cases identical. The polypeptides referred to as variants may differ in amino acid sequence by one or more substitutions, insertions, deletions in any combination. A substituted or inserted amino acid residue may or may not be encoded by the genetic code. This invention also includes variants of each polypeptide of the invention. It also includes polypeptides derived from the subject by conservative amino acid substitutions, such as substitutions by other residues with similar characteristics.

Typically, such substitutions include valine, leucine and isoleucine, serine and threonine, acidic residues aspartic acid and glutamic acid, basic residues lysine and arginine, aromatic residues. It occurs between phenylalanine and tyrosine. Particularly preferred are mutants in which 1-10, 1-5, 1-3, 2-3 or one or more amino acids are substituted, deleted or inserted in all combinations. Variants of polynucleotides or polypeptides can occur naturally, such as allelic variants, or can be variants that are not known to occur naturally. Non-naturally occurring variants of the polynucleotide or polypeptide can be synthesized by mutagenesis techniques, direct synthesis, or other recombinant methods that require skillful skill.

As used herein, the term “fragment”, when used in reference to a polypeptide, is an amino acid sequence that is partly identical, but not partly identical, to the amino acid sequence of a DnaI polypeptide according to the invention. Is a mutant polypeptide having In the case of the S. aureus DnaI polypeptide, the fragment is "
“Independent” (“consisting of”), or fragments thereof, may be contained within a larger polypeptide forming part or region thereof, often within one larger polypeptide It is included as one continuous area.

When the term “isolated” is used in reference to nucleic acids, the native sequence is removed from its normal cellular (eg, chromosomal) environment, or synthesized in a non-natural environment (eg, , Artificially synthesized). Therefore, the sequence may be placed in a cell-free solution or in an environment different from intracellular. The term does not imply that the sequence is the only nucleotide chain present, but is absolutely free of substances other than the nucleotides with which it is naturally associated (at least about
90-95% pure). Therefore, this term is distinguished from segregated chromosomes.

When the term “enriched” is used in reference to a polynucleotide, the specific DNA
Or a fraction whose RNA sequence has a significantly higher amount (2-5 times) of the total amount of total DNA or RNA present in the cell or solution of interest than normal or abnormal cells or cells from which the sequence was originally taken. Means to configure. This is the DNA or other
It can be done artificially by selective reduction of the amount of RNA, or selective increase of specific DNA or RNA sequences, or a combination of the two methods. However, note that the term "enriched" does not mean that no other DNA or RNA sequences are present at all, but merely a relative significant increase in the amount of the sequence of interest. Should.

As used herein, the term “significantly higher fraction” indicates that the degree of enrichment is beneficial to the person performing such enrichment, and that the relative enrichment with other nucleic acids is at least It suggests an increase of about 2 times, or 5 to 10 times or more. The term "purified" as used herein with reference to nucleic acids does not require absolute purity (such as a homogeneous preparation). Instead, it means that the sequence is relatively more pure than in its natural environment (this level is at least 2-5 fold, for example in mg / ml, compared to the natural state). Individual clones isolated from a chromosomal or cDNA library are electrophoretically purified to homogeneity. Claimed DNA molecules obtained from these clones will be obtained directly from total DNA or total RNA.

CDNA clones are not naturally occurring, but rather are obtained through the manipulation of partially purified naturally occurring material (messenger RNA). Construction of a cDNA library from mRNA involves the production of synthetic material (cDNA), and pure individual cDNA clones
It can be separated from the synthetic library by clonal selection of cells carrying the cDNA library. Thus, a process involving the construction of a cDNA library from mRNA and isolation of specific cDNA clones results in purification of approximately 10 ⁶ times the native message in native cells. It is therefore particularly expected to be purified in at least one, preferably two or three, and more preferably four or five purification units. Chromosomal libraries can be used as well, producing approximately the same level of purified product.

The terms “separation”, “concentration” and “purification” as used in connection with the above nucleic acids may also be used to describe relative polypeptide purities and amounts. These are likewise preserved, enriched, screened and selected from libraries using biochemical techniques well known in the art. Such polypeptides are naturally occurring, synthetic or chimeric molecules and can be extracted using a variety of methods such as immunoprecipitation, other "tagging" methods, conventional chromatography or electrophoresis. Some of the above utilize matching nucleic acid sequences.

The term “complementary strand” as used herein, when used with reference to a particular polynucleotide sequence, means a nucleotide sequence capable of forming a heteroduplex, in which that nucleotide All nucleotides included in the sequence form a base pair with the particular polynucleotide sequence by hydrogen bonding with the opposite nucleotide in the heteroduplex. The term may also mean a DNA or RNA sequence that is the complement of another RNA or DNA sequence. The term "hybridize" as used herein refers to the formation of hydrogen bonded heteroduplexes between two nucleic acid molecules. In general, a particular nucleic acid molecule will hybridize to its complementary strand or be sufficiently complementary to that particular molecule to form a hydrogen-bonded heteroduplex between the two molecules. Hybridizes with the molecule.

The term “probe” as used herein, is at least 15 nucleotides (nt) in length.
, 20 nt, 30 nt, 40 nt, 50 nt, 75 nt, 100 nt, 200 nt, 500 nt, 1000 nt, and up to 5000 to 10,000 nt polynucleotides. As used herein, and as is known in the art, the terms "identity" and "similarity" refer to the relationship between two or more polypeptide or polynucleotide sequences, In such cases, it is determined by comparing the sequences.

“Identity” or “similarity” of amino acid or nucleotide sequences is determined from the overall optimal alignment between the two sequences being compared. For example, the overall optimal alignment is the Needleman-Wunsch algorithm (Needleman and Wunsch, 197
0, J. Mol. Biol. 48: 443-453). "Identity" means that an amino acid or nucleotide at a particular position within a first polypeptide or polynucleotide is such that an amino acid or nucleotide is second optimal in its overall optimal alignment with the first polypeptide or polynucleotide. It is meant to be exactly the same as the amino acid or nucleotide to which the nucleotide corresponds.

As opposed to identity, “similarity” is meant to include conservatively substituted amino acids. "Conservative" substitution is the blosum62 substitution matrix (Hentikoff and Hentiko
ff, 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919). The phrase "sequence A is n% identical to sequence B" means that n% of the overall optimal alignment between sequences A and B consists of identical residues or nucleotides. . The following parameters in the Needleman-Wunsch alignment algorithm were used for global optimal alignment in the present disclosure.

For polypeptides: Substitution matrix: blosum62 Gap scoring function: -A -B * LG, A = 11 (gap penalty), B = 1 (gap length penalty), LG is the gap Length For nucleotide sequences: Substitution matrix: 10 match, 0 mismatch Gap scoring function: -A -B * LG, A = 50 (gap penalty), B = 3 (gap length penalty) , LG is the length of the gap

A typical conservative substitution is between methionine, valine, leucine and isoleucine,
It occurs between serine and threonine, residues aspartic acid, glutamic acid and asparagine, residues glutamine, lysine and arginine, and aromatic residues phenylalanine and tyrosine. When calculating the degree of similarity (usually a percentage) between two polypeptide sequences, the degree is only measured between the corresponding amino acid residues in the two polypeptide sequences over the entire length of the two molecules. It is considered as the number of residues showing the given identity and similarity.

As used herein, the term “antibody” is meant to include the binding (variable) region of the antibody and the structure utilizing other antibody modifications. Thus, the antibodies useful in the present invention may include whole antibodies, antibody fragments, multifunctional antibody complexes, or molecules that generally consist of one or more specific binding sites derived from an antibody. Antibody fragments include, for example, variable region antibody fragments (Fv) such as single chain variable region antibody fragments (Fv), antigen-binding fragments (Fab).
) Or F (ab ') ₂ fragment or its derivative. The antibody or antibody fragment may be non-recombinant, recombinant or humanized. This antibody may be composed of immunoglobulin isotypes such as IgG and IgM. In addition, immunoglobulin aggregates, polymers, derivatives or conjugated complexes, or fragments thereof, can be used where appropriate. According to the present invention, neutralizing antibodies are particularly useful in diagnostic, therapeutic, drug screening methods and drug design.

As used herein, the term “specific for an epitope present on a S. aureus DnaI polypeptide”, when used in reference to an antibody, refers to an antibody of the invention By Staphylococcus aureus (S.aureus)
Means to recognize and bind to an antigenic determinant present on a DnaI polypeptide. As used herein, the term "antigenically equivalent derivative" is meant to include the equivalent of a polypeptide, a polynucleotide, or either, as specifically recognized by certain antibodies. This class of antibodies is one which, when raised against a protein, polypeptide or polynucleotide according to the invention, interferes with the immediate physical interaction between the pathogen and the mammalian host.

The term “essential” as used herein, when used in association with a gene or gene product, cannot survive without the host carrying this gene in the absence or depletion of a functional product, or It means that the growth is significantly inhibited.
An "essential gene" is one that is beneficial or even essential for the growth of cells in vitro in cultures suitable for the growth of strains with wild-type alleles that match the particular gene in question. By a gene that encodes a product. Therefore, when the essential gene is inactivated or inhibited, the cells are expected to grow significantly slower or not grow at all compared to the wild type strain.

The growth of the strain in which such a gene is inactivated is 20% or less of the growth rate of the wild type in the growth medium, preferably 10% or less, preferably 5% or less, or completely zero. Is expected to become. At least under culture conditions close to a growth environment in a normal living body, it is expected that the cells will not grow at all or will not be able to survive if the gene product has no activity. For example, the lack of biological activity of certain enzymes involved in bacterial cell wall synthesis can lead to cell lysis under normal osmotic conditions, even if protoplasts survive under osmo-regulated conditions. It is possible to cause.

When such a gene is inhibited, for example by an antibacterial agent or a phage product, the growth rate of the inhibited bacteria is not necessarily 50%, and preferably even more than that of the uninhibited bacteria. Expected to be 30%, more preferably 20% and most preferably less than 10%. As will be appreciated by such skilled artisans, the extent of growth inhibition generally depends on the concentration of the inhibitor. In the context of the present invention, essential genes are generally good targets for antibacterial agents. An essential gene may directly encode the "target" molecule, or it may encode a product involved in the production, modification or maintenance of the target molecule.

As used herein, the term “target” refers to a biomolecule, or biomolecule complex, on which an endogenous substance or compound may act, resulting in regulation, rather inhibition of bacterial cell growth or viability. To do. As used herein, the term "signal produced by activation or inhibition of S. aureus DnaI polypeptide" means a measurable indicator of DnaI activity in a DnaI activity test. For example, tritium thymidine
DNA uptake, plasmid replication, helicase loading, or simply Dna
Is the signal resulting from the binding of 77ORF104 to the I polypeptide.

The term “reference,” as used herein, when used for the activity of a polypeptide, is observed or detected (directly or indirectly) in a particular test performed in the absence of a candidate compound. Activation value. The "reference" serves as a standard value for determining a positive or negative effect on the activity of a polypeptide of a candidate compound.

As used herein, the term “candidate compound” refers to Staphylococcus aureus (
S. aureus) refers to any compound that may modulate the expression or activity of the DnaI polypeptide. As used herein, the term "increased activity" refers to the level of measurable activity of a polypeptide in the presence of a candidate compound in a particular test, as compared to the level of activity measurable in the absence of the candidate compound. It means rising. According to the present invention, as compared with the state without the candidate compound, at least 10% increase, 20% increase, 50% increase, 75% increase, 100% increase or more, 2 times, 5 times, 10 times, 20 times , 50 times, 10
The activity is considered to be increased when the activity is 0-fold or more.

As used herein, the term “reduced activity” refers to a measurable amount of a polypeptide in the presence of a candidate compound as compared to a measurable level of activity in the absence of the candidate compound in a particular test. This means that the activity level is decreasing. According to the invention, there is at least a 10% reduction, preferably a 15% reduction, a 20% reduction, a 50% reduction, a 75% reduction or even a 100% reduction (e.g. If there is (no activity), the activity is considered to have decreased.

As used herein, the term “conditions under which an interaction is possible” means that when used with a S. aureus DnaI polypeptide and a candidate compound, the two components are both When in solution, or when one is immobilized or localized in some way and the other is in solution, the parameters of the solution (e.g., salt, detergent (interface) Activator), protein or candidate compound concentration, temperature, redox potential, and other factors) are conditions that allow the Staphylococcus aureus DnaI polypeptide to physically bind the candidate compound. means. The conditions under which the protein and the candidate compound can interact include, for example, the conditions described here for the phage display method, the surface plasmon resonance method, and the FRET method.

As used herein, the term “detectable change in a measurable parameter of DnaI” refers to a change in a quantifiable characteristic of a S. aureus DnaI polypeptide. As used herein, the term "agent" refers to Staphylococcus aureus (S.
aureus) means a substance or compound that enhances or increases the activity of a DnaI polypeptide or polynucleotide. The agent acts directly on the S. aureus DnaI polypeptide or polynucleotide or enhances the activity of the S. aureus DnaI polypeptide or polynucleotide. It may act on one or more components of the pathway that induce or induce an increase.

As used herein, the term “antagonist” refers to Staphylococcus aureus (S.
aureus) means a substance or compound that diminishes or reduces the activity of a DnaI polypeptide or polynucleotide. The antagonist either acts directly on the S. aureus DnaI polypeptide or polynucleotide or reduces the activity of the S. aureus DnaI polypeptide or polynucleotide. It may act on one or more components of the pathway to reduce or reduce the amount. As used herein, the term "antimicrobial substance" or "antimicrobial compound" means a substance or compound that exhibits a bactericidal or bacteriostatic effect against one or more bacterial strains, rather The substance or compound must be at least Staphylococcus
It is bactericidal or bacteriostatic against S. aureus.

The term “synthesis” as used herein refers to the process of chemically synthesizing a compound. The terms “therapeutically effective amount”, “pharmaceutically effective amount” or “sufficient amount to exert a therapeutic effect” as used in the context of the treatment of a bacterial infection are defined as the Means quantity. This generally means, to some extent, inhibiting the normal cellular function of bacterial cells necessary for subsequent bacterial infection. Further, a therapeutically effective amount, as used herein, means an amount of antimicrobial substance that results in the desired therapeutic effect, as judged by clinical trials or animal models. This amount can be readily determined by certain mature techniques and will vary depending on several factors such as the particular bacterial strain involved and the particular substance used. In the same context, a "sufficient amount to reduce adhesion" of a bacterium to a tissue or tissue surface is an antibacterial substance which has the effect of preventing or reducing the spread of a bacterial infection of a particular tissue or tissue surface. Means the amount of.

As used herein, the term “tissue” refers to an aggregate of one or more types of cells, which collectively serve one or more specific functions in vivo. . As used herein, "tissue surface" means the portion of tissue that forms the boundary between a particular tissue and other tissues or the tissue periphery. The tissue surface may mean the outer surface of the animal, such as the skin or cornea, or the inner surface,
It may also refer to the inner surface of the intestine or the surface exposed to the outside of the animal's perimeter, for example by wounding or surgery.

As used herein, the term “measurement of binding of a candidate compound” refers to Staphylococcus
It refers to the use of a test method that can quantitatively assess the amount of compound that is physically associated with the S. aureus DnaI polypeptide. As used herein, the term "directly or indirectly detectable label" makes a compound directly detectable (eg, an isotope or fluorophore (fluorescent labeling reagent)) or indirectly detectable. Means addition of a molecule, such as a detectable enzyme activity in the presence of a suitable substrate, or a specific antigen or other label detectable by the addition of an antibody or other specific indicator, to a candidate compound To do.

As used herein, the term “small molecule” refers to 3000 Da or less, preferably 2000 or 1500.
, More preferably 1000, most preferably less than 600 Da. It is desirable, but not essential, that the small molecule is not an oligopeptide. As used herein, the term “analog” means a compound that is a natural, synthetic or chimeric molecule, which is structurally and functionally related to the reference compound. The term "peptidomimetic" in the context of the present invention is, for example, a non-peptide compound which resembles the three-dimensional structural characteristics of the peptide or polypeptide associated with its activity, such as the ORF of a phage or a bacterium. A compound similar to the structure of the peptide or active site of a polypeptide.

As used herein, the term “bacteriophage inhibitory protein” means a protein encoded by the nucleic acid sequence of a bacteriophage, which protein inhibits bacterial function in the host bacterium. Therefore, it is a bacterial inhibitory phage product. The term "bacteriophage inhibitory protein" includes fragments, derivatives, or active sites of bacteriophage inhibitory proteins. As used herein, the term "active site" refers to an antigenic determinant, enzyme or regulatory domain, or fragment of a bacteriophage inhibitory protein that contributes to or is an important factor in the inhibition of a bacterial target. The active site can be cleaved from the adjacent sequence, and it is expected to be effective for inhibition even in a separated state.

The term “treatment of a bacterial infection” as used herein refers to the inhibition or elimination of bacterial growth or metabolic activity, or bacterial population, preferably in a host such as a mammal, more preferably a human. Means any treatment. As used herein, the term "bacterial strain" means a single bacterial strain, and includes a single cell and a plurality or groups of cells of that bacterial strain, unless a distinctly different meaning is indicated. When referring to a bacterium or bacteriophage, the term "strain" means a bacterium or phage that has a particular genetic element. Genetic elements include genetic elements such as recombinant vectors. Thus, for example, two or the same bacterial cells containing different vectors (eg, plasmids) each having a different insertion site are meant to be different strains.

As used herein, the term “diagnosis” means the identification of the microorganism or microbial strain responsible for the bacterial infection. The term "Staphylococcus aureus" is used here.
"infection" of a mammal, preferably a human, means the presence, development or proliferation of S. aureus strain cells in or on the body of a mammal. The term "polypeptide encoded by bacteriophage 77ORF104" as used herein refers to the polypeptide encoded by SEQ ID NO: 4, or a fragment or derivative thereof, which is of the sequence disclosed in SEQ ID NO: 5. Contains the active site of the bacteriophage 77ORF104 encoded polypeptide.

The term “DnaC” as used herein refers to the polypeptide of SEQ ID NO: 9 and also includes the polypeptide encoded by the polynucleotide of SEQ ID NO: 7. Alternatively, it means a fragment or derivative of such a polypeptide containing the active site of S. aureus DnaC. In this context, the active site of Staphylococcus aureus DnaC includes a fragment or derivative of Staphylococcus aureus DnaI or Staphylococcus aureus DnaI. A fragment or part of S. aureus DnaC that interacts with or is part of a complex.

As used herein, the term “polypeptide complex” means a group of two or more polypeptides physically associated with each other. Rather, such physical associations are expected to be required for some aspects of the activity exhibited by one or more polypeptides in such polypeptide complexes. The term "physical association" as used herein means an interaction between two components, including contact between the two components.

As used herein, the term “biological sample” means a sample obtained from an individual, or a sample that is infected, invaded by a microorganism, or obtained from a carrier. This can be cells, tissues or excretions such as bone, blood, serum, cerebrospinal fluid, semen, saliva, muscle, cartilage,
Includes, but is not limited to, organ tissue, skin, urine, feces or autopsy samples. As used herein, the term "disease" refers to any disease caused by, or associated with, a bacterial strain, which includes, for example, otitis media, conjunctivitis, pneumonia, bacteremia, myelomeningitis, Includes sinusitis, pyometra and endocarditis, and most typical myelomeningitis, such as cerebral spinal cord infections.

As used herein, the term “fusion protein” means a protein encoded by a gene consisting of amino acid coding sequences from two or more individual proteins that are fused in reading frame. Such proteins have fusion amino acid sequences derived from the individual proteins. As used herein, the term "host cell" is a cell that has been transformed or transduced, or that is capable of being transformed or transduced with an exogenous polynucleotide sequence.

As used herein, the term “immunologically equivalent derivative”, when used as a suitable treatment to produce antibodies in vertebrates, results in the antibody being released between the pathogen and the mammalian host. Of the polypeptide, polynucleotide, or either equivalent, which acts to interfere with the rapid physical interactions in. As used herein, the term "immunospecific" refers to a polypeptide or polynucleotide of the invention to another related polypeptide or polynucleotide, respectively.
In particular, it refers to the characteristic of antibodies to have a significantly greater affinity than their affinity for these polypeptides and polynucleotides in the prior art. As used herein, the term "individual" means a multicellular eukaryote, including, but not limited to, metazoans, mammals, sheep, bovines, apes, primates and humans. .

As used herein, the term “microorganism” includes (i) prokaryotes, including but not limited to: Streptococcus, Staphylococcus, Bordetella. Genus (Bordetella), Corynebacterium (Corynebacterium), Mycobacterium (Mycobacterium), Neisseria (Neisseria), Haemophilus (Haemophilus), Actinomycetes (Actinomycetes), Streptomyces (Streptomycetes), Nocardia (genus) Nocardia), Enterobacter, Yersinia
), Genus Fancisella, genus Pasturella, genus Moraxella, genus Acinetobacter, genus Erysipelothrix, genus Branhamella, actinobacillus, streptobacter Genus (Streptobacillus),

Listeria, Calymmatobacterium
, Brucella, Bacillus, Clostridium (Cl
ostridium), Treponema genus, Escherichia
, Salmonella, Klebsiella, Vibrio (
Vibrio), Proteus (Proteus), Erwinia (Erwinia), Borrelia (Borrelia), Leptospira (Leptospira), Spirrillum (Spirillum),
Campylobacter spp., Shigella spp., Legionella spp., Legionella spp., Pseudomonas spp., Aeromonas spp.
as), Rickettsia, Chlamydia, Mycoplasma, and more particularly, but not limited to:

Those belonging to the following species or genera, Group A Streptoco
ccus), Group B Streptococcus, Group C Streptococcus, Group D Streptococcus
occus), Group G Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactie
reptococcus agalactiae), Streptococcus
faecalis), Streptococcus faecium, Streptococcus durans, Neisseria gonorrheae, Neisseria menenjayitis
meningitidis),

Staphylococcus aureus, Staphylococcus epidermidis, Corynebacterium diptheriae, Gardnerella vaginalis
rdnerella vaginalis), Mycobacterium tuberculosis (Mycobacterium t
uberculosis), Mycobacterium bovis, Mycobacterium ulcerans, Mycobacterium leprae, Actinomyctes i
sraelii), Listeria monocytogenes, Bordetella pertusis, Bordatella parapertusis, Bordetella bronchiseptica
a bronchiseptica),

Escherichia coli and Shigel
la dysenteriae), Haemophilus influenzae
, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonella chifi (S)
almonella typhi), Citrobacter freundii,
Proteus mirabilis, Proteus bulabilis
teus vulgaris), Yersinia pestis, Klebsiella pneumoniae, Serratia marcesen
marcessens), Serratia liquefaciens, Vibrio cholera, Shigella dysenterii
), Shigella flexneri, Pseudomonas aeruginosa, Francisella
tularensis),

Brucella abortis, Bacillus anthorasis
cillus anthracis), Bacillus cereus, Clostridium perfringens, Clostridium tetani, Clostridium botulinum
tulinum), Treponema pallidum, Rickettsia rickettsii, and Chlamydia trachomitis.
mydia trachomitis), (ii) archaeon, including but not limited to Archaebacter, and (iii) protozoan, fungus (fungu
s), and Saccharomyces, Kluveroms
yces) or a member of the genus Candida, and Saccharomyces ceriviseae, Kluveromyces
lactis) or unicellular or filamentous eukaryotes, including but not limited to Candida albicans.

As used herein, the term “recombinant expression system” includes sequences that encode a polypeptide of the invention, or a portion thereof, to produce the polypeptides and polynucleotides of the invention, or A vector containing the polynucleotide of
It means a system for transforming or introducing into host cells or host cell lysates. As used herein, the term "artificial synthesis", when used with reference to a peptide, polypeptide or polypeptide, means chemically linking subunits of amino acids or nucleotides without the use of cells or polymerizing enzymes. Means The chemistry of polynucleotide and peptide synthesis is well known in the art.

In addition to the standard one- and three-letter abbreviations for amino acids, the term "X" or "Xaa" may be used in describing certain polypeptides of the invention. By "X" and "Xaa" is meant that all of the 20 naturally occurring amino acids can be replaced at such a designated position in the polypeptide sequence. In the context of the interaction of two polypeptides, the term "specific binding" as used herein refers to the two polypeptides physically interacting through individual regions or domains on the polypeptide. Meaning, the interaction depends on the amino acid sequence of the interacting domains. Generally, the equilibrium binding concentration of a polypeptide that specifically binds to the other is about 1 μM or less, desirably 100 nM or less, 10 nM or less, 1 nM or less, 100 pM or less. Below, just in the range of 10 pM or less.

As used herein, the term “reduced binding” refers to the decrease in the signal produced by the physical association between two polypeptides in one set of conditions relative to the signal in another reference set of conditions. Means that If the signal decreases, the signal is at least 10% lower than the level in the reference set conditions, preferably 20%, 40%, 50%, 75%, 90%, 95% or even 100% (e.g. Possible interactions) lower.

As used herein, the term “detectable label”, when used in the context of the yeast-to-hybrid method, refers to a polypeptide, which refers to cells expressing the polypeptide. , A polypeptide which imparts a characteristic indicative of a signal indicative of the presence or amount of the expressed polypeptide. The detectable label may be present as an episome or is encoded on a plasmid that can integrate into the host cell chromosome. A detectable label is a polypeptide encoding an enzyme that allows for colorimetric or fluorescence detection (eg, E. coli Lac).
Z, which catalyzes the conversion of the substrate analog X-gal to a blue color), a polypeptide encoding an enzyme that confers antibiotic resistance, and on culture media lacking certain components. It includes, but is not limited to, polypeptides that encode enzymes that confer the ability of yeast to develop (ie, are decisive for degreasing nutrition).

As used herein, the term “resulting expression of a detectable label” refers to the interaction of the factors necessary for expression of the detectable label (eg, a two-hybrid transcription promoting domain and a DNA binding domain fusion protein). ) Causes transcriptional enhancement and promotes detectable levels of transcription of the detectable label. By "detectable level" is meant that background expression that occurs in the substantial absence of one or more factors can be distinguished from expression levels in the conditions required for expression of the label. Detectable levels will vary depending on the nature of the detectable label, but will generally be at least about 10% or more above background level of that label.

As used herein, the term “reduced expression” means a decrease in the expression of a detectable label under certain set conditions as compared to expression under other reference set conditions. If the detectable label expression is reduced, expression is at least 10% lower than the level in the reference setting, preferably 20%, 40%, 50%, 75%,
90%, 95% or just 100% lower (eg not expressed).

How to identify the Staphylococcus aureus dnaI sequence
Or: Using the methodology detailed in Examples 1 and 2, Staphylococcus aureus specifically binds to the bacterial growth inhibitory 77 phage ORF104 protein.
reus) polypeptide was isolated. Briefly, 77ORF104 protein was used as a ligand in the affinity chromatography binding step with S. aureus protein extract. Selected Staphylococcus aureus interacting polypeptides were purified and further analyzed by trypsin digestion and mass spectrometry. http://www.genome.ou.edu/staph.h
A BLAST search for S. aureus nucleotide sequences in the S. aureus genomic database of the University of Oklahoma S. aureus in tml is performed using the trypsin of the S. aureus polypeptide. The digested peptide sequence, GHVPENVTDNDR (SEQ ID NO: 10), was used.

Sequence alignment group having a certain length of 4850 nucleotides (converted to an amino acid sequence, the alignment group 9
81) has a similar amino acid sequence GHVPELYVDNNR (SEQ ID NO: 11; Fig. 5).
) Was included. The results of identification of this temporary candidate protein were further confirmed by in-house tryptic digestion of the open reading frames found in this alignment group (Fig. 5). The resulting tryptic digested PCD / CID spectra of the 1351.6, 1412.5, 1617.8 Da monoisotopic MH ⁺ masses were found in the +3 reading frame of the alignment group 1
The expected PSD / CID fragmentation pattern of tryptic peptides using monoisotopic MH ⁺ masses of 352.622, 1413.538, 1618.7928 Da was very similar.

ORF of the Staphylococcus aureus alignment group that encodes a tryptic digestive peptide similar to that identified in the Staphylococcus aureus phage 77 ORF104 binding assay and public institutions. As a result of comparison with all other sequences in the database, it was revealed that this ORF is associated with the DnaI protein (Table 1, SEQ ID NO: 14-15) derived from Bacillus subtilis involved in chromosome replication. Became. No other significant similarities were found in any other protein in publicly available databases. The degree of association of the identified ORFs with the Bacillus subtilis (B. subtilis) DnaI protein is 41% identity and 63% similarity (Table 1).

Many Bacillus subtilis (B. subtilis) genes involved in DNA replication have been identified by isolation methods by temperature-sensitive mutations. One of these,
dnaI2 affects an unknown chromosomal replication process at a defined temperature (Karam
ata, D. and Gross, JD (1970) Mol. Gene. Genet. 108, 277-287). This gene maps to around 250 ^o on the Bacillus subtilis (B. subtilis) chromosome and is located immediately downstream of the dnaB gene on the Bacillus subtilis (B. subtilis) chromosome. (Bruand, C. and Ehrlich, SD (1995) Mi
crobiology 141, 1199-1200). Previously, the characterization of this dnaI2 mutation has been characterized and resides in the dnaI gene, resulting in nucleotide position 922 that replaces glycine for glutamate at position 307 (Figure 1; SEQ ID NO: 2). (Figure 1;
Made by G to A substitution of SEQ ID NO: 1) (Bruand, C. and Ehrlich, S
.D. (1995) Microbiology 141, 1199-1200).

DnaC was genetically identified as a major component of the DNA helicase in chromosomal replication (Sakamoto, Y., Nakai, S., Moriya, S., Yoshikawa, H., and Ogas.
awara, N. (1995) Microbiology 141, 641-644), which gradually rewinds double DNA and is thought to be able to bind the holoenzyme DNA polypeptide merase III required for replication initiation and DNA synthesis. . One possible function of DnaI is as a helicase carrier that acts to transfer DnaC helicase to oriC. The products of the dnaC and dnaI genes are required for chromosome replication and are all Bacillus subtilis (bacillus
Is essential for DNA replication in B. subtilis (Ceglowski,
P., Lurz, R., Alonso, JCJ (1993) Mol. Biol. 236, 1324-1340).

The database was searched for Staphylococcus aureus genes that are believed to be related to the B. subtilis dnaC gene. Using the DnaC amino acid sequence of Bacillus subtilis (B. subtilis) (Cat. No. P37469), BLAST search of the Staphylococcus database at http://www.tigr.org resulted in a staphylo Coccus aureus (S
.aureus) It has been revealed that there is an ORF encoding a related protein in the genome. The nucleotide sequence and the corresponding protein sequence are shown in the figure.
It is shown in 6A (SEQ ID NO: 7) and FIG. 6B (SEQ ID NO: 9).

Identification of Apparent Interactions on DnaI The present invention is directed, in part, to a growth inhibitory protein encoded by the Staphylococcus aureus bacteriophage 77 chromosomal gene and an essential Staphylococcus aureus (S. aureus 3.) It is associated with specific interactions with proteins. The present invention forms the basis for drug screening tests. The present invention provides methods for identifying DnaI polypeptide fragments, such as those involved in the interaction of DnaI with bacteriophage 77ORF104 described above. Several approaches and techniques known in the art can be used to identify and characterize DnaI fragments that interact with 77ORF104. These fragments are, for example, DnaI
May be a truncated polypeptide having a portion of the amino acid sequence of the protein, such as a contiguous set of residues containing the amino- and / or carboxy-terminal amino acid sequences of, or variants thereof. .

A) Partial proteolysis of proteins in affinity chromatography solutions is one way to reveal boundaries between domains in proteins with multiple domains. By partially digesting the protein, the most susceptible cleavage site is preferentially hydrolyzed. These cleavage sites reside primarily in less structured regions, which include cyclic structures and highly mobile regions, and are typically the binding amino acids between the highly structured domains. For this analysis, the purified recombinant DnaI polypeptide (including the protease-digested DnaI fragment described above, or the DnaI fragment expressed as a fragment in a recombinant nucleic acid vector) is subject to partial proteolysis.

[0112] Proteolysis can be performed in solutions containing low concentrations of proteases including but not limited to trypsin, chymotrypsin, endoproteases Glu-C and Asp-N, resulting in a large number of Clear proteolytic products can be observed by SDS-PAGE. The concentration and reaction time suitable for the conditions are set close to the conditions for converting the full-length protein into a stable proteolytic product almost completely.

Next, to determine the portion of the DnaI polypeptide containing the 77ORF104 binding site,
The partially proteolytic fragment is used for affinity chromatography with immobilized 77ORF104. The interaction domains are identified by mass spectrometry and both the mass of the tryptic digested contiguous fragments is determined to identify the amino acid residues contained within the complete and partial proteolytic fragments. Both sets of data can be used to refine the amino acid sequence of partial proteolytic fragments.

B) Yeast Two Hybrid Analysis The interaction between 77ORF104 and a portion of the DnaI polypeptide can also be evaluated in vivo using the yeast two hybrid system. To perform this experiment, bacteriophage 77ORF104 was fused to the DNA binding domain of the yeast transcriptional transactivator Gal4 and various portions of the DnaI polypeptide were fused to the carboxyl terminus of the Gal4 activation domain. Two plasmids having such a structure can be used in series or in combination, for example, E. coli lacZ in a form introduced into a chromosome.
Pre-designed to have a copy and a selectable HIS3 or ADE2 gene
It can be introduced into yeast strains such as AH109 (Clontech Laboratories).

Expression of the LacZ, HIS and ADE2 reporter genes was induced by promoters each containing a Gal4 binding site, and used to measure protein-protein interactions. If the two recombinant proteins interact in yeast, the resulting protein: protein complex activates transcription from a promoter with a Gal4 binding site. HIS3 expression and the ADE2 gene are revealed by the elimination of histidine and adenine auxotrophy. As in the example below,
Using this system, full-length DnaI as well as the DnaI fragment can be transformed into bacteriophage 77.
It was found to interact with the ORF104 fusion polypeptide.

Furthermore, elucidation of the bacteriophage 77ORF104 interaction domain of DnaI is possible by first introducing a deletion mutation into the full-length DnaI polypeptide, and such a method is well known in the art. . The mutated DnaI polypeptide is then sequenced as described above in order to further limit the amino acid sequence or polypeptide fragment contained in SEQ ID NO: 16 and required for DnaI to bind to bacteriophage 77ORF104. It can be used for hybrid analysis.

S. aureus DnaI Polypeptides As a feature of the invention, “DnaI” and “DnaI polypeptides”, as well as biological, diagnostic, prophylactic, clinical Also provided are therapeutically useful variants thereof, and S. aureus polypeptides, referred to herein as a complex consisting of such.

Among the embodiments of the present invention that are particularly emphasized are the Staphylococcus aureus encoded by the naturally occurring dnaI gene allele.
) For variants of the DnaI polypeptide. The invention presented herein (a) has at least 50% identity, preferably at least 80% identity, more preferably at least 90%, more preferably at least 95% with the sequence over the entire length of SEQ ID NO: 2. , Most preferably at least 97-99% or exactly the same amino acid sequence or (b) over the entire length of SEQ ID NO: 2, at least 70% similarity, at least 80% similarity, at least 90% similarity, at least similarity 95%, at least similarity 97-9
An isolated polypeptide is provided that comprises or consists of an amino acid sequence that is 9% or even 100% similar.

The polypeptides of the invention have the biological activity of polypeptides and fragments, especially DnaI, as in FIG. 1 (SEQ ID NO: 2) (especially the mature polypeptide), and
At least 50% identity to the polypeptide of SEQ ID NO: 2 or an appropriate portion thereof over 20, 40, 50 or more amino acids, preferably at least 60%, 70% identity to the polypeptide of SEQ ID NO: 2 Or 80%, more preferably at least 90% identity, more preferably at least 95% identity to SEQ ID NO: 2, even more preferably at least 99% identity to SEQ ID NO: 2. The polypeptide of the present invention also includes the polypeptide of SEQ ID NO: 2, 10, 20, 25, 30
Or at least 60%, 70%, 80% or over similarity over amino acids
It also includes polypeptides or proteins with 90%, 95% similarity or even 97-99% similarity. The protein of the invention preferably has at least 60% similarity over at least 20 amino acids to the polypeptide of SEQ ID NO: 2.

Most preferably, the polypeptides of the present invention are derived from S. aureus, however, the polypeptides may also be derived from other taxonomically identical organisms. The polypeptides of the present invention may be obtained, for example, from taxonomically identical families or organisms of the order.

DnaI fragments are also included in the invention. These fragments include, for example, the amino terminus and
/ Or like a contiguous set of residues containing a carboxyl-terminal amino acid sequence,
It may include a truncated polypeptide comprising a portion of the amino acid sequence of Figure 1 (SEQ ID NO: 2), or variants thereof. Degradable polypeptides of the present invention, such as those produced by or in host cells, particularly Staphylococcus aureus, are also desirable.

Further desirable are fragments as characterized by structural or functional properties, such as alpha helix and alpha helix formation sites, beta sheets and beta sheet formation sites, inversions and inversion formation sites. , Coil and coil forming site, hydrophilic site, hydrophobic site, alpha amphipathic site,
Examples include beta amphipathic sites, plasticity sites, surface formation sites, substrate binding sites, and high antigen presenting sites. The DnaI fragment can be expressed as a fusion protein with other proteins or protein fragments.

Desirable fragments also include at least 20, 30, 40 from the amino acid sequence of SEQ ID NO: 2.
Also included is an isolated polypeptide consisting of an amino acid sequence with 50, 50, or 100 contiguous amino acids. Also, biologically "active" fragments are desirable, these are those fragments that mediate the activity of S. aureus DnaI and may have similar or even greater activity. Or a fragment with reduced undesirable activity. Further included are fragments that are antigenic or immunogenic in animals, especially humans. Particularly desirable is a fragment containing a domain that confers an essential function on the viability of S. aureus. The polypeptide fragments of the present invention may also be utilized to produce the corresponding full-length polypeptide by peptide synthesis. Therefore, these variants may be used as intermediates for producing the full-length polypeptides of the present invention.

S. aureus Polypeptides A polypeptide encoding a DnaI polypeptide, and more particularly a polypeptide encoding a polypeptide herein designated S. aureus DnaI, is provided. Is one of the purposes. One of the features of the present invention is that Staphylococcus aureus D.
Provided is a polynucleotide comprising a portion encoding a naI polypeptide, which polynucleotide comprises the sequence designed in SEQ ID NO: 1. Such polynucleotides encode the full length DnaI gene or variants thereof. This full-length gene
Organisms that carry this gene, such as Staphylococcus aureus
A) is essential for growth and / or survival.

As a further feature of the present invention there is provided an isolated nucleic acid molecule which encodes or expresses a fragment of a full length DnaI polypeptide, in particular a Staphylococcus aureus DnaI polypeptide or variant thereof. . Further embodiments of the invention are biologically, diagnostically, prophylactically, clinically or therapeutically useful polynucleotides and polypeptides, and variants and conjugates containing these.

The polynucleotides of the present invention are cloned and sequenced from Staphylococcus aureus cells as starting materials, this nucleotide sequence information disclosed in SEQ ID NO: 1, and chromosomal DNA fragments, eg from bacteria. It can be obtained by using standard cloning or sequencing methods for For example, to obtain a polynucleotide sequence of the invention, such as the polynucleotide sequence disclosed in SEQ ID NO: 1, S. aureus was used in E. coli or other suitable host. 3.) Chromosomal DNA clone libraries are probed with radiolabeled oligonucleotides (preferably 17-mer or greater) obtained from partial sequences.

Clones with exactly the same DNA as the probe can be identified by using stringent hybridization conditions. As used herein, the terms "stringent conditions" and "stringent hybridization conditions" mean hybridizations that occur only under conditions of at least 95% and desirably 97% identity between the sequences. For a detailed example of hybridization conditions, incubate the hybridization carrier overnight (eg, with nylon or nitrocellulose membranes).
42 ° C in the following solution: 1x10 ⁶ cpm / ml labeled probe, 50% formamide, 5xS
SC (150 mM sodium chloride, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 μg / ml denatured, crushed salmon sperm DNA) followed by 0.1x SSC, about 65 Wash the hybridization carrier at ° C.

Hybridization and washing conditions are well known in the art for such skilled methods, see Sambrook, et al., Molecular Cloning: A Laboratory Ma.
nual, Second Edition, Cold Spring Harbor, NY, (1989), of which, among others, Chapter 11. It is possible to confirm the identity of the clones by sequencing the individual clones thus confirmed by hybridization.

Alternatively, an amplification process can be used to separate the polypeptides. In this method, the sequence disclosed as SEQ. It is an oligonucleotide located at the codon or downstream thereof. A full-length translated sequence gene is obtained by using the polymerase chain reaction with these oligonucleotides as the starting sites. Maniatis, T., Fritsch, EF
and Sambrook, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Edition, Cold
Spring Harbor, Cold Spring Harbor, NY (1989).

As a further feature, the invention provides (a) at least 60% identity, preferably at least 70% identity, and more preferably at least at least 60% identity over the entire length of polynucleotide sequence SEQ ID NO: 1 and SEQ ID NO: 1. 80% identity, more preferably 90% identity
, And even more preferably at least 95%, most preferably at least 97-9
9% or a polynucleotide sequence having exactly the same identity (b) over the entire length of SEQ ID NO: 2 and SEQ ID NO: 2, or at least 50% identity, preferably at least 60% identity, more preferably at least identity 70%, more preferably at least 80% identity, more preferably at least 90% identity, even more preferably at least 95% identity, most preferably at least 97-99% identity or exactly the same identity. There is provided a polynucleotide sequence encoding a peptide, or an isolated polynucleotide comprising or consisting of the complementary sequence of (a) or (b) above.

The present invention provides a polynucleotide identical to the translated sequence of SEQ ID NO: 1 over its full length. Also, according to the present invention, the translated sequences (eg, including the fragment encoding the polypeptide of SEQ ID NO: 16) encoding those mature polypeptides or fragments may be by themselves or similarly, eg, leader or secretory sequences, pre-sequences. Alternatively, the translated sequence of the mature polypeptide or fragment is provided in reading frame with other translated sequences, such as the sequence encoding the pro or prepro protein sequence. The polynucleotide of the present invention may include at least one untranslated sequence, but is not limited to at least one untranslated 5-terminal and 3-terminal sequences, and includes, for example, a transcribed but untranslated sequence, a termination signal (for example, rho-dependent sequence). Sex and rho independent termination signals), ribosome binding sites, Kozak sequences, mRNA stabilizing or destabilizing sequences, introns, and non-translated sequences such as polyadenylation signals.

The polynucleotide sequence may include additional translated sequences that encode additional amino acids. For example, a label sequence may be encoded that facilitates purification of the fusion protein. In some embodiments of the invention, the labeling sequence is provided by the pQE vector (Qiagen, Inc.), Gentz et al., Proc. Natl. Acad.
Sci. 86: 821-824 (1989), 6 histidine peptides, and
HA peptide tag (Wilson et al., Cell 37: 767 (1984)), both of which are useful for purifying the polypeptide sequences fused thereto. Polynucleotides of the invention also include, but are not limited to, structural genes and sequences naturally associated with them that regulate expression of genes.

The polynucleotide of the present invention is a Staphylococcus aureus.
u aureus), but it may also be derived from other taxonomically identical organisms. The polynucleotide of the present invention may be obtained from, for example, a taxonomic family or an organism of the order. More desirable embodiments include some 2-3, 5-10, 1-5, 1-3, 2, 1 or 0.
S. aureus DnaI of SEQ ID NO: 2 with 4 amino acid residues substituted, modified, deleted and / or added in any of the following combinations:
Staphylococcus aureus (S that has the amino acid sequence of the polypeptide
.aureus) A polynucleotide encoding a dnaI variant. Particularly desirable among these polynucleotides are polynucleotides that encode silent nucleotide mutations that do not alter the translated sequence or activity of the S. aureus DnaI polypeptide that they encode. .

A preferred embodiment is a polynucleotide encoding a polypeptide which retains substantially the same biological function or activity as the mature polypeptide encoded by the DNA of SEQ ID NO: 1. In accordance with some preferred embodiments of the present invention, there is provided a polynucleotide, such as the polynucleotide shown in Figure 1, which hybridizes to a Staphylococcus aureus dnaI polynucleotide sequence under particularly severe conditions. To do.

The polynucleotide of the present invention is useful as a hybridization probe for RNA, cDNA and chromosomal DNA in order to separate full-length cDNA and chromosomal gene having high sequence identity with the dnaI gene. Such probes generally consist of at least 15 to about 100 residues or base pairs, but such probes may rather be about 20 to 50 nucleotide residues or base pairs. Particularly desirable probes are about 20 to about 30 nucleotide residues or base pairs in length.

[0136] The translation portion of the dnaI-related gene derived from a bacterial species other than Staphylococcus aureus is a library using the DNA sequence attached to SEQ ID NO: 1 to synthesize an oligonucleotide probe. Can be separated by screening. A labeled nucleotide having a sequence complementary to the sequence of the gene of the present invention is then used to screen a library of cDNA, chromosomal DNA or mRNA to determine which clone of the library the probe hybridizes to. .

Several methods are available to obtain full length DNA or to extend short DNA, which are well known in the art. For example, these methods include RACE (Rapid Amplification of cDNA ends) (eg, Frohman, et al., P
NAS USA 85: 8998-9002, 1988)). Recent modifications of this technology have significantly simplified the search for longer cDNAs, as exemplified by the MARATHON ^™ technology (Clontech Laboratories Inc.). In the MARATHON ^™ technology, cDNA is made from mRNA extracted from selected cells and "adapter" sequences are ligated at both ends. The nucleic acid is then amplified by PCR using a combination of gene-specific and adapter-specific oligonucleotide primers to amplify the "deletion" 5 ends of the DNA.

The PCR is then repeated using the "nested" primers. This primer is designed to anneal inside the amplification product (generally, an adapter-specific primer that anneals further inside the 3 terminus of the adapter sequence,
And a gene-specific primer that anneals further inside the 5'end of the selected gene sequence). This reaction product can then be analyzed by DNA sequencing, by ligating directly to known DNA to obtain the complete sequence,
Alternatively, full-length DNA is constructed by performing a separate full-length PCR with new sequence information to design the 5-terminal primer.

The polynucleotides and polypeptides of the present invention may be used to develop therapeutic or diagnostic methods for diseases, particularly human diseases, eg, as further discussed herein in connection with polynucleotide assays, In particular, it can be used as a research reagent and a sample. The polynucleotide of the present invention, which is an oligonucleotide derived from the sequence of SEQ ID NO: 1, reacts in bacteria of infected tissues to determine whether the entire length or a part of the polynucleotide identified herein is transcribed. Useful for designing PCR primers for This means that the polynucleotides of the invention are useful in diagnosing infections by bacterial strains carrying these sequences. An array like this
It is also known to be useful in diagnosing the stage of infection and the type of infection the pathogen has acquired.

The present invention also provides a polynucleotide encoding a polypeptide that is the mature protein plus additional amino or carboxyl terminal amino acids, or amino acids within the mature protein. Such sequences may function in the processing of the protein from its precursor to its mature form, among other things, allowing protein transport, increasing or decreasing the half-life of the protein, or testing or producing it. May facilitate protein engineering for In vivo, additional amino acids are commonly cleaved from mature proteins by cellular enzymes.

A precursor protein that comprises a mature polypeptide fused to one or more prosequences may be an inactive form of that polypeptide. Such non-active precursors are generally activated when the prosequence is removed. Often some or all of the prosequences are removed prior to activation. Generally, such precursors are called proproteins.

Accordingly, the polynucleotides of the present invention have one or more mature proteins, mature proteins and leader sequences (sometimes referred to as precursor proteins), prosequences that are not leader sequences of precursor proteins. Encodes a mature protein, or a preproprotein with one or more prosequences, and a leader sequence that may be removed at the processing stage to produce the mature polypeptide that is the precursor of the proprotein and is generally active there's a possibility that.

In addition to the standard nucleotide representations of A, G, C, T / U, the term “N” may be used to indicate a particular polynucleotide of the invention. "N" is DNA or
It means that any of the four DNA or RNA nucleotides can be inserted at the position thus indicated in the RNA sequence. As an exception, it is desirable that N is not a nucleotide that, when read in the correct reading frame in combination with the positions of adjacent nucleotides, could create a false stop codon in such reading frame. Polynucleotides complementary to each and every polynucleotide of the invention are also provided.

Vectors, Host Cells and Expression Systems The present invention comprises a vector containing a plurality of polynucleotides of the invention or a single polynucleotide and genetically engineered with the vectors of the invention by recombinant techniques,
Also involved in genetically engineered host cells that produce the polypeptides of the invention. Cell-free transcription systems are also available for producing such proteins using RNA derived from the DNA construct of the present invention. The recombinant DnaI polypeptides of the present invention can be obtained from host cells equipped with genetically engineered expression systems by methods well known in the art. Consequently, as a further interpretation, the invention relates to expression systems comprising one or more dnaI polynucleotides of the invention, host cells genetically engineered by such expression systems, the invention by recombinant techniques. Involved in the production of the polypeptide.

To produce the recombinant DnaI polypeptides of the present invention, host cells can be genetically engineered to introduce expression systems or portions thereof, or polynucleotides of the present invention. Representative examples of suitable hosts include bacterial cells (gram positive and gram negative), fungal cells, insect cells, animal cells and plant cells. Polynucleotides can be introduced into bacteria using standard chemical treatment methods, such as those that elicit DNA uptake capacity by treatment with calcium chloride (Sambrook et al., Supra). If desired, the polynucleotide can be introduced into a fungal (eg, yeast) host cell by standard chemical methods, eg, transformation with lithium acetate.

A great variety of expression systems are useful for producing the DnaI polypeptides of the present invention. Such vectors include, among others, chromosome, episome, and virus-derived vectors. For example, bacterial plasmid origin, bacteriophage origin, transposon origin, yeast episome origin, insertion factor origin, yeast chromosomal factor origin,
Virus-derived vectors, and vectors derived from these combinations are useful in the present invention.

DnaI polypeptides of the present invention may be ammonium sulfate or ethanol precipitated, acid or urea extracted, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, And is recovered and purified from the recombinant cell culture medium (or cell body) by well known methods including lectin chromatography. Well-known techniques for protein refolding are available to restore the active structure when the DnaI polypeptide is denatured during the separation and / or purification process.

Diagnosis, Prognosis, Serotype, and Mutation Assays The present invention also relates to the use of the dnaI polynucleotides of the invention as diagnostic agents. Detection of the mutant polynucleotide of the invention, preferably SEQ ID NO: 1, as associated with disease or pathogenicity, will provide a diagnostic tool. The diagnostic means can define or be added to the diagnosis of diseases caused by low expression, high expression or alteration of expression of the polynucleotide, prognosis, stage determination, or diagnosis of susceptibility to disease. Microorganisms having mutations in such polynucleotides, particularly infectious microorganisms, can be detected at the polynucleotide level by a variety of techniques, such as the methods described elsewhere in this application.

The dnaI nucleotide sequences of the present invention are also useful in demonstrating the chromosome of a microorganism. This sequence is found in microbial chromosomes, especially Staphylococcus aureus (S. aureus
us) is a sequence that can specifically target a specific site on the chromosome and hybridize with them. Producing a map of the sequence of interest to a chromosome in accordance with the present invention is not only the requirement of the gene for the microorganism, but also the pathogenicity and / or ecological status of the microorganism and / or the drug resistance of the microorganism. It can also be an important step in the mutual relationships. Once a sequence is mapped to a precise chromosomal location, the physical location of that sequence on the chromosome can be correlated with genetic map data.

Such data are in online sequence databases. Next, the relationship between the gene mapped to the same chromosomal site and the disease is identified using known genetic techniques. For example, it is identified through linkage analysis (linkage of physically adjacent genes) or a mating test such as crossing. Differences in polynucleotide and / or polypeptide sequence between microorganisms exhibiting a second phenotype that differs from the first phenotype can also be determined. If there is a mutation
If some or all of the microorganisms exhibiting the first phenotype are observed but none at all in the microorganism exhibiting the second phenotype, then the mutation is probably the causative agent of the first phenotype. Conceivable.

The polypeptides and polynucleotides necessary for prognosis, diagnosis or other analysis are obtained from individual biological samples suspected of and / or infected. Whether obtained from any of these samples, in particular DNA or polynucleotides are directly available for detection, or Staphylococcus aureus dna
P using an amplification oligonucleotide primer derived from the polynucleotide sequence of I
It can also be enzymatically amplified using CR or other amplification techniques. RNA, especially m
RNA or RNA reverse transcribed into cDNA is also useful for diagnosis.

Following amplification of the S. aureus dnaI-related polynucleotide obtained from the sample, the species and strains of the infectious or occult microbial organism are associated with the relevant microorganism (eg, known Staphylococcus aureus ( S. aureus strain)) and can be characterized by analysis of one or more reference polynucleotides, or amplified polynucleotides that correlate with the sequence. Point mutations can be confirmed by hybridizing amplified DNA to known dnaI polynucleotide sequences and by detecting differences in melting temperature or reversion kinetics. Complete or significantly matching sequences can be distinguished from incomplete or less significantly matching duplexes by ribonuclease protection or S1 nuclease mapping (eg, Cotton et al.
., (1985) Proc. Natl. Acad. Sci. USA 85: 4397-4401).

The difference in the polynucleotide sequence can also be detected from the difference in the in-gel electrophoretic mobility of the polynucleotide fragment compared to the reference sequence. This is done in the presence or absence of denaturing agents. Differences in polynucleotides can also be detected by directly decoding the DNA or RNA sequences (eg, Myers et al, (1985) Science).
230, 1242). Sequence changes at specific sites are also revealed by RNase, nuclease protection assays such as V1 and S1 protection assays, or by chemical cleavage methods (see eg Cotton et al., (1988) supra). ).

In another embodiment, an array of oligonucleotide probes containing dnaI nucleotide sequences or fragments thereof can be constructed for efficient screening of, for example, genetic mutation, serotype, taxonomic classification or identification. Is. Array technology methods are well known, versatile, and available to address a variety of questions in molecular genetics, including gene expression, genetic linkage, and genetic variability (
See, eg, Chee et al., (1996) Science 274, 610).

Thus, from a different point of view, the invention comprises (a) a polynucleotide of the invention,
Desirably the nucleotide sequence of SEQ ID NO: 1 or a fragment thereof; (b) a nucleotide sequence complementary to the sequence of (a); (c) the polypeptide of the present invention, preferably the polypeptide of SEQ ID NO: 2 or a fragment thereof; Alternatively, (d) a diagnostic kit comprising an antibody against the polypeptide of the present invention, preferably an antibody against the polypeptide of SEQ ID NO: 2. Such a kit would be used, inter alia, to diagnose a disease or susceptibility to a disease.

The invention further provides a method of diagnosing a bacterial infection such as that caused by Staphylococcus aureus. This method compares a sample obtained from an individual such as a biological sample with a sample obtained from a disease-free individual, and
: 1 and confirming that the expression level of the polynucleotide having the sequence disclosed therein is increased. Elevated or decreased expression of dnaI polynucleotides is well known in the art for quantifying polynucleotides such as PCR, RT-PCR, ribonuclease protection, Northern blotting and other hybridization methods, and spectrometry. It can be measured using any one of the available methods.

In addition, diagnostic tests to detect high expression of DnaI polypeptide compared to normal control tissue samples according to the invention can be used, for example, to detect the presence or absence of infection. Test techniques that can be used to determine the level of S. aureus DnaI polypeptide in a sample obtained from a host, such as a biological sample, are well known in the art. Such assays include radioimmunoassays, binding competition assays, western blot analysis, antibody sandwich methods, antibody detection and ELISA methods.

[0158] Staphylococcus aureus (S.aureus) DNAi polynucleotide of Oyo gridding of chromosomal sequences beauty polynucleotide subtraction present invention, polynucleotide arrays, preferably utilized as a component of high density arrays or grids can do. These high density arrays are particularly useful as methods for diagnostic and prognostic purposes. For example, a set of spots, each containing a different gene, as well as one or more polynucleotides of the invention, were obtained from a biological sample to ascertain the presence of a particular polynucleotide sequence or related sequences in an individual. Alternatively, it can be used for stringent investigations such as hybridization or nucleic acid amplification using probes from biological samples.

[0159] Cells expressing the Staphylococcus aureus (S.aureus) peptide or polypeptide DnaI polypeptides and polynucleotides or a variant thereof singular antibodies present invention, or they may, like this It is useful as an antigen for producing antibodies immunospecific for a polypeptide or a polynucleotide, respectively. In one preferred embodiment of the invention, Staphylococcus aureus (S. aure
us) An antibody against a DnaI polypeptide or a polynucleotide encoding the same is provided. Antibodies to DnaI-polypeptide or dnaI-polynucleotide are
It is useful for treating infections, especially bacterial infections.

Antibodies raised against the polypeptides or polynucleotides of the invention include antigenic determinants bearing fragments of either the polypeptides and / or polynucleotides of the invention, or either or both, either or both. A cell expressing the analog of the above, or either or both of them, is preferably obtained by administering to a nonhuman animal by a predetermined method. To prepare a monoclonal antibody,
Any method known in the art to produce antibodies by serially passaged cells can be used. This is for example Kohler, G. and Milstein, C., Nature 256: 495-4.
97 (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,
Includes a variety of technologies, such as those in Inc. (1985).

To produce single chain antibodies to the polypeptides or polynucleotides of the invention, techniques for making single chain antibodies (US Pat. No. 4,946,778) are available. Also, transgenic mice, or other transgenic mammals, are useful for expressing immunospecific humanized antibodies to the polypeptides or polynucleotides of the invention.

When the antibody is administered therapeutically, it is desirable that the antibody or variant thereof be modified to be less immunogenic in the individual. For example, if the individual is human, the antibody is most preferably a "humanized" antibody, Jones et al. (1986), Nature 321, 522-525 or Tempest et al.,
(1991) Biotechnology 9, 266-273, such antibodies are "humanized" by transferring one or more complementarity determining regions of a hybridoma-derived antibody into a human monoclonal antibody. .

As another method, the phage display method is useful for selecting an antibody gene capable of binding to the DnaI polypeptide of the present invention. One approach is to use Staphylococcus aureus DnaI from an immune library of antibody genes that express fusion proteins with filamentous phage coat proteins.
It is a method of selecting an antibody fragment specific for.

Another method is to screen naive libraries using the phage display method to identify genes encoding antibodies specific for DnaI (McCafferty, et al. , (1990), Nature 348, 552-554; Marks
, et al., (1992) Biotechnology 10, 779-783; As a recent paper, Haard et al.
(1999) J Biol Chem 274: 18218-18230). Immunization is not necessary if antibodies with nanomolar affinity for various targets can be recovered. The affinity of these antibodies can also be improved, for example by chain shuffling (Clacks
on et al., (1991) Nature 352: 62).

The antibodies described above can be used to isolate or identify clones expressing a polypeptide or polynucleotide of the invention, eg, to purify the polypeptide or polynucleotide by immunoaffinity chromatography. Variants of the polypeptides or polynucleotides of the invention, such as antigenically or immunologically equivalent derivatives or fusion proteins of the polypeptides, are also provided for immunizing mice, or other animals such as rats or chickens. It is useful as an antigen.

The fusion protein imparts stability to the polypeptide, which acts as a carrier, or acts as an adjuvant, or both. Another method is to mix an immunogenic carrier protein such as bovine serum albumin, keyhole limpet hemocyanin or tetanus toxin with the antigen, eg by conjugation. Alternatively, when the antibody is administered therapeutically, a complex antigenic polypeptide comprising multiple copies of the polypeptide, or an antigenically or immunologically equivalent polypeptide thereof, enhances immunogenicity. Is considered to be sufficiently antigenic, that it is not necessary to use a carrier.

According to a feature of the invention, the invention may be used for therapeutic or prophylactic purposes, especially genetic immunity.
Provides utilization of dnaI polynucleotides. Utilization of the dnaI polynucleotides of the invention in genetic immunization is desirably accomplished by a suitable transfer method (Wolff et al., Hum Mol Genet (1992) 1: 363, Manthorpe, for example by direct injection of the plasmid into muscle.
et al., Hum. Gene Ther. (1983) 4: 419), introduction method by complex with DNA and specific protein carrier (Wu et al., J. Biol. Chem. (1989) 264: 16985). , DNA coprecipitation with calcium phosphate (Benvenisty & Reshef, PNAS USA, (1986) 83:
9551), a method of encapsulating DNA in liposomes of various shapes (Kaneda et al., S
cience (1989) 243: 375) Particle gun method (Tang et al., Nature (1992)
356: 152, Eisenbraun et al., DNA Cell Biol (1993) 12: 791) or an in vivo infection method using a cloned retovirus vector (Seeger et al., P.
NAS USA (1984) 81: 5849).

Antagonists and Agonists : Assays and Molecules The present invention is based in part on the discovery that DnaI is the target of the bacteriophage 77ORF104 inhibitory factor. Applicants have recognized the utility of their interactions in the development process of antibacterial agents. In particular, the inventor has found that 1) DnaI is an important target for bacterial inhibition; 2) 77ORF104 or a derivative or functional analogue thereof is useful for inhibiting bacterial growth; and 3) It has been recognized that the interaction between S. aureus dnaI and 77ORF104 could be used for drug screening and rational design or as a target for antibacterial drugs. In addition to the method of directly inhibiting DnaI activity, the method of inhibiting DnaI expression is also interesting in terms of antibacterial activity.

In some embodiments of the invention, compounds are identified that bind to, or otherwise interact with or inhibit or activate the activity or expression of the polypeptides and / or polynucleotides of the invention. Provide a way to. This is because in order to determine the binding or other interaction with the compound, the polypeptide and / or polynucleotide of the present invention and the compound of the present invention can be bound to each other under the condition that the compound and the polypeptide and / or the polynucleotide can bind or interact with each other. Another method that includes a method of screening for contacts, for example, such binding or interaction is capable of exhibiting a detectable signal in response to binding or interaction of a compound with a polypeptide and / or polynucleotide. It should be related to the ingredients. In addition, by detecting the presence or absence of a signal generated by the binding or interaction between the compound and the polypeptide and / or polynucleotide, the compound binds or otherwise interacts with the activity of the polypeptide and / or polynucleotide or Also included are methods of determining whether to activate or inhibit expression.

Potential antagonists include, inter alia, small organic molecules, peptides, polypeptides and antibodies, which bind to the polynucleotides and / or polypeptides of the invention so that their activity or expression Hinder or lose. Potential antagonists are also closely related proteins that bind to the same sites on the binding molecule as other binding molecules, or small organic molecules such as antibodies, peptides, polypeptides. Sometimes there is.

Potential antagonists also include small molecules that bind to the polypeptide and occupy its binding site, resulting in impeded binding of cellular binding molecules and impaired normal biological activity. Examples of small molecules include, but are not limited to, small organic molecules, peptides or peptide-like molecules. Other potential antagonists include:
Antisense molecules are also included (see below for details of these molecules; Okano, (1991
) J. Neurochem. 56, 560; OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS O
F GENE EXPRESSION, CRC Press, Boca Raton, FL (1988)).

Potentially more suitable antagonists include compounds related to 77ORF104 and DnaI and variants thereof. Examples of other potential antagonist polypeptides include antibodies, and in some cases, oligonucleotides or proteins closely related to ligands, substrates, receptors, enzymes and the like. Also optionally included are fragments of the polypeptide, eg, ligands, substrates, receptors, enzymes, etc .; or small molecules that bind to the polypeptide of the invention but do not induce a reaction and thus interfere with the activity of the polypeptide. .

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

The compound may also be derived from the polypeptide sequence of 77ORF104 itself. 77ORF1
04 Small overlapping fragments over the entire amino acid sequence of the protein or peptide fragments representing peptides are available for extensive screening. The 77ORF104 fragment can be produced by proteolytic digestion of the full length protein as described above. Alternatively, a suitable 77ORF104-derived peptide or polypeptide fragment, representative of the complete sequence of 77ORF104 protein, may be chemically synthesized. For example, in the multi-pin method, glycolate and 4- (
Peptides are simultaneously synthesized by a small amount of peptide construction on a resin pin based on (hydroxymethyl) benzoic acid (4- (hydeoxymethyl) benzoate) (Maeji et al. (1991) Pept Res , 4: 142-6).

Some polypeptides of the present invention are found in the native Staphylococcus aureus (S.
aureus) DnaI polypeptide biomimetics (biomimetics) and functional analogs. These functional analogues are useful, inter alia, for competing with the activity of S. aureus DnaI polypeptides or as antigens or immunogens according to the methods described above. Functional analogs of the polypeptides of the invention include, but are not limited to, defective polypeptides. For example, substances suitable as functionally similar substances include polypeptides that include the polypeptide sequence shown in SEQ ID NO: 2 lacking 20, 30, 40, 50, 60, 70 or 80 amino acids or carboxyl terminal residues. Be done. The polypeptide also includes fusion proteins that include one or more of these incomplete sequences. Polynucleotides encoding these functionally similar substances can be used as expression cassettes to express the respective similar polypeptides.

Screening Methods According to the Invention It is worth devising a screening method to identify compounds that enhance or inhibit the function of the DnaI polypeptides or polynucleotides of the invention. Accordingly, the invention provides screening methods to identify compounds that modify the function of the polypeptides or polynucleotides of the invention. In general, antagonists are used for therapeutic and prognostic purposes. DnaI agonists are expected to be useful, for example, to increase the growth rate of bacteria in samples cultured for diagnostic or other purposes. Screening methods can generally be divided into two broad categories: binding tests for candidate compounds and tests for functional characteristics of targets.

A) Binding Assays There are many ways to test the binding properties of a candidate compound to a target protein such as DnaI. In the present invention, by directly or indirectly binding a label to a candidate compound, a DnaI polypeptide or polynucleotide, or a cell expressing the DnaI polypeptide or a carrier thereof, or a cell expressing a fusion protein containing the DnaI polypeptide Alternatively, a screening method for measuring the binding property of the candidate compound to the carrier is useful.

This screening method may also include a method of competing for the binding of a labeled competitor such as a fragment capable of binding to 77ORF104 or DnaI. i) Phage display Phage display is a powerful assay for measuring protein: protein interactions. In this method, the protein or peptide is expressed as a fusion protein with the coat or tail proteins of filamentous phage.

A comprehensive paper on this method is Phage Display of Peptides and ProteinSalabor.
atory Manual edited by Kay et al. (1996) Academic Press. In the case of the Ff family of phages including M13 and fd, gene III and gene VIII proteins are most often used as fusion partners with foreign proteins or peptides. A phagemid is a vector that has replication initiation sites for both plasmids and bacteriophages. A phagemid encoding a fusion protein with gene III or gene VIII can be recovered from a bacterial host infected with helper phage,
As a result, the foreign sequence is displayed on the outer shell or tip of the recombinant phage.

In the simplest assay, purified recombinant DnaI protein comprising, for example, the amino acid sequence of SEQ ID NO: 16, or DnaI fragment, was immobilized onto the wells of a microtiter plate and fusion 77ORF104 with gene III protein was displayed. It may be possible to incubate with the phage. Wash to remove unbound phage,
Bound phages are detected using a monoclonal antibody against the phage coat protein (gene VIII protein).

Staining with an enzyme-conjugated secondary antibody allows for quantitative detection of bound fusion protein. Inhibitor screening is performed by incubating the compound with the immobilized target prior to adding the phage. The presence of the inhibitor clearly reduces the signal in a concentration-dependent manner compared to a control containing no inhibitor.

Ii) Surface Plasmon Resonance Another powerful assay for screening inhibitors for protein: protein interactions is surface plasmon resonance. Surface plasmon resonance is caused by a change in mass near the sensor surface caused by the binding of a first protein or other biomolecule from the aqueous phase to a second protein or biomolecule immobilized on the sensor. It is a quantitative method that measures the binding between one (or more) molecules.

This change in mass is measured as resonance units per time after injection or removal of the second protein or biomolecule and is measured using the Biacore Biosensor (Biacore AB). Covalent method (for example, amine in 10 mM sodium acetate [pH 4.5])
Sensor chip (eg, CM5 chip for research; Biaco
It is also possible to immobilize DnaI on re AB). The blinds are set up by activating and deactivating the sensor without protein immobilization.

77ORF1 to DnaI or a DnaI fragment, which comprises, for example, the amino acid sequence of SEQ ID NO: 16
Binding of 04 is measured by injecting purified 77ORF104 onto the chip surface. The measurement is performed at room temperature. The conditions of the assay (for example, the conditions under which they bind) are as follows: 25 mM HEPES-KOH (pH 7.6), 150 mM sodium chloride, 15% glycerol, 1 mM dithiothreitol, 0.001% Tween 20, flow rate. 10 μl / min. Preincubation of the sensor chip with the candidate inhibitor is expected to reduce the interaction between 77ORF104 and DnaI. Reduced binding of 77ORF104 suggests competitive binding by the candidate compound.

Iii) Fluorescence Resonance Energy Transfer (FRET) A method for measuring the inhibition of binding of two other proteins is a method using fluorescence resonance energy transfer (FRET: de Angelis, 1999, Physiological Genomics). FRET is a state in which the fluorescent donor (D) and fluorescent acceptor (A) are in close proximity (usually <
It is a quantum mechanical phenomenon between two molecules that occurs when the emission spectrum of D overlaps with the excitation spectrum of A at a distance of 100 A). In FRET, a green fluorescent protein (GFP) mutant derived from the luminescent aequorea victoria is fused with a polypeptide or protein to measure a protein-protein interaction, and used as a DA pair.

Cyan (CFP: D) and yellow (YFP: A) fluorescent proteins are each coupled with a DnaI polypeptide or DnaI fragment, such as containing the amino acid sequence of SEQ ID NO: 16, and a 77ORF104 protein, respectively. The interaction of 77ORF104 with DnaI or a DnaI fragment comprising, for example, the amino acid sequence of SEQ ID NO: 16 with 77ORF104 under sufficiently close conditions causes a decrease in the fluorescence intensity of CFP with an increase in YFP fluorescence. Addition of a candidate modifier to a mixture of appropriately labeled DnaI and 77ORF104 proteins results in the addition of a particular 77
Inhibits energy transfer, as evidenced by the decrease in YFP fluorescence at ORF104 concentration.

Iv) Fluorescence Polarization Method In addition to surface plasmon resonance and FRET methods, measurement of fluorescence polarization is useful for quantifying protein-protein binding. The degree of fluorescence polarization of fluorescently labeled molecules depends on the rotational correlation time or the inversion rate. For example, a S. aureus DnaI polypeptide or DnaI fragment comprising the amino acid sequence of SEQ ID NO: 16 is formed in association with a fluorescently labeled polypeptide (eg, 77ORF104 or a binding fragment thereof). The protein complex exhibits a higher degree of fluorescence polarization than the simple amount of fluorescently labeled protein. If a candidate inhibitor interferes with or interferes with the interaction of DnaI with its polypeptide binding partner, inclusion of this candidate inhibitor in this DnaI interaction results in a better result than the mixture without the candidate inhibitor. This causes a decrease in the degree of fluorescence polarization. This method is preferably utilized as a method for characterizing small molecules that disrupt the formation of polypeptide or protein complexes.

V) Scintillation Proximity Assays Scintillation Proximity Assays include Staphylococcus aureus DnaI polypeptides or other DnaI fragments, such as those comprising the amino acid sequence of SEQ ID NO: 16. It can be used to characterize the interactions between proteins. For this assay, a method is to covalently attach S. aureus DnaI polypeptide to beads. Addition of radiolabeled 77ORF104 will result in binding of the radioactive source molecule to a site in proximity to the scintillation solution. Thus, binding of the 77ORF104 polypeptide radiates a signal that causes Staphylococcus aureus Dna
Compounds that interfere with the action of the I polypeptide and 77ORF104
Reduce the signal.

Vi) Biosensor Assays ICS biosensors are described in AMBI (Australian Membrane Biotechno
logy Research Institute; http // www.ambri.com.au /). In this technique, for example, a DnaI or DnaI fragment containing the amino acid sequence of SEQ ID NO: 16 or a bacteriophage 77
Association of macromolecules such as ORF104 is linked to the closure of gramicidinergic ion channels in suspended dual substrates and thus correlates with changes in biosensor admittance (similar to impedence). This approach exhibits linearity across magnitudes of admittance change of 10 ⁶ and is well suited for large-scale, high-throughput screening of small molecule combinatorial libraries.

Important points to note in protein-protein interaction assays are:
That is, the substance that modifies the interaction does not always directly interact with the domain of the physically interacting protein. It is also possible that the modifier interacts at a site that is independent of the protein-protein interaction site, resulting in structural changes in, for example, the DnaI polypeptide. Modifiers that work in this way (
Inhibitors or agonists) are interesting because their alterations may induce alterations in the activity of DnaI polypeptides.

B) Assay of functional activity of DnaI i) DNA replication, tritium thymidine incorporation assay To determine the effect of expression of 77ORF104 on DNA replication of Staphylococcus aureus (S. aureus) The level of radiolabeled thymidine incorporated into is measured in the presence or absence of sodium arsenite (5 μM).
Collect the culture sample (0.5 ml) at appropriate time intervals and add 4.5 μl of labeling solution (0.2 μC
i / ml tritiated thymidine (73 Ci / mmol, NEN Life Science Products, Inc) and 7
0 pmol non-radioactive thymidine). After 15 minutes of reaction, uptake is stopped by adding a solution containing 0.2% sodium azide and 1 mM non-radioactive thymidine.

Samples are precipitated with 10% (w / v) trichloroacetic acid and filtered through a glass fiber filter (GF-C, Whatman). Results are normalized by the optical density of the culture and expressed as counts of incorporated tritium thymidine. This assay is performed in the presence of various concentrations of candidate inhibitors in place of 77ORF104 to screen for inhibitors. At least a 10-fold or less reduction in tritiated thymidine incorporation in the presence of 77ORF104 or other inhibitors is suggestive of reduced DnaI activity.

Ii) Plasmid Replication The pC194 plasmid replicates in Staphylococcus aureus by the rolling cycle method. The single-strand initiation site, sso of pC194, is involved in the synthesis of lagging DNA strands. The pADG6406 plasmid is an sso-deficient pC194 derivative. Deficiency of sso causes accumulation of plasmid single-stranded DNA. The single-stranded (ss) replication initiation site, ssiA, is located on the lagging strand of pAM1 and is a primosome assembly site. SsiA was inserted into the pADG6406 plasmid. S. aureus carrying the plasmid is grown to mid-log phase, its total DNA is extracted and analyzed by Southern hybridization using ³² P-labeled plasmid DNA as a probe. The presence of pADG6406 with ssiA is associated with a reduced ratio of ss to double stranded (ds) DNA as compared to a plasmid without ssiA.

In the assay, the S. aureus strain is harbored with the plasmid pADG6406 containing a sequence encoding the 77ORF104 or candidate inhibitory polypeptide downstream of the arsenite-inducible promoter. The ratio of ss DNA to ds of pADG6406 is measured in the presence or absence of sodium arsenite (5 μM). ss D for ds
An increase in the proportion of NA (10% or higher) suggests the effect of candidate modifiers. In another aspect, the invention provides agonists, antagonists, ligands, receptors, substrates, enzymes, etc. for the polypeptides and / or polynucleotides of the invention, as well as the production of such polypeptides and / or polynucleotides. Related to screening kits for identifying compounds that reduce or enhance.

These include, for example: (A) a polypeptide and / or polynucleotide of the present invention; (b) a recombinant cell that expresses the polypeptide and / or polynucleotide of the present invention; (c) expresses the polypeptide and / or polynucleotide of the present invention Cell membranes; (d) antibodies against the polypeptides and / or polynucleotides of the invention; these polypeptides are preferably the polypeptides of SEQ ID NO: 2, and the polynucleotides are the polynucleotides of SEQ ID NO: 1. In all such kits, (a), (b), (c) and (d) are expected to contain substantial components.

The polypeptides and / or polynucleotides of the invention are: (a) as a first instance determining the three-dimensional structure of a polypeptide and / or polynucleotide, or a complex thereof; (b) an agonist, antagonist or Inference of the reaction site, binding site or three-dimensional structure of a potential inhibitor; (c) binding site, reaction site, and /
Or synthesis of a candidate compound predicted to bind or react at a putative motif site; and (d) testing whether the candidate compound is actually an agonist, antagonist or inhibitor Based on that polypeptide and
The availability of methods for designing polynucleotide agonists, antagonists or inhibitors will be greatly appreciated by the skilled artisan.
Furthermore, since such an operation is usually an iterative operation, it is highly appreciated that it can be performed using automation or computer-controlled steps.

Each of the polynucleotide sequences provided herein can be used in the discovery and development of antibacterial compounds. Once expressed, the encoded protein can be used as a target for screening antibacterial agents. In addition, each mRNA
The protein encoded by Escherichia coli or its Shine-Delgarno or other transcription promoting sequences can be used to construct antisense sequences to control the expression of the translation sequence of interest.

The invention also provides a polypeptide of the invention, for the purpose of interfering with the initial physical interaction between a pathogen responsible for the sequelae of infection and a eukaryotic rather than mammalian host.
The use of polynucleotides, agonists or antagonists is also provided. In particular, the molecules of the invention are: bacterial adhesion, in particular Gram-positive and / or Gram-negative bacteria, to eukaryotic rather mammalian extracellular matrix proteins contacting indwelling devices, or to extracellular matrix proteins at the wound site. To prevent adhesion; to mediate tissue damage, to inhibit bacterial adhesion between eukaryotic rather than mammalian extracellular matrix proteins and bacterial DnaI proteins, and / or; implantation of indwelling devices Or could be used to inhibit the normal pathogenic progression in infections caused by other non-surgical routes. Further in accordance with another aspect of the invention there is provided a dnaI antagonist, preferably a bacteriostatic or bactericidal antagonist.

Compositions, kits and administration regimen As a further feature of the invention, dnaI for administration to cells or multicellular organisms.
Polynucleotide and / or Staphylococcus aureus
A composition comprising a DnaI polypeptide is provided. The invention provides a therapeutically effective amount of a polypeptide, such as a water-soluble polypeptide and / or polynucleotide, antagonist peptide or small molecule compound of the invention in combination with, for example, a pharmaceutically acceptable carrier or excipient. And / or polynucleotides are provided.

Such carriers include, but are not limited to, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. Administering the drug component by an effective and convenient method including, for example, topical, oral, anal, vaginal, intravenous, transperitoneal, transmuscular, transdermal, nasal or intradermal administration, among others. You can In treatment or prevention, the active substance will preferably be administered to an individual as an injectable component, preferably as an isotonic sterile diffusion solution.

Alternatively, the components may be formulated for topical use in the form of, for example, ointments, creams, lotions, eye ointments, eye drops, ear drops, mouthwashes, dip dressings or sutures and aerosols. And may include suitable and conventional additives, including, for example, preservatives, solvents to aid drug penetration, and emollients in ointments and creams. Such topical formulations may also contain compatible, commonly used carriers, such as cream or ointment bases and ethanol or oleyl alcohol for lotions.

Such carriers may make up from about 1% to about 98% by weight of the formulation; more usually they will usually comprise up to about 80% by weight of the formulation. Another means of systemic administration includes transmucosal and transdermal administration using penetrants such as bile salts or fusidic acid or other surfactants. In addition, oral administration is also possible when the polypeptides or other compounds of the invention can be formulated as enteric-coated or encapsulated formulations. Administration of these compounds is localized and / or localized in ointments, pastes, gels, and similar shapes.

For administration to mammals and especially humans, the daily dosage of the active substance will be 0.01 m
It is expected to be g / kg to 10 mg / kg, generally around 1 mg / kg. In all cases, the physician will determine the actual dosage which will be most suitable for an individual and it will vary with the age, weight and susceptibility of the particular individual. The above prescribed amounts are exemplary of the standard case. Of course, higher or lower dosages may be convenient, eg for some individuals, and such dosages are within the scope of the invention.

As used herein, the term "internal implantable device" refers to something that is surgically implanted and is an artificial device or catheter, such as a device that is inserted into an individual's body and remains there for an extended period of time. Means Such devices include, but are not limited to, artificial joints, ear valves, pacemakers, vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinary catheters, continuous ambulatory peritoneal dialysis (CAPD) catheters. The components of the present invention can be administered by injection just prior to insertion of an indwelling device to exert a systemic effect on relevant bacteria. Treatment is continued post-operatively while the device is in the body. In addition, this component can also be used in a wide range of intraoperative covers used in all surgical procedures to prevent bacterial wound infections, especially S. aureus wound infections.

Many orthopedic surgeons consider that humans with artificial joints should be considered for prophylaxis with antimicrobial agents before undergoing dental treatments that can cause bacteremia. Severe infections are a serious complication, sometimes leading to the loss of artificial joints and significant morbidity and mortality. Therefore, in such a situation, the scope of application can be extended to the use of this active substance instead of the prophylactic antibacterial substance. In addition to the treatments described above, the components of the present invention can be commonly used as wound healing agents to prevent bacterial adhesion to exposed matrix proteins in wound tissue, and as an alternative or concomitant antimicrobial prophylaxis. It can also be used prophylactically in dental care.

Alternatively, the components of the invention can be used to bathe an indwelling device immediately before insertion. The active substance concentration for bathing wounds or indwelling devices is
Desirably it will be around 1 mg / ml to 10 mg / ml. The vaccine component is conveniently in injectable form. Conventional adjuvants could be used to enhance the immune response. A suitable dosage unit for vaccination is 0.5-5 μg / kg of antigen, this dosage is preferably 1--3 at intervals of 1-3 weeks.
Administer 3 times. It is believed that, within the dosage ranges set forth herein, the components of the present invention do not exhibit toxic side effects that would render them incapable of administration to suitable individuals.

Sequence databases, sequences in tangible media, and algorithmic polynucleotide and polypeptide sequences are used to identify further homologous sequences as well as to determine two-dimensional and three-dimensional structures. Is also a valuable source of information. These approaches store sequences in a computer readable medium and use the stored data in known polymer structure programs,
It can be most easily speeded up by using it for searching a sequence database using a well-known search tool such as GCC. The polynucleotide and polypeptide sequences of the present invention are particularly useful as components of databases that are useful in search analysis as well as in sequence analysis algorithms.

[0208] The terms "polynucleotide of the invention" and "polynucleotide sequence of the invention" as used in this section, and in the claims related to this section, "Sequence Databases, Sequences in Solid Media, and Algorithms". By "is meant a detectable chemical or physical characteristic of all polynucleotides of the present invention, which may be reduced or potentially reduced or converted to a tangible medium, preferably computer readable. Saved as possible. For example, chromatographic scan data or peak data, photographic data or its scan data, mass spectroscopic data, etc. may be mentioned.

The terms “polypeptide of the invention” and “polypeptide sequence of the invention” as used in this section and the claims related to this section, entitled Databases and Algorithms, refer to all polypeptides of the invention. Means a detectable chemical or physical characteristic, which is or may be reduced, or is stored on a tangible medium, preferably in a computer-readable form. For example, chromatographic scan data or peak data, photographic data or its scan data, mass spectroscopic data, etc. may be mentioned.

The invention provides a computer readable medium having stored thereon a polypeptide sequence of the invention and / or a polynucleotide sequence of the invention. Examples of the computer-readable medium include commercially available floppy disks, tapes,
Including chips, hard disks, compact disks, and video disks,
All recording media used to store information or data.

In a preferred embodiment of the invention there is provided a computer readable medium having a portion selected from the group comprising: 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 in at least one of the aforementioned sequences comprising the sequence of SEQ ID NO: 1 or SEQ ID NO: 17; SEQ ID NO: 2 Or a set of polypeptide sequences within at least one of the aforementioned sequences comprising the sequence of SEQ ID NO: 16; a set of data corresponding to a polynucleotide sequence comprising the sequence of SEQ ID NO: 1 or SEQ ID NO: 17; A set of data corresponding to a polynucleotide sequence encoding a polypeptide sequence comprising SEQ ID NO: 2 or SEQ ID NO: 16.

All publications and references cited in this specification include, but are not limited to, patents and patent applications, and individual publications or references are hereby incorporated by reference in their entireties. The whole picture is provided here for reference so that it is clearly and individually suggested. Any patent application to the priority of this application claim is also hereby incorporated by reference in its entirety according to the above-mentioned rules for publications and references.

[0213] Examples Example 1. Identification of inhibitory ORF 104 derived from Staphylococcus aureus bacteriophage 77 Staphylococcus aureus (S. aureus) growth strain 77 (PS 77) was transformed into phage 77 (ACTT number: 27699). -B1) Used as a growth host. Phages from Swanstorm and Adams (Swanstrom et al. (1951) Proc. Soc. Exptl. Biol. & Med. 78: 37
2-375) and propagated using the agar layer method. Phage DNA is Samb
. rook et al (1989) Molecular Cloning: A Laboratory Manual, 2 nd Edition,
Prepared from phage purified by the method described in Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

The phage DNA fragment digested by sonication was blunt-ended and the pKSII vector (St
ratagene). Recombinant clones were sequenced using the ABI 377-36 automated sequencer. To confirm the reciprocal continuity of this sequence with the genome, all sites in the phage genome were sequenced at least once on two separate clones in both directions. Sequence alignment groups are generated by the software Sequencher 3.1 (GeneCode
s) was used (Fig. 2). To identify all predicted ORFs larger than 33 codons, we used the publicly available program SEQUIN, which can be downloaded from ftp://negi.nlm.nih.gov/sequin/ for phage genomic sequences (see Figure 3).

77ORF104 (SEQ ID NO: 4) was cloned from phage genomic DNA (Figure 4) into polymerase
Amplified by chain reaction (PCR). Amplification by PCR used a sense strand primer starting from the start codon after the BamHI restriction site; an antisense strand starting from the last codon (excluding the stop codon) after the SalI restriction site. The PCR product was gel purified and digested with BamHI and SalI. Next, the digested PCR product was ligated to BamHI- and SalI-digested pT vector (Fig. 7A), and Staphylococcus aureus RN4220 strain (Kreiswirth et al. (1983) Natu
re 305: 709-712). 30 μg / m for selection of recombinant clones
Luria-Bertani (LB) agar medium containing 1 kanamycin was used.

Sodium arsenite (NaAsO ₂ ) was used to induce gene expression from the ars promoter / operator. The effect of phage 77ORF expression on bacterial cell growth was then evaluated by functional tests using solid and liquid media. Figure
Induction of phage 77ORF104 expression by culturing transformants on a semisolid medium containing 5 μM sodium arsenite, as shown in 7B, resulted in cultures containing no inducer or control. Non-inhibitory ORF (phage 77 ORF
19) The growth of bacteria on the solid medium was inhibited as compared with the case where the transformant was cultured.

As shown in FIG. 7C, the culture densities of Staphylococcus aureus clones with 77ORF104, as assessed by the colony forming unit (CFU), varied with time in uninduced conditions. To increase. Similar growth rates were found in non-inhibitory ORFs (graph "non-kille
It was also observed in the transformants having "r". After 4 hours of induction, 77ORF104 expression was cytotoxic, resulting in a 0.5 log reduction in the number of CFUs compared to the number of CFUs originally present in the same culture.

Example 2 Staphylococcus aure is a target of bacteriophage 77ORF104
Identification of S. aureus protein To identify a Staphylococcus aureus protein that interacts with the bacterial growth inhibitory Staphylococcus bacteriophage 77ORF104, a GST-fused ORF104 was created and recombined. The protein was purified and used to make a GST / ORF104 affinity column. Incubating a cell extract extracted from Staphylococcus aureus cells with an affinity matrix,
Washing was performed using different detergents with increasing salt concentration, and the protein elution pattern of the washing was evaluated by SDS polyacrylamide gel electrophoresis. A protein of mass ~ 40 Kda was specifically eluted from the affinity matrix. To elucidate the identity of the interacting protein, and 77ORF1 as detailed below.
To confirm the interaction between 04 and its protein, the characteristics of the eluted protein were further analyzed.

A. Production of GST / ORF104 Recombinant Protein Bacteriophage 77ORF104 was transformed into expression vector pGEX 4T-1 (Phar
sub-cloned into macia). The gene encoding ORF104 was obtained by digesting 77pTORF104 (Fig. 7A) with BamHI and SalI. DNA containing ORF104
The fragment was gel purified by QiaQuick spin column (Qiagen) and pGEX 4T / ORF104
Was ligated into pGEX 4T-1 (previously digested with BamHI and SalI) to generate The recombinant expression vector was identified by restriction enzyme analysis of the miniprep method plasmid, a large amount of DNA was prepared using a Qiagen column, and the obtained plasmid was sequenced.

B. Purification of Fusion Protein Cells containing GST / ORF104 fusion protein were treated with 20 m / ml of cultured cells.
l GST lysis buffer (20 mM Hepes pH 7.2, 500 mM sodium chloride, 10% glycerol, 1 mM DTT, 1 mM EDTA, 1 mM benzamidine and 1 mM PMSF) was suspended and homogenized using a French press cell. The mixture was homogenized at 4 ° C. for 20 seconds 3 times using an ultrasonic crusher. Triton X-100 was added to the solution so that the final concentration was 0.1%, and the mixture was mixed for 30 minutes at 4 ° C. Dissolve the solution using a Sorval SS34 rotor at 4 ° C for 10,00
Centrifugation was performed for 30 minutes at 0 rotation.

The supernatant was applied to a 4 ml glutathione Sepharose column previously equilibrated with lysis buffer and allowed to drain by gravity. After washing the column with 10 times the column volume of lysis buffer, GST elution buffer (20 mM Hepes pH 8.0, 500 mM sodium chloride, 10% glycerol, 1 mM DTT, 0.1 mM EDTA and 25 mM reduced glutathione).
Was eluted in 1.5 ml fractions. This fraction was analyzed by 12.5% SDS-PAGE (Laemmli) and visualized by Coomassie Brilliant Blue R250 staining to evaluate the amount of eluted GST / ORF104 protein.

GST / ORF104 (12 mg) was dialyzed overnight against 20 mM Hepes pH 7.5, 150 mM sodium chloride, 10% glycerol, 1 mM DTT, 0.1 mM EDTA 2.5 mM CaCl ₂ to separate the GST domain from the ORF104 domain. To separate, use bovine thrombin to mass ratio 1:10 (
Thrombin: GST / ORF104), digested for 2.5 hours at 28 ° C. Final concentration 1 mM PMSF, 1 mM
The digestion reaction was stopped by adding a stop solution so that benzamidine and 1M sodium chloride were added. The digested protein was loaded onto a 1 ml glutathione column and 1 ml of effluent fraction was collected. This fraction was analyzed by 12% SDS-PAGE (Tricine), which fraction was
It was visualized by Coomassie Brilliant Blue R250 staining to determine if it contained bacterially expressed ORF104 without the GST tag.

C. Affinity column preparation GST and GST / ORF104 fusion proteins were loaded onto affinity chromatography buffer (ACB; 20 mM Hepes pH 7.5, 10% glycerol, 1 mM DTT, and 1 mM) containing 1 M sodium chloride. It dialyzed against EDTA overnight. The ORF104 protein obtained by thrombin digestion of GST / ORF104 was used without dialysis. Protein concentration as Bi
Cross-linked to Affigel 10 resin (Bio-Rad) at 0, 0.1, 0.5, 1.0 and 2.0 mg / ml protein / resin concentrations as determined by o-Rad protein assay. The crosslinked resin was subsequently incubated with ethanolamine and bovine serum albumin (BSA) before loading onto the column and equilibrated with ACB containing 75 mM sodium chloride.

Preparation of D. Staphylococcus aureus Extract Two extracts were prepared from Staphylococcus aureus cell pellets. One eye extract was prepared by French press cell followed by sonication and the other was prepared by lysostaphin digestion followed by sonication. The French press cell lysate contains 3 g of frozen Staphylococcus aureus (S
aureus) cells were suspended in ACB containing 500 mM sodium chloride, 1 mM PMSF and 1 mM benzamidine to prepare the cells. The suspended cells were passed through a French press cell three times, followed by sonication for three times for 20 seconds, adjusting to 0.1% Triton X-100, stirring for 30 minutes, and then using a Ti70 angle fixed Beckman rotor at 50,000 rotations. It was centrifuged for 3 hours. The cell lysis efficiency was low and the resulting lysate (7 ml) contained 2.4 mg / ml protein.

The cell pellet after lysis by the French press cell was cryogenically ground in liquid nitrogen together with a motor and a pestle using the same buffer solution. The lysate was adjusted to 0.1% Triton X-100, stirred for 30 minutes, then centrifuged at 50,000 rpm for 3 hours using a Ti70 angle fixed Beckman rotor, and the lysate containing 2.0 mg / ml protein (10 ml ) Got. SDS-P
Pool the cells by confirming that the cell lysates are similar by AGE and concentrate to 8 ml. 3
Dialysis was performed overnight using a 000 Mr dialysis membrane in an affinity chromatography buffer containing 1 mM PMSF, 1 mM benzamidine and 75 mM sodium chloride. Remove the dialyzed protein extract from the dialysis tube and use a Sorval SS34 rotor.
Centrifuge at 10,000 rpm for 1 hour, protein concentration (using Bio-Rad Protein Assay)
And salt concentration (using a conductivity meter) were checked.

The second lysate was prepared by lysostaphin digestion followed by sonication. 8 ml of Staphylococcus aureus cell pellet (2.9 g)
20 mM Hepes pH 7.5, 150 mM sodium chloride, 10% glycerol, 1 mM DTT, 1
The cells were suspended in mM PMSF, 1 mM benzamidine, and 1000 units of lysostaphin. Incubate the cell suspension for 30 minutes at 37 ° C, cool to 4 ° C, then add a final concentration of 1 mM ED.
Adjusted to TA and 500 mM sodium chloride. The lysate was sonicated on ice three times for 20 seconds.

The lysate was adjusted to 0.1% Triton X-100 and stirred for 30 minutes, then Ti70 angle fixed Beckman
It was centrifuged at 50,000 rpm for 3 hours using a rotor. The supernatant was collected and dialyzed against ACB containing 1 mM PMSF, 1 mM benzamidine and 75 mM sodium chloride overnight using a 3000 Mr dialysis membrane. Remove the dialyzed protein extract from the dialysis tube and use Sorval
Centrifuge at 10,000 rpm for 1 hour using an SS34 rotor to obtain protein concentration (Bio-Rad P
The rotein assay was used) and salt concentration (using a conductivity meter) was examined. The extract was dispensed and frozen at -70 ° C.

E. Affinity Chromatography Staphylococcus aureus extract (400 μl) at 0, 0.1, 0.
Apply to a 40 μl column containing 5, 1.0 and 2.0 mg / ml of ligand and add another 2.0
75 mM sodium chloride (400 μl) was applied to the column containing mg / ml ligand.
The column was washed with ACB (400 μl) containing 75 mM sodium chloride, followed by 1% Triton.
AC containing X-100 and 75 mM sodium chloride (160 μl), 250 mM sodium chloride
Elution was performed with B (160 μl), ACB containing 1 M sodium chloride (160 μl), and 1% SDS (160 μl). 40 μl of each eluate was separated by 12 cm 12.5% SDS-PAGE (Laemmli), and the eluted protein was visualized by silver staining.

F. Identification of Staphylococcus aureus DnaI homologs as ORF104 interacting proteins Obtained from ACB containing 75 mM Natrium chloride and 1% Triton X-100, and 1% SDS, A protein of about 38 kDa was specifically observed in the eluate of the GST / ORF104 and ORF104 (GST-removed) columns (Fig. 8-10; eluted protein is indicated by an arrow). These bands were excised from the SDS-PAGE gel and adjusted to determine trypsin-digested peptide masses by MALDI-ToF mass spectrometry (Qin, J
., et al. (1997) Anal. Chem. 69, 3995-4001). High quality mass spectra were obtained (Figure 11).

The candidate proteins found in the two eluates were found to be exactly the same from trypsin-digested peptide mass (FIG. 11). Collision-Induced Decay (CI
1351.6, 1412.5, and 1617 using Post-Source Decay (PSD) in conjunction with (D).
A tryptic digested peptide fragment spectrum with an 8 Da monoisotopic MH ⁺ mass was obtained. As a result of searching the databases in all public domains using fragment masses, the same was not detected. Next, 1412.5 monoisotopic MH ⁺
The GHVPENVTDNDR peptide sequence (SEQ ID NO: 10) was obtained from the PSD / CID spectrum obtained for the peptide with mass.

This sequence was used in a BLAST search of the S. aureus nucleotide sequence database at http://www.genome.ou.edu/staph.html. One nucleotide sequence in the +3 open reading frame, contig 981, encoded a similar amino acid sequence GHVPELYVDNNR (SEQ ID NO: 11). The results of this transient candidate compound identification were further confirmed by virtual translation of the open reading frame found in contig 981 and computational trypsin digestion.

In addition, the PSD / CID spectra for the trypsin-digested peptides with the resulting 1351.6 and 1617.8 Da monoisotopic MH ⁺ masses were found to be 1352.6221 and 1618.7928 Da monoisotopic MH ⁺ masses found in the contig 981 reading frame. It was similar to the expected PSD / CID spectrum of the tryptic peptide with. Contig 981 is a homologue of Bacillus subtilis DnaI involved in initiation-site-dependent DNA replication when the contig 981 open reading frame is compared to all other sequences in the public domain database. Understood (
42% identity, 62% similarity) (Table 1).

Confirmation of DnaI and ORF104 Interactions by G. Yeast Two Hybrids To confirm the result that the Staphylococcus aureus dnaI homolog is an interaction partner of bacteriophage 77ORF104, yeast was confirmed. The two-hybrid system evaluated the interaction in vivo.
As shown in Figure 12B, bacteriophage 77ORF104 was transformed into yeast Gal4 DNA binding domain (pGBKT7, Clontech Laboratories) or activity domain (pGADT7, Clontec).
h Laboratories) (pGBK77ORF104 and pGAD77ORF104, respectively). The polynucleotide sequence of the DnaI homologue is an oligonucleotide targeting the transcription start codon and stop codon of the dnaI gene (SEQ ID NO: 1).
By PCR using the primers, Staphylococcus aureus (S. aureus
) Obtained from chromosomal DNA.

As shown in FIG. 12A, the sense strand primer targets the start codon and forward
EcoRI restriction enzyme site (5'-gaattc-3 '); The antisense oligonucleotide targets the stop codon and has a BamHI restriction enzyme site (5'-ggatcc-3') in front. Quiag
The PCR product was purified using the en PCR purification kit and digested with EcoRI and BamHI. The digested PCR product was ligated into EcoRI- and BamHI-digested pGADT7 vector (pGADdnaI). DnaI was cloned into the pGBKT7 vector by the same method (pGBK
dnaI).

As shown in FIG. 12D, pGAD and pGBK plasmids (No. 1) -6) with different combinatorial constructions were prepared in advance with chromosomally integrated copies of E. coli LacZ and the selected HIS3 and ADE2 genes. Genetically engineered yeast strains (AH109, Clontech
Laboratories). The co-transformants were cultivated side by side on a synthetic medium (SD) for tryptophan- and leucine-deficient amino acid-added (TL minus) yeast and a synthetic medium for tryptophan-, histidine-, adenine- and leucine-deficient amino acid-added (THAL minus) yeast. .

Co-transformants with the 77ORF104 polypeptide grew only in selective THAL-minus media in the presence of DnaI (right petri dishes, numbers 5 and 6). dnaI and 77ORF1
Both of the 04 plasmids are control plasmids (pGBKT7-53 or pGAKT7-53).
Induction of the reporter HIS3 and ADE2 genes was induced by dnaI and 77ORF104 as no reporter expression was observed when introduced into host yeast with DT7-T) (Nos. 2 and 3).
It can be seen that it depends on protein interactions.

PGADT7-T and pGBKT7-53 are positive controls for protein: protein interactions (No. 1) and pCL1 is the active Gal4 transcription factor (No. 4). 7
The presence of luminescent β-galactosidase activity in the 7ORF104-DnaI cotransformants also demonstrated the interaction of dnaI and 77ORF104 (FIG. 12E: numbers 5 and 6). These results show that Staphylococcus aureus identified here.
Consistent with the interpretation that the DnaI homolog is a host-side target of bacteriophage 77ORF104.

Example 3. To identify Staphylococcus aureus (S.aureus) DNAi Staphylococcus aureus involved in the interaction with the same constant bacteriophage 77ORF104 interaction surface (S.aureus) DNAi specific domains, recombinant The DnaI protein was partially proteolyzed and used on an affinity column containing 77ORF104. Next, the DnaI partial proteolytic fragment that interacts with 77ORF104 was added to SDS-PA.
It was analyzed by GE and mass spectrometry and subsequently characterized using the yeast two hybrid assay to verify the interaction of the DnaI subfragment with 77ORF104 as detailed below.

A. Subcloning of DnaI into a Bacterial Inducible Expressable System The polymerase chain reaction (PCR) was used to amplify full-length DnaI from Staphylococcus aureus chromosomal DNA. For PCR amplification of DnaI, a sense strand primer targeting the start codon and having a BamHI restriction enzyme site (5'-ggatcc-3 ') in the front; a SalI restriction enzyme site (5'-gtcga
An antisense oligonucleotide having c-3 ') was used (SEQ ID NO: 1). The digested PCR product was purified using the Qiagen PCR purification kit and ligated to the BamHI and SalI digested pGEX-6P-1 vector (# 27-4597, Amersham Pharmacia Biotech) to obtain E. coli BL21 strain. Used for transformation.

Expression of the GST-DnaI recombinant protein was induced from the plasmid pGEX-6P-1-DnaI by adding 0.5 mM IPTG to 6 liters of culture medium (OD ⁶⁰⁰ -0.5). After expressing the protein for 3 hours at 30 ° C and collecting the cells by centrifugation, the cell pellet is -7
Stored at 0 ° C. Thaw the frozen cell pellet and add 1 mM PMSF and 1 mM benza.
Resuspend in buffer 1 containing midine (20 mM HEPES pH 7.3, 500 mM sodium chloride, 10% glycerol, 1 mM DTT, and 1 mM EDTA), lyse in a French press cell, then at 20 Ultrasonic disruption was performed 3 times per second.

The cell lysate was centrifuged at 10,000 rpm for 30 minutes at 4 ° C. The supernatant was passed through a 6 ml glutathione sepharose column equilibrated with buffer 1 and 60 ml of 1 mM PMSF and
After washing with buffer solution 1 containing 1 mM benzamidine, 6 ml fractions were eluted with buffer solution 1 containing 50 mM reduced glutathione. Fractions were analyzed by 12% SDS-PAGE and Coomassie
Visualization was performed with Brilliant Blue R-250 staining.

B. Cleavage of GST-fused DnaI and removal of GST, and partial proteolysis of DnaI Elution fraction 5 containing 7.0 mg GST-DnaI was added to buffer 2 (20 mM HEPES pH 7.5, 150 mM
It was dialyzed against sodium chloride, 10% glycerol, and 1 mM DTT) and digested with 40 units of precision proteolytic enzyme (Amersham Pharmacia Biotech) at 25 ° C for 4 hours. The digested GST-DnaI was loaded onto a 1 ml glutathione sepharose column equilibrated with buffer solution 2, and the effluent was collected and eluted with buffer solution 1 containing 25 mM reduced glutathione. Fractions were analyzed by 12% SDS-PAGE and visualized by Coomassie Brilliant Blue R-250 staining.

The effluent containing DnaI was dialyzed against buffer 2 and the proteolytic enzyme / DnaI mass ratio 1: 500.
The protein was digested and decomposed at room temperature for 2 hours in a reaction solution containing (w / w) chymotrypsin or 1:50 (w / w) endoprotease Gluc-C. The partial proteolytic products obtained by chymotrypsin and endoprotease Glu-C digestion were used for affinity chromatography. 1 mM PMSF and 1 mM benzamidine
The protein digestion / degradation reaction was stopped by adding and analyzed by SDS-PAGE (1/10 of the reaction solution was used for analysis).

[0244] The affinity chromatographic I 77ORF104 between C. immobilized 77ORF104 and DnaI proteolytic fragments were cross linked to Affigel 10 (BioRad), and the remaining active sites in ethanolamine, nonspecific in BSA The site was blocked. 1 M column
Equilibration was performed with ACB containing sodium chloride and ACB containing 100 mM sodium chloride. The partial protein digestion digest was diluted with ACB containing 100 mM sodium chloride to a final volume of 120 μl, and purified BSA was added to a final concentration of 0.1 mg / ml.

The partial proteolysis reaction was divided into three fractions, 50 μl of which were added to 77ORF104
Was cross-linked at a concentration of 2.0 mg / ml, 50 μl was passed through the column containing the ligand, and 10 μl was reserved for SDS-PAGE. Column is 10 times column volume of 100 mM
ACB with sodium chloride, 4 column volumes of 100 mM sodium chloride and 1%
The column was washed with ACB containing Triton X-100, followed by elution with 4 column volumes of ACB containing 1 M sodium chloride and 1% SDS. The effluent and eluate are trichloroacetic acid (TCA
) And washed with an equal volume of cold (-70 ° C) acetone. TCA-precipitated samples were developed on 15% SDS-PAGE and proteins were visualized by silver staining (Figure 14A).
And B).

D. Identification of DnaI Partial Proteolytic Fragments That Interact with 77ORF104 The interacting proteolytic fragments were excised, digested with trypsin and analyzed by mass spectrometry. The peptides contained within each interacting proteolytic fragment were analyzed by MALDI-ToF mass spectrometry to determine the universal DnaI site for each proteolytic peptide. The amino and carboxyl termini of the partially proteolytic fragment were determined.

E. DnaI fragment subcloning into a yeast inducible expression system.
The interaction between ORF104 and part of the DnaI polypeptide was evaluated. Two parts of the DnaI polynucleotide sequence were amplified by PCR from Staphylococcus aureus chromosomal DNA with the appropriate pair of oligonucleotides (Figure 15).
. A portion of amino acid residues 64-313 was obtained using the following oligonucleotide: sense strand (including EcoRI cloning site) 5'-ccggaattc TATAAAGATCAACAA
AAAC-3 ', SEQ ID NO: 12: antisense strand (including BamHI cloning site) 5'
-cgcggatccTCAATTGTTTCTGAAATT- 3 ', SEQ ID NO: 13

The polynucleotide sequence encoding amino acid sequence 150-313 of SEQ ID NO: 2 corresponds to nucleotides 448-942 of SEQ ID NO: 1 and is presented herein as SEQ ID NO: 17. Amino acid residues 150-313 were obtained using the following oligonucleotides: sense strand (including EcoRI cloning site) 5'-ccggaattcGCAGCAGATGATATTTGT -3 ',
SEQ ID NO: 14: antisense strand (including BamHI cloning site) 5'- cgcggat
ccTCAATTGTTTCTGAAATT -3 ', SEQ ID NO: 15. The digested PCR product was gel-purified and EcoRI-
And BamHI-digested pGADT7 prey vector and ligated and used to transform E. coli DH10β. The integrity of the clone product sequence was confirmed directly by DNA sequencing.

As shown in FIG. 16, bait and prey vectors (No. 1)
To 6) were introduced into AH109 yeast cells in different combinations. When the appropriate plasmid is introduced into host yeast, it grows on THAL-SD medium (No. 1
And 3), the DnaI portion of amino acid residues 64-313 (referred to herein as SEQ ID NO: 18) and the DnaI portion of amino acid residues 150-313 (referred to herein as SEQ ID NO: 16) Both were found to interact with bacteriophage 77ORF104. Induction of these reporter genes was induced by Dn because expression of the reporter genes was not observed upon introduction of control plasmids expressing non-interacting protein partners (pGBKLam: N # 2 and 4) or 77pGADORF13: # 6.
It depends on the interaction between the aI-related polypeptide and 77ORF104 (Figure 16A).

[0250]

[Table 1]

[0251]

[Table 2]

[0252]

[Table 3]

[Brief description of drawings]

FIG. 1 shows the nucleotide sequence (A; SEQ ID NO: 1) and amino acid sequence (B; SEQ ID NO: 2) of Staphylococcus aureus Dnal.

Figure 2 Figure 2. Staphylococcus aureus bacteriophage 7
The complete nucleotide sequence of the 7-chromosomal gene (SEQ ID NO: 3) is shown.

Figure 3: Figure 3. Staphylococcus aureus bacteriophage 77
The ORF (open reading frame) map of a chromosomal gene is shown.

FIG. 4 shows Staphylococcus aureus bacteriophage 7
7 shows the nucleotide sequence of 7ORF104 (A; SEQ ID NO: 4) and its amino acid sequence (B; SEQ ID NO: 5).

FIG. 5 shows predicted tryptic digested peptide masses confirmed by the Oklahoma University Staphylococcus aureus chromosomal gene database. This is in good agreement with the characteristics of the tryptic digested peptide of the 77ORF104 binding polypeptide.

FIG. 6 shows the alignment of the sequence of B. subtilis DnaC with the homologous sequence from Staphylococcus aureus. A)
Shows an alignment of a S. aureus derived dnac polynucleotide sequence (SEQ ID NO: 7) homologous to the B. subtilis dnac polynucleotide sequence (SEQ ID NO: 6). This Staphylococcus aureus-derived dnac polynucleotide sequence (SEQ ID NO: 7) is h
Sequences confirmed by BLAST search of the S. aureus database at ttp: //www.tigr.org with the B. subtilis dnac sequence. B) is the amino acid sequence of the predicted polypeptide encoded by the B. subtilis DnaC amino acid sequence (SEQ ID NO: 8) and the Staphylococcus aureus dnac polynucleotide sequence shown in Figure 6A. The alignment with the sequence (SEQ ID NO: 9) is shown.

FIG. 7 shows the bactericidal ability of bacteriophage 77ORF104 and Staphylococcus
The expression vector used to induce its expression in S. aureus is shown. A) 77ORF104 in Staphylococcus aureus cells
Schematic diagram of the expression vector 77pTORF104 used for inducing the expression of Escherichia coli. B) Screening results for evaluating the bactericidal ability when 77ORF104 is expressed in Staphylococcus aureus grown in a semi-solid support medium. C) This result shows the ability of 77ORF104 to be suppressed when 77ORF104 is expressed in S. aureus in a liquid medium.

FIG. 8 shows affinity chromatography of S. aureus extracts prepared by French press cell lysis and sonication using GST and ORF104 as ligands. 0, 0.1, 0.5, 1.0 and 2.0
The eluate from the affinity column containing GST and GST / ORF104 ligand at mg / ml resin concentration was developed by 12.5% SDS-PAGE. Proteins were visualized by silver staining. Microcolumn, A) ACB with 1 M sodium chloride, B) 250 mM
Elution was with sodium chloride, C) 1% Triton X-100, and D) 1% SDS. Each molecular weight marker (Mr) is approximately 100 ng. The lane labeled ACB is a 2.0 mg / ml ligand-only buffer containing ACB buffer containing 75 mM sodium chloride.
Represents the eluate from the column.

FIG. 9 shows affinity chromatography of S. aureus extract lysed by lysostaphin digestion and sonication and processed, using GST and GST / ORF104 as ligands. 0, 0.1, 0.5, 1.0 and
The eluate from the affinity column containing GST and GST / ORF104 ligand at 2.0 mg / ml resin concentration was developed by 12.5% SDS-PAGE. Micro column sequentially 1%
Triton X-100, 250 mM sodium chloride, 1 M sodium chloride ACB, and 1% S
Elution was performed with 75 mM ACB containing DS. The elution image obtained by 1% SDS is shown in the figure. Each molecular weight marker (Mr) is approximately 100 ng. The lane labeled ACB represents the eluate from a 2.0 mg / ml ligand column running only ACB buffer containing 75 mM sodium chloride. The lanes labeled C and L are shown in Figure 8, 2.0.
Consistent with the eluate from the column containing mg / ml GST and GST / ORF104. Arrow is GST /
A polypeptide that specifically interacts with ORF104 is shown.

FIG. 10 shows S. aureus extract (Lys extract) dissolved and prepared by lysostaphin digestion and sonication, and staphylo prepared by French press cell lysis and ultrasonication. Coccus aureus (
Fig. 3 shows affinity chromatography of S. aureus) extract (FP / S extract) using ORF104 (excluding GST) as a ligand. 0, 0.1, 0.5, 1.0 and 2.0 mg
Eluate from the affinity column containing ORF104 ligand at a concentration of / ml resin 12.
It was developed by 5% SDS-PAGE and the gel was stained with silver nitrate. The micro column was sequentially eluted with 75 mM ACB containing 1% Triton X-100, 250 mM sodium chloride, 1 M sodium chloride ACB and 1% SDS. The figure shows the elution image obtained by 1% SDS. Each molecular weight marker (Mr) is approximately 100 ng. The lane labeled ACB is 2.0 mg / ml running only ACB buffer containing 75 mM sodium chloride.
Represents the eluate from the ml ligand column. The arrow indicates a polypeptide that specifically interacts with ORF104.

FIG. 11: Relationship between interacting protein eluted by Triton X-100 (FIG. 8C, arrow) and interacting protein eluted by 1% SDS (FIG. 8D, arrow). The result of the trypsin digested peptide mass spectrum analysis is shown. Of note is that trypsin-digested peptides were 1351.6, 1412.5 and 1617.8.
It is to have Da monoisotopic MH ⁺ mass.

FIG. 12 shows the results of a yeast-to-hybrid analysis designed to test the interaction of S. aureus DnaI and 77ORF104. A) Structure of the pGAD dnaI yeast vector expressing the Staphylococcus aureus DnaI fused Gal4 activation domain polypeptide (GAD). B)
P expressing a phage 77 ORF104 fusion Gal4 DNA binding domain (GBK) polypeptide
GBK77ORF104 yeast vector structure. C) East-to-hybrid analysis. D)
Yeasts were co-transformed in the presence and absence of the control vector as indicated by numbers 1 to 6. pGADT7-T and pGBKT7-53 (No. 1) are positive controls for protein-protein interaction, and pCL1 (No. 4) is an active Gal4 transcription factor.
The co-transformants were cultivated side by side on a synthetic yeast medium (SD) containing no tryptophan and leucine (TL minus) and a synthetic yeast medium containing no tryptophan, histidine, adenine and leucine (THAL minus). Co-transformants expressing the 77ORF104 polypeptide grew only in selective THAL-minus medium in the presence of DnaI (numbers 5 and 6). E) Results of luminescent β-galactosidase enzyme assay using protein extracts from the same transformants (Nos. 1-6).

FIG. 13 shows that Staphylococcus by the bacteriophage 77ORF104 protein
5 shows inhibition of S. aureus DNA synthesis.

FIG. 14 shows the interaction between a partial proteolytic fragment of DnaI and ORF 104 from S. aureus bacteriophage 77. Partial proteolytic fragments generated by A) endoproteinase Glu-C or B) chymotrypsin were used for affinity chromatography using columns containing 0 or 2.0 mg / ml 77ORF104 protein. The partial proteolytic fragment that interacts with 77ORF104 and not the control column was excised for peptide mapping. Labels in each lane indicate Mr: molecular weight marker, L: loading solution, FT: effluent, 1: 1 M sodium chloride eluent, 2: 1% SDS eluent, ACB: affinity chromatography buffer. The interacting bands excised for peptide mapping are shown based on apparent molecular weight by SDS-PAGE. Bands with no interaction are shown with (-). C) List of confirmed DnaI proteolytic fragments that interact with 77ORF104. A partial proteolytic fragment that interacts with 77ORF104 was purified by reverse phase and analyzed using MARDI-ToF. The observed high molecular weight fragment was mapped to a position corresponding to the amino acid sequence of SEQ ID NO: 2. .
The minimal domain of Dnal interacting with 77ORF104 determined by partial proteolysis with chymotrypsin is amino acids 131 to 313 of SEQ ID NO: 2, also the minimal domain determined by endoproteinase Glu-C is SEQ ID NO: 2. 2 amino acids
119 to 313.

FIG. 15 shows the amino acid sequence of the DnaI fragment tested for interaction with 77ORF104 in the yeast two hybrid system. SEQ ID NO: 16 is SEQ ID NO:
2 amino acids 150 to 313, SEQ ID NO: 17 is nucleotide 448 of SEQ ID NO: 1.
To 942 matching nucleotides. SEQ ID NO: 18 comprises amino acids 64-313 of SEQ ID NO: 2.

FIG. 16 shows the results of a yeast-to-hybrid analysis designed to test the interaction between the DnaI fragment and 77ORF104. The Staphylococcus aureus DnaI fragment was cloned into the pGADT7 vector. Number for yeast
Cotransformation was performed with the plasmids indicated in 1) to 6). pGBKLam and 77pGADO
RF13 is a control vector that expresses a protein that does not interact.
The cotransformants were cultivated side by side on THAL-SD medium and TL-SD medium. Cotransformants expressing 77ORF104 were selected THAL in the presence of DnaI or DnaI fragments.
Proliferated only in minus medium (numbers 1, 3 and 5). D) interacts with 77ORF104
A description of the DnaI fragment. The minimal domain of DnaI that interacts with 77ORF104 as determined by yeast two hybrid analysis is amino acids 150-313.

[Procedure amendment]

[Submission date] June 27, 2002 (2002.27)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0104

[Correction method] Change

[Contents of correction]

How to identify the Staphylococcus aureus dnaI sequence
Or: Using the methodology detailed in Examples 1 and 2, Staphylococcus aureus specifically binds to the bacterial growth inhibitory 77 phage ORF104 protein.
reus) polypeptide was isolated. Briefly, 77ORF104 protein was used as a ligand in the affinity chromatography binding step with S. aureus protein extract. Selected Staphylococcus aureus interacting polypeptides were purified and further analyzed by trypsin digestion and mass spectrometry. http://www.genome.ou.edu/staph.h
A BLAST search for S. aureus nucleotide sequences in the S. aureus genomic database of the University of Oklahoma S. aureus, located in tml, uses a trypsin of the S. aureus polypeptide for a BLAST search. The digested peptide sequence, GHVPENVTDNDR (SEQ ID NO: 19) was used.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0230

[Correction method] Change

[Contents of correction]

The candidate proteins found in the two eluates were found to be exactly the same from trypsin-digested peptide mass (FIG. 11). Collision-Induced Decay (CI
1351.6, 1412.5, and 1617 using Post-Source Decay (PSD) in conjunction with (D).
A tryptic digested peptide fragment spectrum with an 8 Da monoisotopic MH ⁺ mass was obtained. As a result of searching the databases in all public domains using fragment masses, the same was not detected. Next, 1412.5 monoisotopic MH ⁺
From the PSD / CID spectrum obtained for the peptide with mass, it was interpreted that the GHVPENVTDNDR peptide sequence (SEQ ID NO: 19) was obtained.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C07K 14/31 C12Q 1/02 4C084 C12P 21/02 1/18 4C085 C12Q 1/02 1/68 A 4H045 1 / 18 1/70 1/68 G01N 33/15 Z 1/70 33/50 Z G01N 33/15 33/53 D 33/50 M 33/53 33/543 595 33/566 33/543 595 33/569 E 33/566 A61K 39/395 N 33/569 48/00 // A61K 39/395 C12N 15/00 ZNAA 48/00 A61K 37/02 (81) Designated countries EP (AT, BE, CH, CY, DE, DK) , ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML) , MR, NE, SN, TD, T ), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM) , AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV , MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Dubow, Michael Kana Country, Quebec H3 x2 K8, Montreal, Coolbrook 4901 F-term (reference) 2G045 AA25 AA28 AA29 AA40 CB17 CB21 DA12 DA13 DA14 DA36 DA77 FA11 FB02 FB03 FB07 FB08 FB12 4A024 AA01 AA13 BA80 CA03 DA06 GA11 CA06 DA06 GA06 DA06 GA06 DA06 QA01 QA18 QQ05 QQ06 QQ20 QQ42 QR32 QR38 QR48 QR55 QR74 QR75 QR82 QS25 QS34 QS39 QX01 QX07 4B064 AG01 CA19 CC24 DA02 4C057 BB02 CC01 DD01 MM02 MM04 4C08 A20 A31 A45A32 A45B31 CA32 A14A025A15A03 A4B35 CA04 CA53 CA04 CA53 CA04 CA53 4 FA74

Claims (52)

[Claims] 1. A method of identifying a compound active against a polypeptide comprising the amino acid sequence of SEQ ID NO: 16, wherein a candidate compound is contacted with said polypeptide and said candidate compound is added to said polypeptide. Detecting the binding of the compound, wherein detecting the binding indicates that the compound is active against the polypeptide. 2. The method of claim 1, wherein said detecting comprises measuring the binding of a candidate compound to said polypeptide, wherein said compound is directly or indirectly detectably labeled. The method described. 3. The method of claim 1, wherein the detecting comprises measuring by phage display. 4. The method of claim 1, wherein the detecting comprises measuring by surface plasmon resonance. 5. The method of claim 1, wherein the detecting comprises measuring by FRET. 6. The method of claim 1, wherein said detecting comprises measuring by fluorescence polarization change. 7. The method of claim 1, wherein said detecting comprises measurement by scintillation proximity assay. 8. The method of claim 1, wherein the detecting comprises measuring by a biosensor assay. 9. The method of claim 1, wherein the compound is selected from the group consisting of small molecules, peptidomimetics, and fragments or derivatives of bacteriophage inhibitory proteins. 10. The method according to claim 1, wherein the active compound is a peptide synthesized and purified by a recombinant expression system or artificially synthesized. 11. A method of identifying a compound active against a polypeptide comprising the amino acid sequence of SEQ ID NO: 16, comprising contacting the first and second polypeptides in the presence and absence of a candidate compound. Wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 16, or a fragment or variant thereof that specifically binds to phage 77ORF104, and the second polypeptide is phage 77ORF104, or SEQ ID NO: 16. Of said domain, which specifically binds to said polypeptide, and comprises the step of detecting the binding of said first and second polypeptides to each other, wherein said binding is carried out in the absence of said candidate compound as described above. When the binding of the first and second polypeptides is reduced in the presence of the candidate compound, the candidate compound is compared with the polypeptide containing the amino acid sequence of SEQ ID NO: 16. Is identified as an active compound. 12. The method of claim 11, wherein the first or second polypeptide is detectably labeled, either directly or indirectly. 13. The method of claim 11, wherein the detecting step comprises measurement by phage display. 14. The method according to claim 11, wherein the detecting step includes measurement by surface plasmon resonance. 15. The method according to claim 11, wherein the detecting step comprises measurement by FRET. 16. The method of claim 11, wherein said detecting step comprises measurement by fluorescence polarization change. 17. The scintillation proximity
The method according to claim 11, which comprises measurement by an assay. 18. The method of claim 11, wherein the detecting step comprises measurement by a biosensor assay. 19. The compound is selected from the group consisting of small molecules, peptidomimetics, and fragments or derivatives of bacteriophage inhibitor proteins.
The method of claim 11. 20. The method of claim 11, wherein the compound is a peptide that has been synthesized and purified by a recombinant expression system or is artificially synthesized. 21. An agent or antagonist for the activity of a DnaI polypeptide or a gene encoding the polypeptide. 22. A method of identifying a compound active against a DnaI polypeptide, comprising contacting a cell expressing a polypeptide comprising SEQ ID NO: 16 with a candidate compound,
And detecting the DnaI activity in the cells, wherein the decrease in activity compared to the DnaI activity in cells not in contact with the candidate compound indicates that the candidate compound is active against the DnaI polypeptide. A method characterized by: 23. A method for producing an antibacterial compound, which comprises detecting whether or not the candidate compound is active against a polypeptide containing the amino acid sequence of SEQ ID NO: 16 or a gene encoding the polypeptide; And synthesizing or purifying said candidate compound in an amount sufficient to exhibit a therapeutic effect when the polypeptide comprising the amino acid sequence of SEQ ID NO: 16 is administered to an organism infected with a naturally occurring bacterium. A method consisting of. 24. The method of claim 23, wherein the antibacterial compound is selected from the group consisting of small molecules, peptidomimetics, and fragments or derivatives of bacteriophage inhibitor proteins. 25. The method of claim 23, wherein the antibacterial compound is a peptide that has been synthesized and purified by a recombinant expression system or is artificially synthesized. 26. A method for inhibiting a bacterium, which comprises contacting the bacterium with a polypeptide comprising the amino acid sequence of SEQ ID NO: 16 or a compound active against a gene encoding said polypeptide. How to include. 27. The method of claim 26, wherein said contacting is performed in vivo. 28. The method of claim 26, wherein said contacting occurs in vivo in an animal. 29. The method of claim 26, wherein said compound is selected from the group consisting of small molecules, peptidomimetics, and fragments or derivatives of bacteriophage inhibitor proteins. 30. The method of claim 26, wherein said compound is a peptide synthesized and purified by a recombinant expression system, or artificially synthesized. 31. A method of treating a bacterial infection in an animal suffering from an infection, which comprises a compound active against a polypeptide comprising the amino acid sequence of SEQ ID NO: 16 or a gene encoding the polypeptide. , Administering to the animal in an amount sufficient to exert a therapeutic effect. 32. The method of claim 31, wherein the compound is selected from the group consisting of small molecules, peptidomimetics, and fragments or derivatives of bacteriophage inhibitor proteins. 33. The method of claim 31, wherein said compound is a peptide synthesized and purified by a recombinant expression system or artificially synthesized. 34. A bacterial infection, which comprises contacting an indwelling device with a compound active against the polypeptide comprising the amino acid sequence of SEQ ID NO: 16 before implanting the indwelling device in a mammal. A method of preventive measures to prevent
Such contact causes Staphylococcus aureus (S.au) at the implantation site.
reus) A method characterized by being sufficient to prevent infection. 35. A method of prophylactic treatment for preventing infection of an animal by a bacterium, comprising Staphylococcus aureus comprising the amino acid sequence of SEQ ID NO: 16.
reus) A method comprising administering to an animal a compound active against a DnaI polypeptide or a gene encoding the polypeptide in an amount sufficient to reduce the adsorption of bacteria on the surface of the animal tissue. . 36. Staphylococcus aureu
s) the method of diagnosing an individual infected with the method, which comprises determining the presence of a polypeptide comprising the amino acid sequence of SEQ ID NO: 16 in the individual, wherein the presence of said polypeptide is Staphylococcus aureus ( S. aureus) A method of diagnosing an infection. 37. The determining step comprises contacting the biological sample from the individual with an antibody specific for an antigenic determinant present on a polypeptide comprising the amino acid sequence of SEQ ID NO: 16. 37. The method of claim 36. 38. A method for diagnosing an individual infected with Staphylococcus aureus, which comprises a method for confirming the presence of a nucleic acid sequence encoding a polypeptide containing the amino acid sequence of SEQ ID NO: 16 in an individual body. . 39. The step of determining, wherein the nucleic acid sample of the individual is SEQ ID NO:
39. The method of claim 38, comprising contacting the sequence of 1 with a nucleic acid probe that is hybridized under stringent hybridization conditions, at least 15 nucleotides in length, purified, enriched, or a complementary strand probe thereof. . 40. An isolated, purified or enriched polynucleotide comprising a polynucleotide sequence that exhibits at least 55% identity to the sequence of SEQ ID NO: 1, or a complementary sequence to said polynucleotide sequence. 41. An isolated, purified or enriched polynucleotide comprising a sequence encoding the amino acid sequence of SEQ ID NO: 16, or a complementary sequence to said polynucleotide sequence. 42. An isolated, purified or enriched polynucleotide comprising SEQ ID NO: 17, or the complementary sequence of said polynucleotide sequence. 43. An isolated, purified or enriched polynucleotide consisting of the sequence of SEQ ID NO: 17. 44. A isolated, purified or concentrated polypeptide which exhibits at least 55% identity to the amino acid sequence of SEQ ID NO: 16. 45. An isolated, purified or enriched polypeptide of at least 50 amino acids in length which exhibits at least 50% identity to the amino acid sequence of SEQ ID NO: 16. 46. An isolated, purified or enriched polypeptide which exhibits at least 70% similarity to the amino acid sequence of SEQ ID NO: 16. 47. An isolated, purified or enriched polypeptide of at least 20 amino acids in length which exhibits at least 60% similarity to the amino acid sequence of SEQ ID NO: 16. 48. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 16. 49. An isolated polypeptide consisting of the amino acid sequence of SEQ ID NO: 16. 50. An antibody specific for the isolated, purified or concentrated polypeptide of SEQ ID NO: 16. 51. A bacteriophage 77 ORF104 polypeptide and a phage 77.
A composition comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 16 or a variant thereof which specifically binds to an ORF104 polypeptide. 52. A composition comprising a nucleic acid encoding a nucleic acid comprising bacteriophage 77ORF104 and SEQ ID NO: 17.