JP6045025B2 - Antimicrobial screening method - Google Patents

Description

  The present invention relates to a technique for finding a novel antibacterial agent.

  Numerous bacteria such as Staphylococcus aureus are known as pathogens causing various infectious diseases in the city and hospitals. Many antibacterial agents have been marketed so far, but the number of strains that have acquired resistance to various antibacterial agents, as represented by methicillin-resistant Staphylococcus aureus (MRSA), is increasing. Since nosocomial infections caused by these resistant bacteria often become serious, they are a serious clinical problem. Therefore, it is desired to find a drug whose action mechanism is different from existing antibacterial drugs and the like.

  E. coli FtsW is a 10-transmembrane protein and is known to be essential for the cell division mechanism (Non-patent Document 1).

  E. E. coli FtsQ (divlB) is a single transmembrane protein and is known to be involved in cell division. It has been reported that FtsQ binds to FtsW, FtsL, FtsB, or FtsK (Non-patent Documents 2 and 3).

  E. E. coli FtsK (spoIIIE) is a four-transmembrane protein and a DNA translocase involved in chromosome distribution. FtsK is known to interact with FtsI, FtsL, FtsQ, or FtsZ (Non-patent Document 3).

  However, an antibacterial screening method using FtsW, FtsQ or FtsK is not known.

FEMS Microbiology Letters, Volume 216, Issue 1, 23-32, 2002 Molecular Microbiology, Vol 67, 1143-1155, 2008 Journal of Bacteriology, vol. 188, no. 1, 19-27, 2006

  An object of the present invention is to provide a novel technique for finding a novel antibacterial agent.

The present inventors have paid attention to stalobacin having a wide antibacterial spectrum and strong antibacterial activity, and conducted research.
Starovacin is a Pseudomonas sp. It is a natural compound derived from PBJ-5360 (Japanese Patent No. 2614920, Japanese Patent No. 3542150, Japanese Patent No. 3568978). It is known to have a broad antibacterial spectrum and strong antibacterial activity. If the present inventors can elucidate the mechanism of action of stalovacin, it is possible to screen for an antibacterial agent having the same mechanism of action as stalovacin. Like stalobacin, the substance has a broad spectrum and strong antibacterial activity. I thought that it would be possible to obtain As a result, it was found that FtsW is a target protein of stalobacin. Furthermore, in particular, it has been found that the 9/10 loop of FtsW is important for binding to stalobacin.
Moreover, it discovered that the complex formation of FtsW, FtsQ, or FtsK was inhibited by stalobacin.
Furthermore, it discovered that FtsW and FtsQ were essential genes in S. aureus.
From the above, FtsW, FtsQ, FtsK, and their partial peptides can be used to screen for antibacterial agents with a novel mechanism of action.
In addition, since FtsW is strongly associated with the strong antibacterial activity of stalovacin, a protein that binds to FtsW can be searched using stalovacin. The found proteins can be used for screening antibacterial agents with a novel mechanism of action.

That is, the present invention relates to the following.
(1-1) A method for screening an antibacterial agent, comprising the steps of bringing FtsW or a partial peptide thereof into contact with a test substance, and selecting a test substance that binds to the FtsW or a partial peptide thereof.
(1-2) contacting one or more polypeptides selected from the group consisting of FtsQ, FtsK and their partial peptides in the presence of a test substance with FtsW or a partial peptide thereof; and A method for screening an antibacterial agent, comprising a step of selecting a test substance that inhibits binding to FtsW or a partial peptide thereof.
(1-3) The screening method according to (1-1) or (1-2), wherein the partial peptide of FtsW is a peptide containing a 9/10 loop.
(1-4) The screening method according to any one of (1-1) to (1-3), wherein the FtsW, FtsQ, and FtsK are polypeptides derived from Staphylococcus aureus.
(1-5) A kit for screening an antibacterial agent, comprising one or more polypeptides selected from the group consisting of FtsW, FtsQ, FtsK and partial peptides thereof.
The present invention further includes the following.
(1-6) The screening method according to (1-1) or (1-2), wherein the partial peptide of FtsW is a peptide containing a 1/2 loop.
(1-7) The screening method according to any one of (1-1) to (1-4), wherein the contact is performed in the presence of stalovacin or an anti-FtsW antibody.
(1-8) A kit for screening an antibacterial agent, which contains an anti-FtsW antibody or stalobacin.

The present invention also includes the following.
(2-1) A screening method for an antibacterial agent, comprising a step of searching for a gene encoding FtsW of S. aureus or a substance that suppresses the expression of FtsW.
(2-2) contacting the test substance with a cell capable of expressing a gene encoding FtsW of Staphylococcus aureus,
A step of measuring the expression level of the gene in a cell contacted with a test substance, and selecting a test substance whose measurement level is smaller than the expression level of the gene in a control cell not contacting the test substance The screening method of (2-1) description including the process to carry out.
(2-3) a step of contacting a test substance with a cell capable of producing S. aureus FtsW or a cell fraction prepared from the cell,
A step of measuring the expression level of the FtsW of the cell contacted with the test substance or the cell fraction, and the amount of measurement comprising the expression level of the FtsW of the control cell not contacting the test substance or the cell fraction The screening method according to (2-1), comprising a step of selecting a small amount of test substance in comparison.
(2-4) A kit for screening an antibacterial agent, comprising a cell capable of expressing a gene encoding FtsW of S. aureus, a cell capable of producing FtsW of S. aureus, or a cell fraction prepared from the cell.

(3-1) A screening method for an antibacterial agent, comprising a step of searching for a gene encoding FtsQ of S. aureus or a substance that suppresses the expression of FtsQ.
(3-2) contacting a test substance with a cell capable of expressing a gene encoding FtsQ of Staphylococcus aureus,
A step of measuring the expression level of the gene in a cell contacted with a test substance, and selecting a test substance whose measurement level is smaller than the expression level of the gene in a control cell not contacting the test substance The screening method according to (3-1), comprising the step of:
(3-3) A step of contacting a test substance with cells capable of producing S. aureus FtsQ or a cell fraction prepared from the cells,
A step of measuring the expression level of the FtsQ in a cell contacted with a test substance or a fraction of the cell; and the measurement amount includes an expression level of the FtsQ in a control cell not contacting the test substance or the cell fraction. The screening method according to (3-1), comprising a step of selecting a small amount of test substance in comparison.
(3-4) A kit for screening an antibacterial agent, comprising a cell capable of expressing a gene encoding FtsQ of S. aureus, a cell capable of producing FtsQ of S. aureus, or a cell fraction prepared from the cell.

Further, the present invention includes the following.
(4-1) In the presence of the test substance,
A method for screening an FtsW binding protein, comprising the steps of contacting stalovacin with FtsW or a partial peptide thereof, and selecting a test substance that inhibits the binding of stalovacin and FtsW or a partial peptide thereof.
(4-2) The screening method according to (4-1), wherein the partial peptide of FtsW is a peptide containing a 1/2 loop or a 9/10 loop.
(4-3) The screening method according to (4-1) or (4-2), wherein the FtsW is a polypeptide derived from S. aureus.

  By the screening method of the present invention, an antibacterial agent having a novel action mechanism can be screened. By this method, a novel antibacterial agent having a broad spectrum and strong antibacterial activity can be obtained.

Lane 1 from the left side of electrophoresis image of stalovacin binding protein. Molecular weight marker, lane 2. 2. Protein purified with carrier immobilized Mock's compound, Lane 3. 3. Protein purified with a carrier on which stalovacin is immobilized, Lane 4. A protein purified on a carrier on which stalovacin is immobilized in the presence of free stalobacin. The MSMS spectrum horizontal axis of trypsin digested 35 kDa protein represents m / z, and the vertical axis represents ionic strength. The spectrum pattern which carried out MSMS of the peptide of m / z 915.9 (bivalent) obtained by digesting 35kDa protein with a trypsin. Cell-free synthesis SA0962 stalovacin binding evaluation Lane 1 from the left. Conditions under which the binding protein was purified with a carrier on which a Mock compound was immobilized, Lane 2. 2. Conditions for purifying the binding protein with a carrier on which stalovacin is immobilized, Lane 3. Conditions for purifying a protein that binds to a carrier on which stalovacin is immobilized in the presence of free stalobacin. Observation of morphological change of Staphylococcus aureus in the presence of stalovacin The photograph on the right side shows an inoculated slant medium dish. In the middle is a paper disc for antibiotic testing, a transparent area indicates that the bacteria are killed, and a translucent area indicates that the bacteria are alive. The six types of photos on the left are magnified forms of the bacteria present at the border. Of these six types, the left lane has no stalovacin and the right lane has stalovacin. Observation of morphological change using SA0962 temperature-sensitive strain The left lane is a condition in which SA0962 is not induced and expressed without xylose. The right lane shows conditions for inducing SA0962 expression with xylose. The upper row is a wild strain, and the lower row is a strain capable of inducing and expressing SA0962 in a SA0962 temperature sensitive strain. Staphylococcus aureus sensitivity analysis The horizontal lane shows the concentration of stalovacin. The vertical lane indicates the conditions of the strain. From the top, the wild strain, the SA0962 temperature-sensitive strain, the strain in which an empty vector is introduced into the SA0962 temperature-sensitive strain, and the SA0962 temperature-sensitive strain are capable of inducing and expressing SA0962. In the lowest condition, SA0962 is induced and expressed by xylose. Experiments on the inhibition of binding between SA0962 and stalobacin by the SA0962 antibody The horizontal axis represents the antibody concentration, and the vertical axis represents the binding inhibition rate. The rhombus is 11E8, the white triangle is 23C7, the American mark is 11B11, the black triangle control antibody. Amino acid sequence of variable region in SA0962 antibody (11E8) Amino acid sequence of variable region in SA0962 antibody (23C7) Amino acid sequence of variable region in SA0962 antibody (11B11) Experiments on the binding inhibition of SA0962 and stalobacin with synthetic peptides The horizontal axis represents competitive inhibition conditions, and the vertical axis represents binding activity. Quantitative results of SA0962 binding protein in the presence of stalovacin The horizontal axis represents the presence / absence of stalovacin, and the peak area on the vertical axis represents the protein expression level. The quantitative values of SA0962, FtsQ, and FtsK are shown from the top. The binding experiment none of SA0962 and SA1027 shows the condition in the absence of FtsQ, and FtsQ shows the condition in the presence of FtsQ. Furthermore, SA0962 is detected under the condition with / without stalobacin. Growth experiment of SA1027 gene expression-regulated strain under non-xylose addition conditions Conditions where SA1027 expression-regulated strain is not induced to express SA1027. The horizontal axis indicates the culture time, and the vertical axis indicates the amount of cells. Growth experiment of SA1027 gene expression-regulated strain under xylose addition conditions Conditions under which SA1027 expression is induced in the SA1027 gene expression-regulated strain. The horizontal axis indicates the culture time, and the vertical axis indicates the amount of cells.

The terms used in the present specification are used in the meaning normally used in the art unless otherwise specified.
In the present invention, unless otherwise specified, gene recombination techniques, recombinant protein production techniques in cells, separation and purification methods for expressed proteins, analysis methods, molecular biological techniques, immunological techniques, etc. are known. The method is adopted. For example, Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press (2001), Current Protocols in Molecular Biology, 200

  The “antibacterial agent” means a compound that functions to suppress bacterial growth or kill bacteria. Examples of bacteria include gram-positive bacteria (staphylococci, streptococci, pneumococci, enterococci, diphtheria, tuberculosis, leprosy, anthrax, Bacillus subtilis, Clostridium perfringens, tetanus, botulinum, etc.) And the like (eg, Neisseria gonorrhoeae, Neisseria meningitidis, Salmonella, Escherichia coli, Pseudomonas aeruginosa, Shigella, Haemophilus influenzae, Bordetella pertussis, Vibrio parahaemolyticus, Vibrio parahaemolyticus, Acinetobacter, Campylobacter, Legionella, Helicobacter, etc.). The bacteria that are the targets of the antibacterial agent obtained by the screening method of the present invention are not particularly limited. The antibacterial agent is expected to be a substance having a wide antibacterial spectrum and strong antibacterial activity, such as stalobacin.

  “Starovacin” refers to Pseudomonas sp. It is a natural compound derived from PBJ-5360, and nine types of compounds from A to I have been identified. Details of the compounds and methods for obtaining the compounds are described in Japanese Patent No. 2614920, Japanese Patent No. 3542150, and Japanese Patent No. 3568978. Preferably, it is stalobacin I described in Japanese Patent No. 3568978.

“FtsW” is a 10-transmembrane protein possessed by bacteria. It may be a protein having an amino acid sequence derived from bacteria, or a derivative having a function equivalent to that of the protein. Examples of bacteria include Escherichia coli, M. tuberculosis, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus anthracis, Streptococcus (Nat Rev Drug Discov., 7 (4), 324-38, 2008).
As FtsW used in the present invention, S. aureus FtsW is particularly preferable. S. aureus FtsW is sometimes referred to as SA0962. Specifically, it is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 48 or a derivative thereof.

The “FtsW partial peptide” is a fragment of the FtsW. 5 or more consecutive amino acid residues in the amino acid sequence of FtsW, for example, 5, 8, 10, 12, 15, 20, 25, 30, 50, 100 A polypeptide consisting of an amino acid sequence consisting of, etc. Particularly preferred is a fragment of FtsW of Staphylococcus aureus.
FtsW is a 10-fold transmembrane protein, and it is known that there are five extracellular loops from the N-terminal side. Each loop is defined as 1/2 loop, 3/4 loop, 5/6 loop, 7/8 loop, and 9/10 loop from the N end side. The position of the extracellular loop on the amino acid sequence of FtsW of each bacterium is determined by a known protein structure prediction method (for example, SOSUI, Internet <URL: http://bp.nuap.nagoya-u.ac.jp/sosui/ >).
As a partial peptide of FtsW used in the present invention, a peptide containing an extracellular loop of S. aureus FtsW (1/2 loop SEQ ID NO: 6, 3/4 loop SEQ ID NO: 20, 5/6 loop SEQ ID NO: 21, 7/8 loop SEQ ID NO: 22, 9/10 loop SEQ ID NO: 23). More preferred is a peptide containing 1/2 loop (SEQ ID NO: 6) or 9/10 loop (SEQ ID NO: 23) of FtsW of S. aureus. Particularly preferred is a peptide comprising the amino acid sequence of SEQ ID NO: 24, a peptide comprising the amino acid sequence of SEQ ID NO: 13, for example, a peptide comprising the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 13.

“FtsQ” is a single-transmembrane protein possessed by bacteria. It may be a protein having an amino acid sequence derived from bacteria, or a derivative having a function equivalent to that of the protein. Examples of bacteria include Escherichia coli, Mycobacterium tuberculosis, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus anthracis, and Streptococcus.
As FtsQ used in the present invention, S. aureus FtsQ is particularly preferable. S. aureus FtsQ is sometimes referred to as SA1027. Specifically, it is a polypeptide consisting of SEQ ID NO: 25 (GenBank: NP — 374300) or a derivative thereof.

  The “FtsQ partial peptide” is a fragment of the FtsQ. 5 or more consecutive amino acid residues in the amino acid sequence of FtsQ, for example, 5, 8, 10, 12, 15, 20, 25, 30, 50, 100 A polypeptide consisting of an amino acid sequence consisting of, etc. Particularly preferred is a fragment of S. aureus FtsQ.

“FtsK” is a four-transmembrane protein possessed by bacteria. It may be a protein having an amino acid sequence derived from bacteria, or a derivative having a function equivalent to that of the protein. Examples of bacteria include Escherichia coli, Mycobacterium tuberculosis, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus anthracis, and Streptococcus.
As FtsK used in the present invention, S. aureus FtsK is particularly preferable. S. aureus FtsK is sometimes referred to as SA1119. Specifically, it is a polypeptide consisting of SEQ ID NO: 26 (GenBank: NP — 374392) or a derivative thereof.

    The “FtsK partial peptide” is a fragment of the above FtsK. 5 or more consecutive amino acid residues in the amino acid sequence of FtsK, for example, 5, 8, 10, 12, 15, 20, 25, 30, 50, 100 A polypeptide consisting of an amino acid sequence consisting of, etc. Particularly preferred is a fragment of FtsK of Staphylococcus aureus.

Examples of the “derivative having an equivalent function of a protein” include a polypeptide comprising “an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added” in the amino acid sequence of the protein. “One or several amino acids” means 1 to 5, preferably 1 to 3, more preferably 1 or 2 amino acids. The polypeptide can be used in the present invention as long as it has a function equivalent to that of a bacterium-derived protein even by deletion, substitution, insertion or addition.
For example, as a result of intensive studies, the present inventors have found that S. aureus FtsW and FtsQ are essential genes for survival. Therefore, the derivatives have the same function as FtsW or FtsQ of S. aureus. In order to confirm whether or not it has, it is sufficient that the derivative complements the growth function in the complementation test of the bacterial growth function.
The complementation test of the bacterial growth function is performed by introducing a gene encoding the derivative into a temperature-sensitive mutant of FtsW or FtsQ by a conventional gene introduction method, and transforming the obtained transformant with wild-type S. aureus. Can grow by culturing under conditions where the temperature-sensitive mutant cannot grow. Here, when the transformant grows, the derivative can be used in the present invention.

“Anti-FtsW antibodies” include antibodies that bind to FtsW and fragments thereof. As long as it has an ability to specifically bind to FtsW or a partial peptide thereof, it may be a polyclonal antibody or a monoclonal antibody. The antibody can be obtained by a known antibody production method. Further, after purification of the obtained antibody, treatment with a peptidase or the like yields an antibody fragment. Furthermore, derivatives of the above-mentioned antibodies, for example, chimeric antibodies, humanized antibodies, Fab fragments, single chain antibodies and the like, and antibodies modified by known techniques can also be used. Such an antibody or fragment thereof may be labeled with a conventional enzyme (for example, peroxidase), a fluorescent dye, a radioactive substance, avidin or biotin.
Examples of the anti-FtsW antibody of the present invention include antibodies against 1/2 loop.
Examples of monoclonal antibodies against the 1/2 loop include
The immunoglobulin heavy chain variable region (V _H ) comprises complementarity determining regions CDR1, CDR2 and CDR3, where CDR1 has the amino acid sequence GDAMS (SEQ ID NO: 27), and CDR2 has the amino acid sequence GMSSGGGSYYPPDTVKG (sequence CDR3 is the amino acid sequence RFGSDDEWFAMDY (SEQ ID NO: 29) and comprises CDR1, CDR2 and CDR3 which are complementarity determining regions in the immunoglobulin light chain variable region (V _L ), where CDR1 is An antibody (11E8) having the amino acid sequence KSSQSLLNSGNRKNYLT (SEQ ID NO: 30), CDR2 having the amino acid sequence WASTRES (SEQ ID NO: 31), and CDR3 having the amino acid sequence QNDYPYPYT (SEQ ID NO: 32);
The immunoglobulin heavy chain variable region (V _H ) comprises complementarity determining regions CDR1, CDR2 and CDR3, where CDR1 has the amino acid sequence SYWIN (SEQ ID NO: 33) and CDR2 has the amino acid sequence NINPGSSSDYNE (sequence CDR3 is the amino acid sequence DRGH (SEQ ID NO: 35) and comprises CDR1, CDR2 and CDR3 which are complementarity determining regions in the immunoglobulin light chain variable region (V _L ), where CDR1 is An antibody having the amino acid sequence KASQSVSHAVA (SEQ ID NO: 36), CDR2 having the amino acid sequence SASNRFT (SEQ ID NO: 37), and CDR3 having the amino acid sequence QQDYSSSPYT (SEQ ID NO: 38), (23C7),
The immunoglobulin heavy chain variable region (V _H ) comprises CDR1, CDR2 and CDR3, which are complementarity determining regions, where CDR1 has the amino acid sequence NYLIIE (SEQ ID NO: 39), and CDR2 has the amino acid sequence VINPVRGVTYYNEKFKD (sequence CDR3 is the amino acid sequence DNSGFF (SEQ ID NO: 41) and comprises CDR1, CDR2 and CDR3 which are complementarity determining regions in the immunoglobulin light chain variable region (V _L ), where CDR1 is An antibody (11B11) having the amino acid sequence KSSQSLLAGDGKTYLN (SEQ ID NO: 42), CDR2 having the amino acid sequence LVSELDS (SEQ ID NO: 43), and CDR3 having the amino acid sequence WQGSSHFPRT (SEQ ID NO: 44);
Etc.

  As long as the sequence of the CDR region is within the range in which the desired biological activity of the present invention (for example, affinity, cytotoxicity inhibiting activity, etc.) is retained, one or more of the three CDRs can be used. A variant having three CDRs consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the CDR is also included in the present invention.

  The method for screening an antibacterial agent of the present invention is characterized by being a screening method using FtsW, FtsQ, FtsK, a partial peptide thereof, an anti-FtsW antibody, or stalovacin. This will be described in detail below.

(A) Screening method of antibacterial agent binding to FtsW Specifically,
(1-1) Means a method for screening an antibacterial agent, comprising a step of bringing FtsW or a partial peptide thereof into contact with a test substance, and a step of selecting a test substance that binds to the FtsW or a partial peptide thereof.

  By this screening method, an antibacterial agent exhibiting antibacterial activity by binding to FtsW can be screened. According to the method, since a substance that inhibits the function of FtsW can be selected through binding to FtsW, the selected antibacterial agent directly binds to the protein and inhibits its function. Has characteristic properties.

  Any compound can be used as the “test substance”. For example, organic compounds (amino acids, polypeptides or derivatives thereof, nucleic acids or derivatives thereof, low molecular compounds, sugars, high molecular compounds, etc.), inorganic compounds, and the like can be used. Such a test substance may be a natural substance or a non-natural substance. Examples of polypeptide derivatives include modified polypeptides obtained by adding modifying groups, variant polypeptides obtained by altering amino acid residues, and the like. Examples of nucleic acid derivatives include modified nucleic acids obtained by adding modifying groups, variant nucleic acids obtained by modifying bases, peptide nucleic acids, and the like. Examples of the nucleic acid include siRNA, antisense, ribozyme, aptamer, decoy nucleic acid and the like. As the test substance of the present invention, for example, a commercially available library of compounds may be used.

  The “FtsW partial peptide” is preferably a fragment of S. aureus. More preferred is the extracellular loop of S. aureus FtsW. Specifically, 1/2 loop (SEQ ID NO: 6), 3/4 loop (SEQ ID NO: 20), 5/6 loop (SEQ ID NO: 21), 7/8 loop (SEQ ID NO: 22), 9/10 loop ( And a peptide containing SEQ ID NO: 23). More preferred is a peptide containing 1/2 loop (SEQ ID NO: 6) and 9/10 loop (SEQ ID NO: 23) of S. aureus FtsW. Particularly preferred is a peptide comprising the amino acid sequence of SEQ ID NO: 24, for example, a peptide consisting of the amino acid sequence of SEQ ID NO: 24 or 13.

  The method for preparing FtsW or a partial peptide thereof is not particularly limited. For example, a polynucleotide encoding FtsW is incorporated into an appropriate expression vector, and prokaryotic organisms such as Escherichia coli, eukaryotic organisms such as yeast, insect cells, and mammalian cells are used. Examples thereof include a method for expressing and isolating the polypeptide in cells or in vitro. Those skilled in the art can appropriately select and implement these methods.

  The “step of bringing FtsW or a partial peptide thereof into contact with a test substance” comprises mixing the test substance and FtsW in a solution that does not interfere with the original function of FtsW, and appropriate reaction conditions such as reaction temperature, It can be carried out by maintaining the reaction time and the like.

  Examples of the “solution that does not interfere with the original function of FtsW” include phosphate buffered physiological saline (PBS), HEPES buffer, Tris buffer, and the like.

  The “reaction conditions” are not particularly limited. For example, in a solution such as PBS, HEPES buffer, Tris buffer, pH 6.0 to 10.0, preferably pH 7.0 to 9.0, more preferably pH 7 0.5 to 8.5, more preferably pH 8.0, 10 ° C. to 50 ° C., preferably 20 ° C. to 40 ° C., more preferably 25 ° C. to 37 ° C., more preferably 25 ° C., 1 minute to 1 hour, preferably 3 to 30 minutes, more preferably 5 to 20 minutes, more preferably 10 minutes, or 0 to 20 ° C, preferably 2 to 10 ° C, more preferably It is maintained at 3 ° C to 8 ° C, more preferably at 4 ° C for 20 minutes to 24 hours, preferably 30 minutes to 12 hours, more preferably 40 minutes to 2 hours, more preferably 1 hour, etc. Mentioned That. More specifically, 10 mM Hepes, 80 mM KCl, 5% glycerol, 10 mM DTT, 0.1 mg / ml polyIdC, 50 ng / ml herring sperm DNA, 1 mg / ml BSA, 10% reticulocyte lysate are maintained under the above conditions. Can be mentioned.

  The “step of selecting a test substance that binds to FtsW or a partial peptide thereof” can be performed, for example, by a binding assay of the test substance to a carrier holding FtsW.

  In the screening method, a competition experiment can be performed using stalovacin or an anti-FtsW antibody. That is, when FtsW or a partial peptide thereof is brought into contact with a test substance in the presence of stalovacin or an anti-FtsW antibody, the binding force to FtsW or the partial peptide is higher than that of stalovacin or an anti-FtsW antibody used. A strong test substance can be selected.

(B) Screening method of antibacterial agent that inhibits complex formation of FtsW and FtsQ or FtsK Specifically,
(1-2) contacting one or more polypeptides selected from FtsQ, FtsK, and their partial peptides with FtsW or a partial peptide thereof in the presence of a test substance, and the polypeptide, FtsW or its It means a method for screening an antibacterial agent, comprising a step of selecting a test substance that inhibits binding with a partial peptide.

  In E. coli, it is known that FtsW and FtsQ directly bind, and that FtsW, FtsQ, FtsK, FtsI, FtsN, FtsL, FtsB, FtsX, FtsE, and FtsZ function and function (Journal). of Bacteriology, vol. 188, no. 1, 19-27, 2006). By this screening method, the binding between FtsW and FtsQ is detected, the binding between FtsW and FtsK is detected, or the binding of FtsW and FtsQ / FtsK is detected to inhibit the complex formation, thereby inhibiting the antibacterial activity. Antibacterial agents exhibiting can be screened. Instead of each protein, a partial peptide of each protein can also be used.

  “Test substance” and “partial peptide of FtsW” are the same as in the screening method (A).

  The “partial peptide of FtsQ” or the “partial peptide of FtsK” is not limited to a length or a site as long as it can bind to FtsW or a partial peptide thereof. Particularly preferred is a fragment of FtsQ or FtsK of S. aureus. Whether or not a partial peptide of FtsQ or FtsK can bind to FtsW or a partial peptide thereof can be confirmed by a known binding assay or the like.

  “The step of bringing one or more polypeptides selected from FtsQ, FtsK and their partial peptides into contact with FtsW or its partial peptides in the presence of a test substance” interferes with the original functions of FtsW and FtsQ or FtsK. FtsW or a partial peptide thereof and one or more polypeptides selected from FtsQ, FtsK and partial peptides thereof (for example, FtsQ, FtsQ partial peptide, FtsK, FtsK partial peptide, FtsQ / FtsK complex) FtsQ partial peptide / FtsK complex, FtsQ / FtsK partial peptide complex, FtsQ and FtsK mixture, etc.) and maintained under appropriate reaction conditions such as reaction temperature, reaction time, etc. Can be done.

  The “solution that does not interfere with the original function of FtsW and FtsQ or FtsK” is the same as the “solution that does not interfere with the original function of FtsW” in the screening method (A).

  “Reaction conditions” are the same as in screening method (A).

“Detection of binding” between FtsW and FtsQ can be performed by, for example, a test substance and a FtsQ binding assay to a carrier holding FtsW, a test substance to a carrier holding FtsQ, and a binding assay of FtsW. Even if any of the proteins is a partial peptide, the same procedure can be performed.
“Detection of binding” between FtsW and FtsK can be performed, for example, by a test substance and FtsK binding assay to a carrier holding FtsW, a test substance to a carrier holding FtsK, and a binding assay of FtsW. Even if any of the proteins is a partial peptide, the same procedure can be performed.
“Binding detection” of a complex of FtsW and FtsQ / FtsK includes, for example, a binding assay of a test substance to a carrier holding FtsW and a complex of FtsQ / FtsK, and a carrier holding a complex of FtsQ / FtsK. The test substance and FtsW binding assay can be used. Even if any of the proteins is a partial peptide, the same procedure can be performed.

In the screening method, a competition experiment can be performed using stalovacin or an anti-FtsW antibody. That is, by contacting one or more polypeptides selected from FtsQ, FtsK and their partial peptides with FtsW or its partial peptides in the presence of stalovacin or anti-FtsW antibody and a test substance, FtsW or its partial peptides It is possible to select a test substance having a stronger inhibitory power against the formation of a complex between a partial peptide and FtsQ or FtsK than stalovacin or an anti-FtsW antibody used.
In addition, a competition experiment between a test substance and a partial peptide can be performed using the full length FtsW and the partial peptide of FtsW.

(C) Screening method of antibacterial agent that suppresses expression of FtsW gene or protein Specifically,
(2-1) It means a method for screening an antibacterial agent, comprising a step of searching for a gene encoding FtsW of S. aureus or a substance that suppresses the expression of FtsW.

  The present inventors have found that S. aureus FtsW is a vital protein. By this screening method, an antibacterial agent exhibiting antibacterial activity can be screened by suppressing the expression of a gene encoding FtsW of S. aureus or FtsW.

More specifically, the following method is mentioned.
(2-2) contacting the test substance with a cell capable of expressing a gene encoding FtsW of Staphylococcus aureus,
A step of measuring the expression level of the gene in a cell contacted with a test substance, and selecting a test substance whose measurement level is smaller than the expression level of the gene in a control cell not contacting the test substance The screening method of (2-1) description including the process to carry out.
(2-3) a step of contacting a test substance with a cell capable of producing S. aureus FtsW or a cell fraction prepared from the cell,
A step of measuring the expression level of the FtsW of the cell contacted with the test substance or the cell fraction, and the amount of measurement comprising the expression level of the FtsW of the control cell not contacting the test substance or the cell fraction The screening method according to (2-1), comprising a step of selecting a small amount of test substance in comparison.

  The “test substance” is the same as the screening method (A).

  “A cell capable of expressing a gene encoding S. aureus FtsW” or “a cell capable of producing S. aureus FtsW” is not particularly limited as long as it is an expressible cell. Any prokaryotic cell, yeast, animal cell, plant cell, insect cell, etc. can be used as long as it can express the FtsW gene encoding FtsW of S. aureus or FtsW. It is also possible to use S. aureus.

  The contact between the “cell capable of expressing the gene encoding S. aureus FtsW” or “cell capable of producing S. aureus FtsW” and the test substance is carried out while culturing under the condition that the cell can grow. It can be carried out. The conditions are not particularly limited, but the culture conditions (pH, temperature, medium, etc.) can be selected so that the cells do not die.

The number of cells when contacting with the test substance is, for example, 1.0 × 10 ^{3 to} 1.0 × 10 ⁴ cells / well, preferably 1.0 × 10 ⁴ when using a 98-well plate. It is -1.0 * 10 < ⁵ > piece / well, More preferably, it is 1.0 * 10 < ⁵ > -1.0 * 10 < ⁶ > piece / well.

  The term “control cell” means a cell that is not brought into contact with a test substance in “a cell capable of expressing a gene encoding S. aureus FtsW” or “a cell capable of producing S. aureus FtsW”. . When the test substance is not contacted, the control substance that does not affect the expression of the gene encoding FtsW or the production of FtsW when adding the same amount of solvent (blank) as the test substance instead of the test substance May be added.

  The method for measuring the expression level of the gene encoding FtsW used in the screening method described in (2-2) above is not particularly limited. For example, RNA prepared from the cell or complementary transcribed from RNA is used. In-situ hybridization using polynucleotides, Northern blotting, dot plots, RNase protection assays, and other methods, methods using microarrays, real-time PCR methods such as PCR, and methods for directly measuring the RNA strand The method which can perform a measurement is mentioned.

  Based on the expression level of the gene encoding FtsW detected and quantified as described above, a substance that suppresses the expression of the gene encoding FtsW can be selected. That is, the expression of a gene encoding FtsW in a cell to which a test substance is added is 70% or less, preferably 50% or less, more preferably 10%, compared to the expression level in a control cell to which the test substance is not added. If it is below, the said test substance can be selected as an expression inhibitory substance.

  The method for measuring the expression level of FtsW used in the screening method described in (2-3) above is not particularly limited, and examples thereof include an immunological measurement method using an antibody specific for FtsW. Examples of the immunological measurement method include Western blot, ELISA, and sandwich ELISA. The antibody can be prepared by a conventionally known method using the FtsW, and may be a polyclonal antibody or a monoclonal antibody.

(D) Screening method of antibacterial agent that suppresses expression of FtsQ gene or protein Specifically,
(3-1) A method for screening an antibacterial agent, comprising a step of searching for a gene encoding FtsQ of S. aureus or a substance that suppresses the expression of FtsQ.

  The present inventors have found that S. aureus FtsQ is an essential protein for survival. By this screening method, an antibacterial agent exhibiting antibacterial activity can be screened by suppressing the expression of a gene encoding FtsQ of S. aureus or FtsQ.

More specifically, the following method is mentioned.
(3-2) contacting a test substance with a cell capable of expressing a gene encoding FtsQ of Staphylococcus aureus,
A step of measuring the expression level of the gene in a cell contacted with a test substance, and selecting a test substance whose measurement level is smaller than the expression level of the gene in a control cell not contacting the test substance The screening method according to (3-1), comprising the step of:
(3-3) A step of contacting a test substance with cells capable of producing S. aureus FtsQ or a cell fraction prepared from the cells,
A step of measuring the expression level of the FtsQ in a cell contacted with a test substance or a fraction of the cell; and the measurement amount includes an expression level of the FtsQ in a control cell not contacting the test substance or the cell fraction. The screening method according to (3-1), comprising a step of selecting a small amount of test substance in comparison.

  The details are the same as the screening method (C).

  The test substance obtained by the screening methods (A) to (D) of the present invention is, for example, a method of administering the test substance to a bacterium and evaluating the growth inhibition life of the bacterium using the number of cells as an index, etc. It can be done by.

  Antibacterial agents obtained by the screening methods (A) to (D) of the present invention are expected to exhibit a broad spectrum and strong antibacterial activity.

  The content of the compound in the antibacterial agent can be adjusted as appropriate depending on the disease to be treated, the age, weight, etc. of the patient, and may be any therapeutically effective amount. In the case of a low molecular compound or a high molecular compound, for example, In the case of a polypeptide or a derivative thereof, for example, 0.0001 to 1000 mg, preferably 0.001 to 100 mg is desirable.

  The above pharmaceutical composition may further contain various auxiliaries capable of stably holding the compound. Specifically, pharmaceutically acceptable auxiliaries, excipients, binders, and stabilizers that exhibit the property of inhibiting the active ingredient from degrading before reaching the site where the active ingredient is to be delivered. Agents, buffers, solubilizers, isotonic agents and the like.

  The administration form of the above pharmaceutical composition is appropriately selected according to the type of active ingredient; individual, organ, local site, tissue to be administered; age, weight, etc. of the individual to be administered. Examples of the administration form include subcutaneous injection, intramuscular injection, intravenous injection, and local administration.

  The dosage of the above pharmaceutical composition is also appropriately selected according to the type of active ingredient; the individual, organ, local site, tissue to be administered; the age, weight, etc. of the individual to be administered. Although it does not specifically limit as administration, When an active ingredient is a low molecular compound or a high molecular compound, as an amount of the said active ingredient, it is 0.0001-1000 mg / kg body weight, Preferably, it is 0.001-100 mg. / Kg body weight, in the case of a polypeptide or a derivative thereof, for example, 0.0001 to 1000 mg / kg body weight, preferably 0.001 to 100 mg / kg body weight, multiple times per day For example, it may be administered 1 to 3 times.

In addition, the present invention includes a method for searching for a protein that binds to FtsW using stalobacin.
(E) Method for searching for a protein that binds to FtsW Specifically,
(4-1) In the presence of the test substance,
It means a method for screening an FtsW binding protein, comprising the steps of bringing stalobacin into contact with FtsW or a partial peptide thereof, and selecting a test substance that inhibits the binding between stalovacin and FtsW or a partial peptide thereof.

  Our study revealed that FtsW and stalovacin form a complex when stalobacin is administered to bacteria. Therefore, a protein derived from a bacterium that inhibits the formation of the complex can be screened by detecting the binding of stalobacin and FtsW by the above screening method. As the protein obtained by the method, a protein that functions in bacteria can be selected through binding to FtsW. Therefore, it can utilize for the screening of the antibacterial agent of a novel action mechanism.

  “Partial peptide of FtsW” is the same as the screening method (A).

  Examples of the “test substance” include bacteria-derived proteins. For example, proteins such as Escherichia coli, Mycobacterium tuberculosis and Staphylococcus aureus can be mentioned.

  The “step of bringing stalobacin into contact with FtsW or a partial peptide thereof” is performed by mixing a test substance and FtsW in a solution that does not interfere with the original function of FtsW, and appropriate reaction conditions such as reaction temperature, reaction time, etc. This can be done by maintaining

  The “solution that does not interfere with the original function of FtsW” and the “reaction conditions” are the same as in the screening method (A).

  “Detection of binding” between stalobacin and FtsW or a partial peptide thereof can be performed, for example, by a binding assay of a test substance and staobacin to a carrier holding FtsW or a partial peptide thereof.

  As a protein obtained by the screening method, a protein that functions in bacteria through binding to FtsW can be selected. Therefore, similarly to the screening method (B), it can be used for screening an antibacterial agent having a novel mechanism of action, that is, a screening method for an antibacterial agent that inhibits complex formation of the obtained protein with FtsW.

  FtsW or a partial peptide thereof, anti-FtsW antibody, FtsQ or a partial peptide thereof, FtsK or a partial peptide thereof, stalobacin or the like used in the screening method of the present invention is also provided as a kit for performing the screening method of the present invention. Therefore, such a screening kit is also included in the present invention.

Examples of the screening kit of the present invention include the following.
(1-5) A kit for screening an antibacterial agent, comprising one or more polypeptides selected from the group consisting of FtsW, FtsQ, FtsK and partial peptides thereof.
(1-8) A kit for screening an antibacterial agent, which contains an anti-FtsW antibody or stalobacin.
(2-4) A kit for screening an antibacterial agent, comprising a cell capable of expressing a gene encoding FtsW of S. aureus, a cell capable of producing FtsW of S. aureus, or a cell fraction prepared from the cell.
(3-4) A kit for screening an antibacterial agent, comprising a cell capable of expressing a gene encoding FtsQ of S. aureus, a cell capable of producing FtsQ of S. aureus, or a cell fraction prepared from the cell.

  The screening kit of the present invention may further contain a detection reagent, a buffer solution, a standard substance, a standard curve, and the like.

  According to the screening kit of the present invention, the screening method exhibits an excellent effect that it can be carried out simply and rapidly, at a high throughput and with high reliability.

  EXAMPLES The present invention will be described in more detail below with reference to examples, reference examples, and test examples of the present invention, but the present invention is not limited thereto.

Example 1 Identification of a binding protein (SA0962) of stalobacin Production of a S. aureus SA1888 deletion strain Using the genomic DNA of Aureus RN4220 strain as a template, each of the upstream and downstream sequences of SA1888 was amplified. At this time, primers with EcoRI and SalI at both ends of the upstream sequence and SalI and PstI restriction enzyme sites at both ends of the downstream sequence were used. The upstream sequence amplified fragment and pUC19 were digested with EcoRI and SalI and ligated, E. coli. Transformation into E. coli DH5α strain was performed. Ampicillin, X-Gal, IPTG-containing LB agar medium [Composition: 1.0 wt% tryptone, 0.5 wt% yeast extract, 0.5 wt% sodium chloride, 1.5 wt% agar (pH 7.0 ± 0. As a result of blue-white selection using 2)], white colonies were found, and insertion of the insert was confirmed by PCR. This colony was cultured in ampicillin-containing LB liquid medium, and the plasmid was extracted and purified by the miniprep method to obtain a plasmid in which the upstream sequence of SA1888 was incorporated into pUC19. Subsequently, this plasmid and the downstream sequence amplified fragment were digested with restriction enzymes with SalI and PstI, and ligated, E. coli. Transformation into E. coli DH5α strain was performed. It was seeded on an ampicillin-containing LB agar medium, and PCR was performed on the grown colonies to confirm insertion of the insert. This colony is cultured in ampicillin-containing LB liquid medium, and the plasmid is extracted and purified by the miniprep method, whereby a plasmid in which the upstream sequence and downstream sequence of SA1888 are continuous on pUC19 (sequence for deletion of SA1888) is mounted. Got. About the obtained plasmid, the sequence analysis was conducted and it confirmed that it had the correct arrangement | sequence. This plasmid was subjected to restriction enzyme treatment with EcoRI and PstI, followed by electrophoresis, and a fragment of the SA1888 deletion sequence was excised from the gel and purified. Subsequently, the temperature sensitive plasmid pCL52.1 was cleaved with EcoRI and PstI, and ligated with the SA1888 deletion sequence fragment. Transformation into E. coli DH5α strain was performed. It was seeded on a spectinomycin-containing LB agar medium, and PCR was performed on the grown colonies to confirm insertion of the insert. This colony was cultured in a spectinomycin-containing LB liquid medium, and a plasmid for SA1888 deletion was constructed by extracting and purifying the plasmid by the miniprep method. About the obtained plasmid, the sequence analysis was conducted and it confirmed that it had the correct arrangement | sequence.
This plasmid was electroporated into S. cerevisiae. It was introduced into the aureus RN4220 strain. The obtained transformant was transformed into tetracycline-containing TSA [composition: pancreatic juice digest of 1.5 wt% casein, papain digest of 0.5 wt% soybean, 0.5 wt% sodium chloride, 1.5 wt% agar ( pH 7.3 ± 0.2)] was cultured overnight at 44 ° C. on the medium. Since pCL52.1 has a temperature-sensitive replication origin, homologous recombination occurs under high temperature conditions of 44 ° C., and only strains in which the plasmid is integrated into the genome can grow well. As a result of the culture, large colonies that were thought to have undergone homologous recombination appeared. PCR was performed on these colonies to confirm that the target strain had the plasmid integrated into the genome.
Subsequently, the cells were cultured overnight in a tetracycline-free medium at 30 ° C., and a strain in which the deletion sequence of SA1888 caused homologous recombination on the genome and became tetracycline sensitive was selected. SA1888 deletion type strains were confirmed by colony PCR for these strains, and SA1888 deletion strains were obtained.

Cultivation of S. aureus SA1888 deletion strain S. aureus deficient in the SA1888 gene is overnight in 500 mL of LB liquid medium (0.5% yeast extract, 1% tryptone, 1% NaCl, pH 7.2-7.5). After shaking culture, the culture solution was added to 15 L of LB liquid medium containing 30 μg / mL chloramphenicol and cultured at 37 ° C. with a jar fermenter. The culture was terminated when the absorbance (625 nm) of the medium reached 1.0, and the cells were collected by centrifugation at 4 ° C., 10,000 × g for 15 minutes. Next, as a washing operation of the bacterial cells, suspension with 50 mM Tris-HCl, pH 7.8 and centrifugation at 4 ° C., 10,000 × g for 15 minutes were repeated three times alternately.

Preparation of membrane fraction The washed cells were resuspended in 50 mM Tris-HCl, pH 7.8, lysostaphin having a final concentration of 10 μg / mL was added, and the mixture was incubated at 30 ° C. for 30 minutes with stirring. The suspension was then set in a cell disrupter and disrupted at 8000 psi. The disrupted solution was centrifuged at 4 ° C., 20,000 × g, 15 minutes, and the supernatant was ultracentrifuged at 4 ° C., 100,000 × g, 35 minutes. The resulting precipitate was collected as a membrane fraction.

Solubilization of membrane fraction The membrane fraction was suspended in TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.4) containing a protease inhibitor (Roche Diagnostics, complete mini EDTA free), and the Bradford method ( BioRad) was used for protein quantification. Next, 1 mg / mL protein concentration, 1% DDM (n-dodecyl-beta-D-maltoside), solubilization operation by stirring at 4 ° C. for 1 hour, followed by ultracentrifugation at 4 ° C., 100,000 × g for 30 minutes The solubilized supernatant was collected.

Purification of stalobacin-binding protein Hereinafter, the experimental operation was performed at 4 ° C., and the fraction of beads and supernatant was centrifuged at 2000 × g for 30 seconds. The solubilized supernatant and monomeric avidin beads (Thermo Fisher Scientific) were mixed by inverting for 30 minutes, and then the supernatant was collected. Next, a final concentration of 400 ng / mL biotinylated stalovacin and monomeric avidin beads were added to the supernatant and mixed by inverting for 12 hours. For Mock conditions, a compound consisting of biotin and a spacer arm was used in place of biotinylated stalobacin. In a competitive inhibition experiment with stalovacin, about 500 times the amount of stalovacin coexisted with biotinylated stalovacin. After removing the supernatant from the bead suspension, the beads were washed with TBS containing 0.2% DDM. Subsequently, TBS containing beads, 5 mM biotin, and 0.2% DDM was mixed by inverting for 30 minutes, and the supernatant was recovered as an elution fraction.
In this example, “stalobacin” means a compound represented by the following structural formula (stalovacin I).

SDS polyacrylamide electrophoresis SDS polyacrylamide electrophoresis followed the widely used Remley method. SDS sample buffer (62.5 mM Tris-HCl, 25% glycerol, 2% SDS, 0.01% BPB, 50 mM DTT, pH 6.8) and elution fraction were mixed at a ratio of 1: 1, 37 ° C., 30 Incubated for minutes. Subsequently, electrophoresis was performed using a polyacrylamide 10-20% gradient separation gel.

Protein Detection The gel after electrophoresis was immersed in an immobilization solution (50% methanol, 7% acetic acid) and shaken for 30 minutes, and then immersed in a YPRO-Ruby staining solution (Invitrogen) and shaken for 1 hour. Finally, it was immersed in a decolorizing solution (10% methanol, 7% acetic acid) and shaken for 1 hour. For protein detection, a fluorescence scanner (GE Healthcare) was used, and excitation at 457 nm was performed to detect 510 nm fluorescence. The results are shown in FIG. Although non-specific binding protein was also detected, it was not detected under Mock or stalobacin competitive inhibition conditions, but a protein with a molecular weight of about 35000 was detected specifically under biotinylated stalovacin conditions.

Protein Identification In-gel digestion was according to the method of Morita et al. (Proteomics 2006, 6, 6, 5880-5890). The target protein band was excised from the electrophoresis gel and digested in gel with trypsin and lysyl endopeptidase. The collected digested peptide was dissolved in 0.1% formic acid and subjected to mass spectrometry using a high performance liquid chromatograph mass spectrometer (Bruker Daltonics). Peptides were prepared using 5% B (0-5 min), 5-60% B (5-35 min) using solvent A: 0.1% formic acid, solvent B: 0.1% formic acid, 80% acetonitryl. ), 60-100% B (35-35.1 minutes), 100% B (35.1-45 minutes), and 100-5% B (45-45.1 minutes). Subsequently, the protein was identified from the obtained mass spectrum using protein identification software Mascot (Matrix Science). The search conditions were as follows: database: NCBInr, species: staphylococcus aureus, enzyme: trypsin, number of permissible uncut sites: one. The Mascot search results are shown in Table 1, and the MSMS mass spectrum is shown in FIG. As a result, SA0962 (SEQ ID NO: 1; GenBank: NP — 374231) was identified as a stalovacin binding candidate molecule.

Cloning of SA0962 S. cerevisiae registered in KEGG (Kyoto Encyclopedia of Genes and Genomes). Based on the nucleotide sequence of Aureus NCTC8325 strain, S. aureus Oligonucleotide primers NFR0962-S (SEQ ID NO: 2) and 0962-TCS-AS (SEQ ID NO: 3) were prepared so as to amplify the full length of the Aureus SA0962 gene. Using these primer DNAs, S. PCR was performed using aureus RN4220 genomic DNA as a template. The first PCR was performed using 50 pmol of each of NFR0962-S primer and 0962-TCS-AS primer, and then separation and purification by agarose electrophoresis were performed to cut out a region containing a 1,227 bp DNA fragment. The PCR reaction was carried out in 30 cycles, with 94 ° C, 15 seconds, 60 ° C, 30 seconds, 68 ° C, 30 seconds as one cycle.

Vector construction of SA0962-His tag for cell-free synthesis (1) Gene fragment RTS-Nfr containing T7 promoter and Shine-Dalgarno sequence upstream of MCS of E. coli cell-free protein synthesis vector pIVEX3.1 amplified by PCR (2) Gene fragment RTS-TCS-Cfr-His each containing 5 ng of thrombin recognition sequence (TCS), 6x His tag sequence, T7 terminator downstream of MCS and 40 ng of SA0962 gene obtained in the above-mentioned "cloning of SA0962", A fragment for cell-free synthesis was prepared by overlap extension PCR. In PCR, oligonucleotide primers RTS-Nfr-S (SEQ ID NO: 4) and RTS-Cfr-AS (SEQ ID NO: 5) were designed and used so as to amplify the entire length of the cell-free synthesis fragment containing the SA0962 gene. The above PCR reaction was performed under the conditions of a total of 35 cycles, with 94 ° C., 15 seconds, 55 ° C., 30 seconds, 68 ° C. and 2 minutes as one cycle. The gene fragment for cell-free synthesis was separated and purified by agarose electrophoresis, and a region containing a 1,582 bp DNA fragment was excised. Although introduced into the pCR-BluntII-TOPO (registered trademark) vector by TA cloning, a single-base mutation was found upstream of the protein translation region. Therefore, the mutation was corrected by a mutation-introducing PCR method, and the recombinant plasmid Nfr having an ampicillin resistance gene was used. -SA0962-TCS-His-pCR-BluntII-TOPO was obtained.

Western blotting After electrophoresis according to the description of “SDS polyacrylamide electrophoresis” above, the separated protein was transferred to a PVDF membrane. Next, Western blotting was performed by partially modifying the method of Ohta et al. (Biochimica et Biophysica Acta 1788 (2009) 1099-1107). After blocking with TBS containing 5% skim milk and 0.1% Tween 20, for detection of His tag fusion protein, rabbit anti-His tag polyclonal antibody (Santa Cruz) as primary antibody and horseradish peroxidase as secondary antibody An anti-rabbit IgG antibody (GE Healthcare) bound with a chemiluminescent reagent ECLplus (GE Healthcare) was used as a detection reagent. LAS3000 (Fuji Film) was used for the measurement of the amount of chemiluminescence.

Cell-free synthesis of SA0962-His tag Cell-free synthesis was performed using the E. coli cell-free protein synthesis kit RTS100 E. coli. The in vitro cell-free translation system batch method was performed using E. coli HY kit (5 Prime). In addition to the Escherichia coli concentrated cell extract attached to the kit, reaction mix, and 20 amino acids, the translation reaction solution contains a cell-free synthesis plasmid vector, protease inhibitor cocktail (Roche Applied Science), and MembraneMax (Invitrogen). The reaction was carried out by shaking at 200 rpm at 30 ° C. for 16 hours in a 25 μL scale in a dedicated incubator (Roche Diagnostics). The expression of the target gene was confirmed by Western blotting using an anti-His × 5 antibody. In the following assay, a translation reaction supernatant centrifuged at 15,000 × g for 5 minutes at 4 ° C. was used.

Cell-free synthetic SA0962-His tag stalovacin binding evaluation Using cell-free synthetic SA0962-His tag and biotinylated stalovacin, a stalovacin-binding fraction was prepared according to the method described in "Purification of stalobacin-binding protein" above. The bound SA0962-His tag was detected by the method described in the above “Western blotting”. The results are shown in FIG. Compared to the Mock condition, the SA0962-His tag was significantly detected under the biotinylated stalobacin condition. Moreover, since the binding was inhibited by stalovacin, it was confirmed that SA0962 specifically binds to stalovacin.

Construction of SA0962 expression induction plasmid
S. A full-length PCR amplification of the SA0962 ORF was performed using the genomic DNA of the Aureus RN4220 strain as a template. At this time, primers provided with restriction sites for SmiI and NotI were used at both ends. This PCR fragment and the vector pSR1000 having xylR and xylO were cleaved with restriction enzymes SmiI and NotI, respectively, and ligated. After purification, S. cerevisiae was electroporated. It was introduced into the aureus RN4220 strain. PCR was performed on colonies that had grown on the chloramphenicol-containing TSA medium to confirm insertion of the insert. This colony was cultured in a chloramphenicol-containing TSB liquid medium, and the plasmid was extracted and purified by the miniprep method to obtain a SA0962 (SEQ ID NO: 48) expression-inducing plasmid. The obtained plasmid was subjected to sequence analysis and confirmed to have the correct sequence.

Observation of morphological changes in Staphylococcus aureus aureus RN4220 strain, SA1888 deletion strain, SA0962 temperature sensitive strain TS3021 HIA (Heart Infusion Agar) [Composition: 1.0 wt% 500 g bovine heart exudate, 1.0 wt% tryptose, 0.5 wt% chloride Sodium, 1.5% by weight agar (pH 7.4 ± 0.2)] Inoculated into slant medium, RN4220 strain and SA1888-deficient strain were cultured at 37 ° C., and TS3021 was cultured at 30 ° C. for 48 hours. Suspension of bacteria for 1 ase in 10 mL of sterilized water, and surface dried CGPY containing 0.5% by weight glucose, 1% by weight bactopeptone, 0.01% by weight yeast extract, 0.6% by weight phosphorus Sodium oxyhydrogen, 0.3 wt% sodium chloride, 0.3 wt% potassium dihydrogen phosphate, 0.01 wt% magnesium chloride hexahydrate, 0.2 wt% ammonium chloride, 0.015 wt% sulfuric acid Sodium, 0.12% by weight sucrose, 0.2% by weight agar (pH 7.0)] was poured over the entire surface of the plate, and the excess bacterial solution was removed. The surface was dried for 15 minutes in a 37 ° C. incubator, and stalovacin (20 mL of a 10,000 μg / mL DMSO solution was soaked in a 6 mm thin paper disc for antibiotic testing and dried in a 37 ° C. incubator for 30 minutes). And incubated at 37 ° C. Twenty-four hours later, the bacteria around the filter paper / inhibitory circle under the influence of stalovacin and the edge of the plate not affected by stalovacin were transferred with an agar film and placed on a slide glass, and the morphology of the bacteria was observed with a microscope. As a result, in the RN4220 and SA1888-deficient strains, the cells were enlarged under the influence of stalovacin near the inhibition circle, and remained unchanged in the portion not affected by stalovacin around the plate. On the other hand, TS3021 showed the same form as that at the time of stanovasin action because the cells were enlarged even in the absence of stanovasin (FIG. 4).

Subsequently, SA0962 expression-inducing plasmid was introduced into TS3021 by electroporation, and PCR was performed on colonies that had grown on the chloramphenicol-containing TSA medium to confirm insertion of the insert.
The RN4220 strain was inoculated into an HIA plate, and the SA0962 expression-inducing plasmid-introduced TS3021 strain was inoculated into a chloramphenicol-containing HIA plate at 37 ° C. and cultured at 30 ° C. for 24 hours. Prepare a normal CGPY plate medium and a 0.5% xylose-containing CGPY plate medium, and suspend the bacteria for 1 ase in 10 mL of sterilized water, and then dry the surface of the CGPY plate medium and the xylose-containing CGPY plate medium respectively. Rinse the entire surface and remove the excess fungus. After culturing in a 37 ° C. incubator for 24 hours, the cells on the plate were transferred with an agar film and placed on a slide glass, and the morphology of the bacteria was observed with a microscope.

  As a result, the cell morphology of the RN4220 strain was unchanged regardless of the presence or absence of xylose. On the other hand, the TS0921 expression-introduced plasmid-introduced plasmid TS3021 strain was enlarged in a xylose-free medium and exhibited the same morphology as that of stalovacin action, but no cell enlargement was observed in a xylose-containing medium. It returned to the almost same form (FIG. 5). Mutation of the SA0962 gene, which brings temperature sensitivity to the cells, became the same phenotype as stalovacin action on the wild type strain. In addition, normal SA0962 was complemented to the SA0962 temperature-sensitive strain to return to the normal expression system. From this, it was shown that the action point of stalobacin may be SA0962.

Analysis of Staphylocin Susceptibility of Staphylococcus aureus Aureus RN4220 strain, SA0962 temperature-sensitive strain TS3021 was applied to TSA medium, mock strain in which vector pSR1000 was introduced into TS3021, and strain in which SA0962 expression-inducing plasmid was introduced into TS3021, was applied to TSA medium containing chloramphenicol. Each of the other 3 strains was cultured overnight at 30 ° C. at C. C., and each of the strains was cultured overnight. CAMHB (cation adjusted Mueller-Hinton broth) [Composition: 22.5 mg / LCa ²⁺ , 11.25 mg / L Mg ²⁺ , 0.2 % By weight beef extract, 1.75% by weight acid digest of casein, 0.15% by weight soluble starch (pH 7.3 ± 0.1)] and diluted to 5 × 10 ⁶ CFU / mL. On the other hand, a 2-fold dilution series of stalobacin was prepared on a 96-well plate. In each well of a 96-well plate, 80 μL of 0.5% xylose-containing CAMHB, 10 μL of 5 × 10 ⁶ CFU / mL of a 2-fold diluted solution of stalobacin, and well mixed, TS3021 can grow and high temperature Incubated at 37 ° C. for about 17 hours, which is expected to be affected by

  As a result, the TS3021 strain and the mock strain showed markedly increased stalovacin sensitivity, and the difference in MIC value from the wild strain RN4220 strain was 8 times and 16 times, respectively. On the other hand, the MIC of the strain in which the SA0962 expression-inducing plasmid was introduced into TS3021 was the same as that of RN4220 (FIG. 6). That is, by complementing SA0962, the stanovasin sensitivity of the SA0962 TS strain returned to the same level as the wild strain. This strongly suggested that SA0962 may be a target for stalobacin.

Example 2 Preparation of anti-SA0963 antibody Preparation and immunization of immunogen A peptide in which a cysteine was introduced into the C-terminal of a partial peptide of SA0962 (41-65: 1/2 loop of SEQ ID NO: 48) was synthesized (SEQ ID NO: 6). 10 mg of the synthesized peptide dissolved in 0.1 M phosphate buffer (pH 6.0) containing 5 mM EDTA and 10 mg of Immediate-Activated mcKLH (manufactured by Thermo) 0.1 ml containing 1 ml of 5 mM EDTA What was dissolved in an acid buffer solution (pH 6.0) was mixed and reacted at room temperature for 4 hours and further at 4 ° C. overnight. The above mixture was dialyzed against distilled water and then lyophilized to obtain SA0962 partial peptide-KLH complex. This was used as an immunogen. 100 μg of the prepared SA0962 peptide-KLH complex was intraperitoneally administered to 5 4-week-old A / J Jms Slc female mice together with Freund's complete adjuvant for initial immunization. Thereafter, 100 μg of SA0962 partial peptide-KLH complex was administered together with Freund's incomplete adjuvant 21 days, 42 days, and 63 days later for booster immunization. Thereafter, a solution in which 100 μg of SA0962 partial peptide-KLH complex was suspended in 0.1 ml of physiological saline was intraperitoneally administered to mice in which an increase in antibody titer was observed, and final immunization was performed.

Biotin labeling of SA0962 partial peptide 0.4 mg of synthesized SA0962 partial peptide dissolved in 0.4 ml of 0.1 M phosphate buffer (pH 6.0) containing 5 mM EDTA, and 160 μg of Maleimide-PEO2-biotin (Thermo Was dissolved in 80 μl of distilled water and reacted at room temperature for 2 hours, and then biotinylated SA0962 partial peptide was purified by reverse phase HPLC.

Preparation of hybridoma Three days after the final immunization, the spleen was removed and spleen cells were collected. Spleen cells and mouse myeloma cells (p3 × 63-Ag8.U1, Tokyo Mass Research Institute) were fused with 50% polyethylene glycol 4000 and selected in a medium containing hypoxanthine, aminopterin, and thymidine.

Selection of SA0962 antibody Specific antibody-producing cells were screened 10 days after cell fusion. The immunoassay used for the screening is as follows. 35 μl of Tris buffer (50 mM Tris-HCl, pH 7.5) containing 0.35 μg of anti-mouse IgG antibody (Jackson ImmunoResearch) was added to each well of a 384-well microtiter plate (Nunk) at 4 ° C. Fixed for 16 hours. These wells were washed once with 90 μl of a washing solution (saline containing 0.01% Tween 20), and then 200 μl of Block Ace (Dainippon Pharmaceutical Co., Ltd.) was added and left at room temperature for 2 hours for blocking. (Anti-mouse IgG antibody-immobilized plate). Each well was washed once with 90 μl of washing solution, and then 15 μl of hybridoma culture supernatant was added and reacted at room temperature for 2 hours. Next, after each well was washed three times with 90 μl of washing solution, 15 μl of DELFIA Assay Buffer (PerkinElmer) containing 0.05 ng of biotin-labeled SA0962 partial peptide and 2 ng of Eu-labeled Streptavidin (manufactured by PerkinElmer) was added. The reaction was carried out at 4 ° C. for 16 hours. Next, after each well was washed three times with 90 μl of washing solution, 25 μl of DELFIA Enhancement solution (manufactured by PerkinElmer) was added, and time-resolved fluorescence was measured by EnVision (PerkinElmer). From the results of screening, clones that bind to the SA0962 protein prepared by cell-free synthesis were obtained from the hybridomas that reacted with the SA0962 partial peptide.

SA0962 antibody binding inhibition test of SA0962 and stalobacin 15 μl of phosphate buffered saline (pH 7.2) containing 0.15 μg of anti-C2 tag antibody was added to each well of a 384-well microtiter plate (manufactured by Nunk). Fix at 16 ° C. for 16 hours. These wells were washed once with 90 μl of a washing solution (saline containing 0.01% Tween 20), and then 200 μl of Block Ace (Dainippon Pharmaceutical Co., Ltd.) was added and left at room temperature for 2 hours for blocking. It was. Each well was washed once with 90 μl of washing solution, and then 15 μl of SA0962-C2 tag was added and reacted at room temperature for 2 hours. Next, each well was washed three times with 90 μl of washing solution, and then buffer A (0.5% bovine serum albumin, 0.01% Tween 80, 0.05% Proclin 150, 0.15 M) containing biotin-labeled stalobacin and SA0962 antibody. A 50 mM Tris buffer containing NaCl, pH 7.4) and a buffer A containing HRP-labeled streptavidin were added and reacted at 4 ° C. for 16 hours. Next, after each well was washed three times with 90 μl of washing solution, 15 μl of TMB + -Substrate-Chromogen (manufactured by DAKO) was added to develop color for 30 minutes at room temperature, and then 15 μl of 0.05 M sulfuric acid was added. The reaction was stopped and the absorbance at 450 nm was measured. As a result of screening, the antibodies that showed binding inhibition activity of 40% or more at an antibody concentration of 100 μg / ml were 11E8, 23C7, and 11B11, with the binding between SA0962 and stalovacin being 100% in the presence of Control IgG. The result is shown in FIG.

Amino Acid Sequence of Anti-SA0962 Antibody For the anti-SA0962 antibody (11E8, 23C7, 11B11), the sequence of the variable region was determined using a conventional method.
The heavy chain (SEQ ID NO: 7) and light chain (SEQ ID NO: 8) of 11E8 are shown in FIG.
The heavy chain (SEQ ID NO: 9) and light chain (SEQ ID NO: 10) of 23C7 are shown in FIG.
The heavy chain (SEQ ID NO: 11) and light chain (SEQ ID NO: 12) of 11B11 are shown in FIG.

Example 3 Synthesis of SA0962 Peptide Experimental Peptides 344-355 (C-terminal amide: part of 9/10 loop) of SEQ ID NO: 48, which is the amino acid sequence of SA0962, by peptide solid phase synthesis by Fmoc method (SEQ ID NO: 13) Was purified by reverse-phase HPLC, and then lyophilized to obtain the desired product. Yield 8.9 mg (10% yield), ESI-MS: Found [M + H] ⁺ = 1187.6 (Theoretical [M + H] ⁺ = 1187.7).

Construction of SA0962-C2 tag vector for cell-free synthesis The SA0962 gene to which a collagen type 2 neoepitope tag (C2 tag) was added was constructed. First, Nfr-SA0962-TCS-His-pCR-BluntII-TOPO (registered trademark) was linearized by PCR using a template. For this PCR, primers with SA0962VP3 (SEQ ID NO: 14) on the forward side and SA0962VP2 (SEQ ID NO: 15) on the reverse side were used. PCR was performed in a total of 30 cycles, with 98 ° C. for 10 seconds, 60 ° C. for 15 seconds, and 72 ° C. for 45 seconds. Next, the Col2 tag region was amplified by PCR using pET15b-p15 (HXB2) -Col as a template. For this PCR, primers with Col2ifF2 (SEQ ID NO: 16) on the forward side and Col2ifR (SEQ ID NO: 17) on the reverse side were used. PCR was performed in a total of 30 cycles, with 98 ° C. for 10 seconds, 60 ° C. for 15 seconds, and 72 ° C. for 45 seconds. The linearized vector and the insert fragment were fused with In-Fusion Advantage to obtain SA0962-TCS-C2-pCR-BluntII-TOPO.

Cell-free synthesis of SA0962-C2 tag The SA0962-C2 tag gene was cell-free synthesized according to the description in “Cell-free synthesis of SA0962-His tag” above.

Binding Inhibition Experiment A 96-well plate on which streptavidin was immobilized was used to evaluate the binding between biotinylated stalovacin and SA0962. The experimental conditions per hole are shown below.
First, for the immobilization of biotinylated stalovacin, 100 μL of TBS containing 0.05% Tween 20 and 25 pmol of biotinylated stalovacin were added and incubated at room temperature for 1 hour to immobilize biotinylated stalovacin. In order to remove unfixed biotinylated stalobacin, it was washed 3 times with TBS containing 0.05% Tween20. After blocking with TBS containing 2% skim milk and 0.05% Tween 20 at room temperature for 1 hour, add 100 μL of TBS, 5 μL of cell-free synthetic SA0962-His tag diluted 25-fold with TBS, 50 μg of Stalovacin or SA0962 partial synthetic peptide, Incubated for hours. After washing the plate with TBS containing 0.2% DDM, 0.25 μg of horseradish peroxidase-fused anti-C2 tag antibody, 0.5% skim milk, TBS containing 0.2% DDM was added and incubated at room temperature for 1 hour. . After washing again with TBS, 100 μL of 3,3 ′, 5,5′-tetramethylbenzidine (TMB) substrate was added and incubated at room temperature, protected from light. After 15 minutes, 100 μL of 0.2 mol / L sulfuric acid was added to terminate the reaction. The amount of bound SA0962-C2 tag was quantified by absorbance at 625 nm. The results are shown in FIG.

Since the binding activity of stalovacin and SA0962 decreased to 1.4 when the synthetic peptide coexists, the synthetic peptide (SEQ ID NO: 13) inhibits the binding of stalobacin and SA0962. confirmed. This result suggests that stalobacin interacts with the SA0962 9/10 loop and its vicinity.
Similarly, peptides were synthesized for 1/2 loop, 3/4 loop, 5/6 loop, and 7/8 loop, and a binding inhibition experiment was conducted, but the peptide showed no binding inhibition.

Example 4 Verification of SA0962 and complex candidate molecule Vector construction of SA0962-C2 tag for intracellular expression
PCR was performed using plasmid Nfr-SA0962-TCS-His-pCR-Blunt II-TOPO having a sequence with a C2 tag added to the C-terminal side of SA0962, and the C2 tag sequence was fused to the C-terminal side of SA0962 ORF. A fragment was obtained. At this time, primers provided with SmiI and NotI restriction enzyme sites at both ends were used. This PCR fragment and the vector pSR1000 having xylR and xylO were cleaved with restriction enzymes SmiI and NotI and ligated. After purification, S. cerevisiae was electroporated. It was introduced into the aureus RN4220 strain. PCR was performed on colonies that had grown on the chloramphenicol-containing TSA medium to confirm insertion of the insert. This colony was cultured in a chloramphenicol-containing TSB liquid medium, and a plasmid for SA0962-C2 tag expression induction was obtained by extracting and purifying the plasmid by the miniprep method. The obtained plasmid was subjected to sequence analysis and confirmed to have the correct sequence.

Construction of SA0962 temperature-sensitive strain forcibly expressing SA0962-C2 tag The SA0962-C2 tag expression-inducing plasmid was introduced into the SA0962 temperature-sensitive strain by electroporation to construct a C2-tag fusion SA0962 expression temperature-sensitive strain. Since the introduced plasmid has xylR and xylO, C2 tag fusion SA0962 was forcibly expressed by culturing this strain in the presence of xylose.

Cultivation of SA0962 temperature-sensitive strain forcibly expressing SA0962-C2 tag Cultivation was carried out by partially modifying the method described in “Cultivation of S. aureus SA1888-deficient strain” above. First, the strain was cultured with shaking in 500 mL of LB liquid medium overnight, and then the culture solution was added to 15 L of TSB liquid medium (3% Tryptic Soy Broth) containing 30 μg / mL chloramphenicol. Culture was performed. When the absorbance at 625 nm reached 1.0, the cells were collected and collected after washing the cells.

Preparation of Fab of anti-C2 tag antibody and preparation of biotin-labeled antibody 2.7 mg of antibody against type 2 collagen neo-epitope fragment (C2 tag) (Clinica Chimica Acta, 2012, in press, development of the immunoassay for the medicine. II To 60 ml of pepsin (manufactured by Roche) was added to acetate buffer (pH 4.2) containing collagen neoepitope generated by collagenase cleavage, and reacted at 37 ° C. for 24 hours. A fraction containing F (ab ′) 2 was separated from the reaction solution by gel filtration HPLC. 40 μl of 0.1 mM 2-mercaptoethylamine hydrochloride (manufactured by Nacalai Tesque) was added and reacted at 37 ° C. for 2 hours. A fraction containing Fab ′ was separated from the reaction solution by gel filtration HPLC. Then, Maleimide-PEO2-biotin (manufactured by Thermo) was added and reacted at 4 ° C. for 24 hours. A fraction containing a biotin-labeled Fab ′ was collected from the reaction solution by gel filtration HPLC to obtain an anti-C2 tag antibody-Fab-biotin body.

Preparation of anti-C2 tag Fab antibody-immobilized beads 2 μg of biotinylated anti-C2 tag Fab antibody was immobilized by mixing with streptavidin magnetic beads by inverting at room temperature for 15 minutes. After removing the supernatant using a magnet, beads and a biotin solution (2 mM biotin, 1.47 mM KH ₂ PO ₄ , 8.1 mM Na ₂ HPO ₄ , 2.68 mM KCl, 136.89 mM NaCl, pH 7.4) were added. Remixed, and unreacted streptavidin was blocked by inversion mixing at room temperature for 15 minutes. The beads were washed 3 times with TBS containing 0.2% DDM and then stored at 4 ° C. until use.

Purification of SA0962-binding protein Membrane fraction preparation and solubilization were performed according to the descriptions of “Preparation of membrane fraction” and “Solubilization of membrane fraction” above. Subsequent fractionation of the magnetic beads and supernatant was performed using a magnet. First, the solubilized supernatant and the anti-C2 tag Fab antibody-immobilized beads were mixed by inversion at 4 ° C. for 12 hours. Under the binding inhibition condition by stalovacin, stalovacin was allowed to coexist so that the final concentration was 0.1 mg / mL. After removing the supernatant, the beads were washed 3 times with TBS containing 0.2% DDM. Next, the beads were mixed with 100 mM ammonium bicarbonate containing 0.1 unit thrombin (Sigma Aldrich) and 0.1% Rapigest (Waters), mixed by inverting at 4 ° C. for 12 hours, and the supernatant was obtained as a SA0962 binding protein fraction. It was collected.

Enzymatic digestion of SA0962 binding protein in solution Add final concentration of 5 mM dithiothreitol to the SA0962 binding protein fraction, reduce at 60 ° C for 30 minutes, and then add final concentration of 15 mM iodoacetamide, alkylate for 30 minutes at room temperature, protected from light Went. Next, 0.5 μg lysyl endopeptidase was added and incubated at 37 ° C. for 12 hours. Subsequently, 0.5 μg trypsin was added and incubated at 37 ° C. for 3 hours, followed by enzyme digestion.

Purification of Peptide A final concentration of 0.5% TFA was added to the digested peptide solution and incubated at 37 ° C. for 45 minutes, followed by centrifugation at 15,000 × g for 5 minutes to decompose and remove Rapigest. Next, the peptide was purified from the supernatant according to the method of Juri Rapsilber et al. (Anal. Chem. 2003, 75, 663-670). The obtained peptide solution was dried by a centrifugal evaporator.

Identification of SA0962 binding protein The content of the above "protein identification" was partially modified. The recovered digested peptide was dissolved in 0.1% formic acid and subjected to mass spectrometry using a high performance liquid chromatograph mass spectrometer. Peptide separation conditions were as follows: solvent A: 0.1% formic acid, solvent B: 0.1% formic acid, 80% acetonitryl, 5% B (0-5 minutes), 5-60% B (5 -125 minutes), 60-100% B (125-125.1 minutes), 100% B (125.1-135 minutes), and 100-5% B (135-15.1 minutes). After identifying the protein from the obtained mass spectrum using the protein identification software Mascot, a differential analysis in the presence or absence of stalovacin was performed based on the identified peptide sequence using Microsoft (Microsoft). . Table 2 shows a list of proteins whose binding is inhibited in the presence of stalobacin obtained from the difference analysis results.

Based on the above results, 17 molecules were identified as candidate molecules whose binding is inhibited in the presence of stalobacin.
Since this list includes SA1027 and SA1119, which may interact with SA0962 as the cell division device divisome, it was expected that the mechanism of action of stalobacin was inhibition of cell division device disome formation. .

Quantitative comparison of SA0962 binding protein in the presence of stalovacin Quantitative comparison was performed on SA0962, SA1027, and SA1119 involved in the formation of the cell division apparatus divome obtained from “Identification of SA0962 binding protein” with or without staobacin. The analysis method was carried out by partially changing the content of “Identification of SA0962 binding protein”. First, in order to quantify SA0962, the product ion m / z 1017.5 obtained by MSMS of the precursor ion m / z 916.0 of the peptide identified as SA0962 (SEQ ID NO: 45) was measured. Similarly, in SA1027, the product ion m / z 933.5 obtained from the precursor ion m / z 1075.6 of the identified peptide (SEQ ID NO: 46), SA1119 is the precursor ion m / z of the identified peptide (SEQ ID NO: 47). The product ion m / z 1223.7 obtained from z902.4 was measured.

  As a result, the amount of SA0962 did not change in correlation with the presence or absence of stalobacin. On the other hand, SA1027 and SA1119 were not detected under the condition with stalobacin. From this, it was considered that the binding of SA0962 and SA1027, and SA0962 and SA1119 was inhibited by stalovacin. (Fig. 12)

SA1027 cloning and cell-free synthesis vector construction According to sequence information (GTOP BAB422279.1), primers NdeI-SA1027-Fwd (SEQ ID NO: 18) and 3 ′ end containing 5 ′ end sequence of SA1027 protein translation region and SwaI recognition sequence A primer XhoI-SA1027-Rev (SEQ ID NO: 19) containing the sequence of I and a NotI recognition sequence was designed. Next, using these primer DNAs, S. cerevisiae. PCR was performed using aureus RN4220 genomic DNA as a template. PCR was performed in a total of 30 cycles, with 98 ° C. for 10 seconds, 60 ° C. for 15 seconds, and 72 ° C. for 45 seconds. The DNA fragment amplified by this PCR reaction was treated with NdeI and XhoI at 37 ° C. for 120 minutes, then subjected to separation and purification by agarose electrophoresis, and a region containing a DNA fragment of 1,323 bp was excised. And introduced into an open pET21a (+) vector prepared using XhoI to obtain a recombinant plasmid SA1027-pET21a (+) having an ampicillin resistance gene.

Cell-free synthesis of SA1027-His tag The cell-free synthesis of SA1027-TCS-His protein was carried out by partially modifying the method described in “SA0962-His tag cell-free synthesis” above. First, E. coli concentrated cell extract attached to the kit, reaction mix, 20 amino acids, plasmid SA1027-pET21a (+), protease inhibitor cocktail, 0.67% Brij58 (registered trademark) (polyethylene glycol monocetyl ether), The reaction was carried out by shaking at 200 rpm for 4 hours at 30 ° C. In the following assay, a translation reaction supernatant centrifuged at 15000 × g for 15 minutes at 4 ° C. was used.

Binding experiment of SA0962-C2 tag and SA1027-His tag The interaction between SA0962 and SA1027 was confirmed by pull-down assay using both molecules synthesized without cell. In the pull-down assay, first, 1 μg of mouse anti-pentaHis monoclonal antibody (Qiagen) and 10 μg of Protein G magnetic beads (Dynabeads (registered trademark) Protein G, Invitrogen) were mixed to immobilize the antibody. Subsequently, antibody-immobilized beads, SA0962-C2 tag and SA1027-His tag cell-free translation reaction supernatant, 4 μL each, and TBS buffer containing 0.2% DDM were mixed, and the final scale was 250 μL at 4 ° C., 16 Mix by inversion for an hour. The beads were collected, washed with TBS buffer containing 0.2% DDM, and then subjected to heat treatment at 37 ° C. for 30 minutes in an SDS-polyacrylamide electrophoresis sample buffer containing DTT to remove SA0962-C2 bound to the beads. Eluted. In a competition experiment with stalovacin, 25 ng of stalovacin was allowed to coexist when SA0962-C2 and SA1027-His were mixed. The amount of SA0962-C2 contained in the eluted fraction was determined by Western blotting using a primary antibody as a mouse anti-C2 tag monoclonal antibody. The result is shown in FIG.

  It was confirmed that SA0962 interacted with SA1027 and coprecipitated only under the pull-down assay conditions in which SA1027 was immobilized. In addition, since the binding between the two is weakened in the presence of stalovacin, it was considered that stalovacin inhibits the formation of the cell division apparatus divome by inhibiting the interaction between SA0962 and SA1027, and exerts a strong antibacterial activity.

Example 5 Confirmation of Survival Essentiality of SA0962 Complementation Test with Temperature Sensitive Mutant To confirm the essentiality of survival of SA0962, the following experiment was conducted using a temperature sensitive mutant TS3021 of S. aureus RN4220 strain. Went. A temperature-sensitive mutant is specifically a mutant that can grow at 30 ° C but cannot grow at 44 ° C.
S. A full-length PCR amplification of the SA0962 ORF was performed using the genomic DNA of the Aureus RN4220 strain as a template. The amplified fragment was treated with restriction enzymes SmiI and NotI, and inserted into the shuttle vector pSR1000 to construct an SA0962 expression-inducing plasmid. Since this plasmid has xylR and xylO, expression of SA0962 can be induced by xylose.
The above plasmid was introduced into TS3021 by electroporation. B2 broth [composition: 1 wt% casamino acid, 2.5 wt% yeast extract, 0.1 wt% dipotassium hydrogen phosphate, 0.5 wt% glucose, 2.5 wt% sodium chloride (PH 7.5)] after culturing at 30 ° C. for 1 hour, chloramphenicol-containing TSA (Tryptic Soy Agar) [Composition: 1.5 wt% casein pancreatic juice digest, 0.5 wt% soybean papain Digested product, 0.5 wt% sodium chloride, 1.5 wt% agar (pH 7.3 ± 0.2)] was applied to the medium and cultured at 30 ° C. for 1 day. The formed colonies were isolated on the above plate medium to obtain a TS3021 recombinant strain into which the SA0962 expression-inducing plasmid was introduced.
For control purposes, a strain in which pSR1000 was introduced into TS3021 was prepared in the same manner as described above.
Two suspensions of the prepared two types of recombinant strains were seeded on a chloramphenicol and xylose-containing TSA medium, and one of them was cultured at 30 ° C. and the other at 44 ° C. for 1 day.

  As a result, the strain into which pSR1000 was introduced, that is, the strain not induced to induce expression of SA0962 grew at 30 ° C, but did not grow at 44 ° C. On the other hand, the recombinant strain in which SA0962 expression was induced could grow under both conditions of 30 ° C and 44 ° C. This confirmed that the temperature sensitivity of TS3021 disappeared by complementing SA0962 (Table 3).

  From the above, it was found that the temperature sensitivity of TS3021 was caused by the mutation generated on the SA0962 gene, and it was revealed that SA0962 is an essential gene in the growth of S. aureus. .

Example 6 Confirmation of SA1027 Survival Essentiality Construction of SA1027 Gene Deletion Plasmid Chromosomal DNA was isolated from S. aureus RN4220 strain by a conventional method. A part of SA1026 and SA1028 was amplified by PCR method using the obtained chromosomal DNA as a template, and each of the obtained fragments was incorporated into a temperature sensitive vector plasmid pCL52.1 to obtain a SA1027 gene deletion plasmid on S. aureus RN4220 chromosome. .

Construction of SA1027 Gene Deletion Plasmid Insertion Strain into Chromosome The resulting SA1027 gene deletion plasmid was introduced into S. aureus RN4220 according to the method of Steve Schenk et al. (FEMS Microbiol Lett., 94, page 133-138). . A colony of the obtained transformant was picked up, suspended in physiological saline, and tetracycline-containing NYE agar medium [Composition: 1% by weight casamino acid, 0.5% by weight yeast extract, 0.5% by weight sodium chloride, 1. 5% by weight agar] and cultured at 42 ° C. About the obtained colony, it confirmed that the SA1027 gene deletion plasmid was inserted on the chromosome by SA1026 side by PCR method.

Construction of S. aureus SA1027 gene expression control plasmid The SA1027 gene was amplified by PCR using chromosomal DNA as a template, and the resulting fragment was incorporated into an expression control plasmid vector pSR1000 to obtain a SA1027 gene expression control plasmid of S. aureus RN4220.

Construction of Staphylococcus aureus SA1027 gene expression control strain The obtained SA1027 gene expression control plasmid was transformed into the SA1027 gene deletion plasmid insertion strain in the same manner as in the above-mentioned "construction of SA1027 gene deletion plasmid insert into the chromosome". Introduced. A colony of the obtained transformant was picked up, and LB liquid medium containing chloramphenicol and xylose [Composition: 1% by weight bactotryptone, 0.5% by weight yeast extract, 1% by weight sodium chloride (pH 7.0)] The cells were cultured at 42 ° C. in a medium, and applied to a chloramphenicol and xylose-containing NYE agar medium and cultured. Then, the resulting colonies were inoculated by replica method on NYE agar medium containing chloramphenicol and xylose and NYE agar medium containing tetracycline and xylose. Subsequently, the replica plate was cultured at 42 ° C., and tetracycline sensitive strains were selected and purified. As a result, a strain in which deletion of the SA1027 gene on the chromosomal DNA was confirmed by the PCR method was obtained as a S. aureus SA1027 gene expression regulatory strain. At the same time, a strain in which the SA1027 gene on the chromosome was not deleted and had the same genotype as that of the RN4220 strain was obtained as a revertant strain.

Confirmation of Essentiality of S. aureus SA1027 Protein The obtained SA1027 gene expression-regulated strain and revertant strain were linearly cultured overnight at 37 ° C. on a chloramphenicol- and xylose-containing NYE agar medium, and then Mueller Hinton liquid medium [ Composition: 0.2 wt% beef extract, 1.75 wt% caseinate hydrolyzed peptone, 0.15 wt% soluble starch]. On the other hand, the parent strain RN4220 was similarly cultured on a xylose-containing NYE agar medium and then diluted in Mueller Hinton liquid medium. Each of the obtained suspensions was diluted and inoculated into a 96-well plate into which Mueller Hinton liquid medium and xylose-containing Mueller Hinton liquid medium were dispensed, and cultured at 35 ° C. The growth of the fungus was confirmed by turbidity at a wavelength of 595 nm. The result of the xylose non-addition condition is shown in FIG. 14, and the result of the xylose addition condition is shown in FIG.

  It was confirmed that under the condition where xylose was not added, the growth of the SA1027 gene expression-regulated strain was clearly suppressed as compared to the RN4220 strain and the reversion strain. On the other hand, under the xylose addition conditions, the SA1027 gene expression-regulated strain showed the same growth as the RN4220 strain and the reverted strain. The slight growth of the SA1027 gene expression-regulated strain was considered to be because the SA1027 gene was slightly expressed from the SA1027 gene expression-regulating plasmid even in the absence of xylose to complement the gene deletion. From the above results, it was confirmed that SA1027 protein is an essential protein for growth in Staphylococcus aureus.

  By the screening method of the present invention, an antibacterial agent having a novel action mechanism can be screened. By this method, a novel antibacterial agent having a broad spectrum and strong antibacterial activity can be obtained.

Claims (5)

A method for screening an antibacterial agent, comprising the steps of bringing FtsW or a partial peptide thereof into contact with a test substance, and selecting a test substance that binds to the FtsW or a partial peptide thereof. Contacting one or more polypeptides selected from the group consisting of FtsQ, FtsK and their partial peptides with FtsW or its partial peptides in the presence of a test substance, and said polypeptide, FtsW or its partial peptides A method for screening an antibacterial agent, the method comprising a step of selecting a test substance that inhibits binding to the antibody. The screening method according to claim 1 or 2, wherein the partial peptide of FtsW is a peptide containing a 9/10 loop. The screening method according to any one of claims 1 to 3, wherein the FtsW, FtsQ, and FtsK are polypeptides derived from Staphylococcus aureus. Fts W or containing that part peptide, the screening kit of the antimicrobial agent.

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