WO2009154016A1 - Procédé d’évaluation de la pathogénicité de bactérie pathogène, kit pour évaluer la pathogénicité et gène pour évaluer la pathogénicité - Google Patents

Procédé d’évaluation de la pathogénicité de bactérie pathogène, kit pour évaluer la pathogénicité et gène pour évaluer la pathogénicité Download PDF

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WO2009154016A1
WO2009154016A1 PCT/JP2009/052647 JP2009052647W WO2009154016A1 WO 2009154016 A1 WO2009154016 A1 WO 2009154016A1 JP 2009052647 W JP2009052647 W JP 2009052647W WO 2009154016 A1 WO2009154016 A1 WO 2009154016A1
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gene
pathogenicity
evaluating
function
ability
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PCT/JP2009/052647
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Japanese (ja)
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和久 関水
力 垣内
洋 浜本
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株式会社ゲノム創薬研究所
国立大学法人東京大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)

Definitions

  • MRSA Methicillin-resistant Staphylococcus aureus
  • This MRSA has been considered to be not particularly strong in pathogenicity other than drug resistance when transported from a hospital to a city such as a home, and to be easily destroyed.
  • MRSA has been detected in outpatients, and the presence of community-acquired MRSA with high pathogenicity has been reported, and a new response to MRSA with high pathogenicity has been demanded. It was.
  • a method for identifying a certain bacterium is roughly divided into a method for analyzing the external characteristics of the bacterium, a method for analyzing the biological activity of the bacterium, and a method for analyzing the characteristics of the genotype. So far, pathogenicity has been identified and evaluated by hemolytic activity, vomiting activity, lethal strength, etc. derived from the toxin produced by the bacterium. In addition, the characteristics of genotypes related to pathogenicity have been identified by the presence or absence of genes related to the toxin (for example, Non-Patent Documents 1 to 3).
  • Non-patent Document 4 the inventor's research has revealed that Staphylococcus aureus, which has been conventionally considered not to have a migration ability on a solid surface, has the ability to slide on a soft agar medium. Furthermore, when the present inventor investigated the correlation between the staphylococci of S. aureus and the virulence of S. aureus, it was found that S. aureus having a low slidability has a clearly lower pathogenicity than a higher one. I found it.
  • Patent Document 1 Japanese Patent Application No. 2008-158176
  • JP 2008-148669 A Tatsuo Yamamoto et al., Japanese Society of Chemotherapy, 52 (11), 635-652 (2004) Teruyo Ito et al., Journal of Infectious Diseases, 78 (6), 459-469 (2004) Shigemo Katsuhiko, Food Sanitation, 51 (4), 81-90 (2005) C. Kaito and K. Sekimizu J. Bacteriol., 189 (6), 2553-2557 (2007)
  • the present invention has been made in view of the above, and its problem is to find characteristics of a pathogenic bacterial gene that exhibits pathogenicity but has not yet been clarified about the gene, and targets that characteristic. It is an object of the present invention to provide a method for evaluating pathogenicity of pathogenic bacteria, a primer for evaluating pathogenicity, a kit for evaluating pathogenicity including the primer, and the like.
  • MSSA methicillin-sensitive Staphylococcus aureus
  • the fudh gene suppresses the gliding ability and the pathogenicity also decreases accordingly. It has been confirmed that the present invention has been completed.
  • the present invention (1) A method for evaluating the pathogenicity of a pathogenic bacterium, the presence or absence of a gene encoding a protein having a function of controlling the gliding ability of the pathogenic bacterium, or the function of the preceding protein in the gene A pathogenicity evaluation method characterized by confirming whether or not there is at least one mutation which gives (2) A method for evaluating the pathogenicity of a pathogenic bacterium, wherein the presence or absence of a gene encoding a protein having a function of suppressing the gliding ability of the pathogenic bacterium, or the function of the preceding protein in the gene is suppressed.
  • the pathogenicity evaluation method according to (1), wherein whether or not there is at least one mutation to be confirmed is confirmed.
  • (4) A method for evaluating the pathogenicity of methicillin-resistant Staphylococcus aureus, the presence or absence of a gene encoding a protein having a function of suppressing the gliding ability of methicillin-resistant Staphylococcus aureus, or SEQ ID NO: 1 in the gene The pathogenicity evaluation method according to (3), wherein whether or not there is a mutation in the base sequence corresponding to amino acid number 29 described in (3) is confirmed.
  • the gene encoding a protein having a function of suppressing the gliding ability of pathogenic bacteria is a DNA comprising the base sequence set forth in SEQ ID NO: 1 (1) to (4) Evaluation method of pathogenicity.
  • a kit for evaluating the pathogenicity of pathogenic bacteria characterized by using the pathogenicity evaluation method according to any one of (1) to (5).
  • At least a primer for detecting a gene encoding a protein having a function of suppressing the gliding ability of pathogenic bacteria, or a primer for detecting a mutation that suppresses the function of a previous protein in the gene The kit for evaluating pathogenicity of pathogenic bacteria according to (6), which has one.
  • a gene comprising the following DNA (a) or (b): (A) DNA consisting of the base sequence described in SEQ ID NO: 1 and having a function of suppressing the gliding ability of pathogenic bacteria and encoding a protein involved in the pathogenicity of the fungus; (B) a DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1; (9) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 and having a function of suppressing the gliding ability of pathogenic bacteria and involved in the pathogenicity of the fungus.
  • a method for evaluating pathogenicity by the genotype of drug-resistant bacteria can be provided. That is, the degree of pathogenicity of a pathogenic bacterium, the presence or absence of a new gene that controls the gliding ability of the pathogenic bacterium, or at least one mutation present in the new gene that affects the function of the gene By confirming the presence or absence of “,” it is possible to make a reliable and quick evaluation. As a result, a treatment policy such as selection of an appropriate drug for the patient can be determined, and the spread of infection can be prevented. In addition, since it is possible to accurately evaluate the high pathogenicity of drug-resistant bacteria such as MRSA that have been difficult to identify, it is possible to bring about appropriate use of drugs that are sensitive to MRSA such as vancomycin. is there.
  • FIG. 4 A diagram showing plasmids introduced with these regions, which were prepared in order to examine in which region in the SCCmec region a gene that suppresses gliding ability exists.
  • B It is the figure which showed the state of the colony when the sliding ability of the strain
  • C A graph summarizing the results of comparing the gliding ability of strains into which each plasmid was introduced. It is the figure which compared the base sequence of the fudoh gene in 40 clinically isolated MRSA strains, and the glide ability which each strain
  • the present invention confirms the presence or absence of a newly found gene related to the control of gliding ability of a pathogenic bacterium, or whether or not there is a mutation that affects the previous factor in the gene. It is a method for evaluating the high pathogenicity of, and can be carried out by executing a normal gene detection means and a means for detecting the mutated portion thereof.
  • a gene amplification method such as a polymerase chain reaction (PCR) method, a real-time PCR method, a Loop-Mediated Isothermal Amplification (LAMP) method is executed,
  • PCR polymerase chain reaction
  • LAMP Loop-Mediated Isothermal Amplification
  • Non-Patent Documents 1 to 3 the evaluation of bacterial pathogenicity based on the genotype has been carried out mainly by evaluating the gene related to the “toxin” possessed by the bacteria (see Non-Patent Documents 1 to 3). Evaluation of pathogenicity by a gene relating to “sliding ability” has not been performed conventionally. Therefore, the fact that the pathogenicity of pathogenic bacteria can be evaluated by a gene related to “sliding ability”, which is the basis of the present invention, will be described with respect to the examination content that is the basis thereof.
  • sliding ability refers to the ability of bacteria to move on a soft agar medium
  • size refers to the size of the colony spread on the soft agar medium under certain conditions.
  • high pathogenicity refers to the strength of damage to a host under the condition that the pathogen infects the host, and is evaluated separately from “presence or absence of drug resistance”.
  • Staphylococcus aureus is also a Gram-positive bacterium that does not have flagella, and until recently was thought to have no gliding ability.
  • the present inventors have found that this S. aureus has the ability to slide on a soft agar medium (Non-Patent Document 4).
  • the gliding ability of bacteria with distinct motor organs such as flagella is thought to play an important role in the pathogenicity of the bacteria.
  • the relationship between the gliding ability of Staphylococcus aureus found in the soft agar medium discovered by the present inventor and the high pathogenicity of the bacterium has been unknown.
  • the present inventor considers that the ability of S. aureus gliding to play a role in its pathogenicity, and the relationship between the magnitude of the gliding ability of the strain in the soft agar medium and the pathogenicity of the strain against the host. As a result of confirming whether or not there is a silkworm as a host, it has been found that the magnitude of gliding ability correlates well with its pathogenicity, and a patent application has already been filed (Japanese Patent Application No. 2006-342713).
  • the invention according to the already filed application is to evaluate the characteristics that appear on the outer surface among various pathogenicity evaluation methods, whereas the present invention clarifies genes related to the gliding ability of pathogenic bacteria, It is characterized by evaluating pathogenicity according to the characteristics of its genotype.
  • the gliding ability of 10 clinical isolates MSSA and 40 MRSA was compared.
  • See Test Example 1 the average value of the running distance of the MSSA strain was 67.7 mm, whereas the average value of the running distance of the MRSA strain was 31.7 mm.
  • 29 of the 40 MRSA strains had a sliding distance of 35 mm or less, and it was found that most of the MRSA strains reduced the sliding ability (FIG. 1).
  • the sliding ability of the SCCmec region is low.
  • the SCCmec region was removed from the MRSA strain and its sliding ability was confirmed (see Test Example 2).
  • the PCR method confirmed that the mecA gene was not present in these strains (FIG. 2), and the resistance to methicillin was reduced. In addition, all of these strains were found to have increased gliding ability (FIGS. 3 (A) and 3 (B)).
  • a plasmid having a region containing the mecA gene, mecR1 gene, and mecI gene in the SCCmec region is methicillin-sensitive yellow. It was inserted into the chromosome of a Newman strain, which is a staphylococcus, and its sliding ability in a soft agar medium was examined (see Test Example 3). As a result, the strain into which the plasmid having the region containing the previous 3 genes and the vicinity thereof had been introduced had a lower glide ability than the strain into which the empty vector was introduced (FIGS. 4B and 4C). ).
  • the mutated fudoh gene was introduced into the Newman strain, which is an MSSA strain, and the gliding ability was examined (Test Example 4).
  • the previous mutant fudoh gene did not inhibit the sliding ability of the Newman strain at all (FIGS. 6A and 6B). Therefore, the previous mutant fudoh gene has lost the ability to suppress gliding ability, and the high gliding ability of 10 out of 11 MRSA strains is explained by the fact that it has lost its function due to mutation of the fudoh gene. It became clear that we could do it.
  • the present invention is a gene comprising the following DNA (a) or (b).
  • the gene “fudh” comprising such DNA is a gene having a function newly found in the present invention.
  • NI-5 strain whose parent strain is MRSA strain, whose gliding ability was increased by dropping of the SCCmec region, and vice versa
  • the pathogenicity to mice and silkworm larvae (hereinafter abbreviated as “Silkworm”) of the Newman strain whose parent strain is the MSSA strain, whose gliding ability was suppressed by introduction was examined.
  • a characteristic feature of the present invention is not only that the fudhoh gene has been found, but if the function of fudoh gene is mutated to suppress gliding ability, the gliding ability increases and that pathogenicity In contrast, the presence of the fudhoh gene suppresses gliding ability, thereby reducing pathogenicity.
  • the fudh gene was inactivated by some new mutations such as point mutations, deletions, duplications, inversions, insertions, translocations, etc. other than single nucleotide mutations with amino acid substitutions specifically found by the present invention.
  • some new mutations such as point mutations, deletions, duplications, inversions, insertions, translocations, etc.
  • the pathogenic bacterium applicable to the present invention completed by the examination described above is a bacterium having gliding ability and a pathogenic bacterium having a gene encoding a protein having a function of controlling gliding ability. Either may be used.
  • MRSA MRSA
  • the present invention was made by using MRSA, it is apparent in principle that pathogenic bacteria are not limited to MRSA from the relationship between gliding ability and pathogenicity.
  • the pathogenic bacterium applicable to the present invention is a bacterium having a gliding ability and having a fudh gene, and the gliding ability is suppressed by the fudh gene, naturally, MRSA and Similarly, the pathogenicity can be evaluated. Examples of such bacterial candidates include staphylococcus epidermidis other than Staphylococcus aureus whose presence of the fudh gene has been clarified by disclosure of genomic information, but the present invention is not limited thereto. is not.
  • the bacteria in the clinical specimen are Staphylococcus aureus, the presence or absence of methicillin resistance, the presence or absence of the fudhoh gene found this time and the gene Check if there is a mutation in it.
  • Staphylococcus aureus for example, a pair of forward and reverse primers that amplify the protein A gene spa can be used.
  • mecA-F as a forward primer for amplifying the mecA gene shown in Table 2
  • mecA-R as a reverse primer
  • the present invention is an evaluation kit for using the above pathogenicity evaluation method, wherein a gene encoding a protein having a function of suppressing the gliding ability of pathogenic bacteria such as methicillin-resistant Staphylococcus aureus is obtained. It is also a “kit for evaluating pathogenicity of pathogenic bacteria” characterized by having at least one primer for detecting or detecting a mutation that suppresses the function of the previous protein in the gene. .
  • a pair of fudh-F and fudhoh-R primers shown in Table 2 can be used.
  • PCR amplification was performed using a pair of primers S2 and S3 shown in Table 2, and the nucleotide sequence of the PCR product was determined. Determine using S2 and S3 primers. Since the mutation of the fudh gene cannot be detected directly with this primer, the base sequence of the PCR product amplified using this primer is determined using the same primer. As a result, it can be seen whether or not the mutation exists.
  • a primer suitable for ARMS Amplification Refractory Mutation System
  • Primers used in the present invention for confirming the presence or mutation of the fudhoh gene and its mutation are not limited to the above primers, and detection of the fudhoh gene and loss of gene function in the gene are the same as the above primers. Any material can be used as long as it can be used for the purpose of detecting a mutation that brings about activity. Such primers include DNA in which a part of the previous primer has been modified by deletion, substitution, insertion, addition, or the like as long as the same function is exhibited. Further, in the future, when a new mutation causing inactivation of gene function is found, an invention using a newly designed primer for detecting the mutation is also within the scope of the present invention.
  • Identification of the bacterial species in the clinical specimen, confirmation of drug resistance, presence of the fudhoh gene, and confirmation of whether or not there is a mutation in the gene may be performed by means of detecting each separately. , All of them may be detected simultaneously.
  • each of the previous confirmations may be carried out by ordinary PCR with a set of primers used for each in an independent system.
  • Multiplex PCR can be performed in which all primer sets are mixed and the PCR reaction is performed in the same reaction system.
  • the presence or absence of a PCR amplification product corresponding to each gene can be confirmed by confirming the presence or absence of a band obtained by electrophoresis by agarose gel electrophoresis or capillary electrophoresis.
  • Nucleic acids are extracted from patient-derived specimens such as blood, sputum, pus, and pharyngeal swabs, or materials such as gauze used for patients, or bacterial fluids obtained by culturing them. Extraction can be performed by heat treatment, enzyme treatment, or using a commercially available DNA extraction kit. In the case of staphylococci, DNA extraction efficiency can be increased by using an enzyme suitable for degradation of staphylococcal membranes such as lysostaphin and achromopeptidase. Moreover, the sensitivity of a test
  • inspection can be raised by processing the obtained extract by nucleic acid purification methods, such as a phenol process, and also adding concentration operation.
  • PCR may be performed under the usual conditions, for example, using Taq DNA polymerase, a heat denaturation step at 92 ° C. for 15 seconds, an annealing step at 60 ° C. to 65 ° C. for 15 seconds, and an extension step at 72 ° C. for 5 seconds to 15 seconds. Can be performed under the condition of repeating the above.
  • Determination of DNA amplification by PCR is performed by confirming the size of the generated amplified DNA by electrophoresis, or by immobilizing a reaction product on a nitrocellulose membrane or the like and using a labeled probe having a sequence complementary to the sequence of the target amplified DNA.
  • a method of performing hybridization is possible. If you want to determine DNA amplification more easily, perform PCR using a primer with a identifiable label, capture the amplified DNA labeled with digoxigenin, etc. on the solid phase and identify it. Good.
  • Other methods for capturing and identifying amplified DNA include a method using a solid phase in which a substance that specifically binds to the labeled product of the primer is immobilized, and a capture probe having a sequence complementary to the sequence of the target amplified DNA. There is a method of preliminarily fixing to a solid phase and performing specific binding by hybridization, etc.
  • a microtiter plate, beads, magnetic particles or the like can be used as the solid phase for that purpose. The use of microtiter plates is preferred when processing multiple samples at once.
  • the specificity and rapidity of the reaction can be enhanced by using hybridization with a capture probe immobilized on a microtiter plate or the like for capturing amplified DNA. Therefore, complementary DNA and DNA fragments that hybridize to the fudoh gene can be used as materials for capture probes and the like, so that the specificity and rapidity of the reaction can be improved.
  • the combination of the primer for detecting the fudoh gene and the mutation site detecting primer used in the present invention as described above is provided as a primer set for evaluating the pathogenicity of pathogenic bacteria, as well as an enzyme for DNA amplification and reaction.
  • a kit for evaluating pathogenicity optimized for evaluating pathogenicity by being set with a DNA synthesis reagent such as a buffer, a labeled capture probe, or a microwell plate on which the capture probe is immobilized. it can.
  • a primer set or kit for performing an amplification method other than normal PCR by separately designing and combining primers suitable for the LAMP method or the like.
  • the present invention is useful in that the pathogenicity of pathogenic bacteria can be evaluated from an unconventional viewpoint.
  • a so-called community-acquired MRSA with high pathogenicity in Japan has not been found a method capable of evaluating the pathogenicity of MRSA with high pathogenicity as a genotype.
  • the fudoh gene which is thought to encode a protein having a function of suppressing the gliding ability of pathogenic bacteria found by the present inventors, and a loss of function due to mutation of the gene, MRSA
  • MRSA The fact that the pathogenicity is increased together with the increase in gliding ability makes it possible to clearly distinguish MRSA having high pathogenicity from ordinary MRSA by targeting the fudhoh gene and its mutation.
  • the “kit for evaluating pathogenicity of pathogenic bacteria” of the present invention is a method for confirming the presence or absence of a marker gene that can be evaluated for pathogenicity of pathogenic bacteria, or a marker gene thereof, It is assembled so that the presence or absence of mutation can be confirmed. That is, whether or not a gene encoding a protein that controls or suppresses the gliding ability of a pathogenic bacterium is present in the pathogenic bacterium to be evaluated, and if that gene exists, It is characterized by being a “kit for evaluating pathogenicity of pathogenic bacteria” that can confirm whether or not there is at least one mutation that affects or suppresses the function of the protein. .
  • the “kit for evaluating pathogenicity of pathogenic bacteria” of the present invention only needs to contain at least a primer set designed for confirming the presence or absence of the marker gene. This is because the presence or absence of a mutation in the marker gene can be confirmed together with the presence of the marker gene by determining the base sequence of the PCR product amplified by the primer set using the same primer. Further, when the mutation to be detected is known, it is preferable that a primer set prepared for confirming the presence of the mutation in the marker gene is in the kit configuration. This is because the presence or absence of mutation can be confirmed more easily.
  • the above-mentioned “primer set prepared for confirming the presence of the mutation” includes, for example, a primer set compatible with ARMS (Amplification Refractory Mutation System).
  • ARMS is a method that utilizes the fact that the function of PCR as a primer strongly depends on the matching between the 3 'end of the primer and the template DNA. Therefore, it is a feature of ARMS that the primer is designed so that the target mutation site comes to the 3 'end.
  • annealing is often possible and amplification reaction often proceeds. At this time, at least one more upstream of the 3 ′ end of the primer It is preferable to design the primer so as to artificially introduce a mismatch. This is because the annealing cannot be performed well, the amplification reaction is impossible, and the specificity of mutation detection is improved (Kwok S. et al., Nucleic Acids Res 18, 999-1005 (1990)).
  • a primer that has only an artificially introduced mismatch and can be annealed is created as a normal primer, and the primer introduced with the previous artificial mismatch is mutated in addition to the mismatch due to mutation.
  • the primer introduced with the previous artificial mismatch is mutated in addition to the mismatch due to mutation.
  • the present invention is not limited to the following specific operation. That is, the test sample is divided into half amounts, PCR is performed by adding a normal primer and its counter primer to one, and PCR is performed by adding a mutation primer and its counter primer to the other.
  • the normal primer hybridizes with the normal gene and PCR proceeds, but does not hybridize with the mutant gene, so the mutant gene is not amplified.
  • the mutation primer hybridizes with the mutant gene and proceeds with PCR, but does not hybridize with the normal gene, so the normal gene is not amplified. Therefore, it is possible to determine whether or not there is a target mutation on the DNA of the pathogenic bacterium to be evaluated by examining each band of amplified DNA by performing agarose electrophoresis after PCR.
  • the marker gene encoding a protein that controls or suppresses the gliding ability of the pathogenic bacterium is “fudhoh” found by the present inventors, and its presence is confirmed.
  • a set of fudoh-F and fudoh-R primers shown in Table 2 can be used.
  • a mutation that loses the function of suppressing the sliding ability of the fudhoh gene a single base mutation (K29R) at the site shown in FIG. 5 has been found. If incorporated in a kit, mutations can be detected more easily.
  • the kit of the present invention contains a primer set for identifying pathogenic bacteria, an internal control DNA for confirming that PCR was normally performed, and a primer set for detecting the DNA.
  • a primer set for identifying pathogenic bacteria Is preferred.
  • the pathogenic bacterium is Staphylococcus aureus
  • Plasmid E. coli strain JM109 was used for the preparation of pND50, pCK20 plasmids and their derivatives. E. coli transformed with the above plasmid was cultured at 37 ° C. in LB (Luria-Bertani) liquid medium containing 25 ⁇ g / mL chloramphenicol.
  • S. aureus genomic DNA Extraction of S. aureus genomic DNA was performed using QIAamp DNA Blood Kit (Qiagen).
  • SCCmec region dropout strain The ccrAB gene encoding the SCCmec region excision enzyme was inserted into the pND50 plasmid to obtain a pcccAB plasmid.
  • This plasmid was introduced into the MRSA strains NI-3, NI-4 and NI-5, which have low gliding ability, to obtain a chloramphenicol resistant strain, 5 ⁇ L of which was added with 5 mL of chloramphenicol.
  • the cells were cultured overnight in a TSB (Tryptic Soy Broth) liquid medium. After repeating the same operation, a single colony was obtained on a TS (Tryptic Soy) agar medium. This strain was used as a SCCmec region-removed strain in the subsequent studies.
  • the Newman strain is a laboratory-owned strain of the University of Tokyo graduate School of Microbial Medicine Chemistry, which is a MSSA strain and a high gliding ability.
  • a plasmid having regions containing the mecA gene, mecR1 gene, and mecI gene in the SCCmec region was inserted into the chromosome of the Newman strain and used for further studies.
  • Silkworm Incubated from fertilized eggs in the laboratory, and grown to 5th instar larvae with artificial diet silk mate (manufactured by Katakura Kogyo) was used for pathogenicity evaluation of genetically modified strains.
  • mice Eight-week-old female CD-1 mice (Charles River Laboratories) were used to evaluate the pathogenicity of the strain in the same manner as silkworms.
  • Table 1 shows a list of strains and plasmids used in Examples and Test Examples, and Table 2 shows a list of primers.
  • MSSA methicillin-sensitive Staphylococcus aureus
  • the SCCmec region is a region where the mecA gene, which is a gene conferring methicillin resistance, is present.
  • the SCCmec region was dropped from the MRSA strain, and the gliding ability was examined.
  • the ccrAB gene encoding the excision enzyme of the SCCmec region was expressed in NI-3, NI-4, and NI-5 strains with low sliding ability, and the SCCmec region was dropped.
  • the PCR method confirmed that the mecA gene was not present in these strains (FIG.
  • the strain into which the region containing three genes and the vicinity thereof (pIntmecAR1I-fudhoh) was introduced had reduced gliding ability compared to the strain into which the empty vector pInt was introduced (FIGS. 4B and 4C). .
  • Example 1 Suppression of gliding ability by introduction of fudoh gene Since the result of Test Example 3 suggests the presence of a gene not known so far, the ORF on the SCCmec region was examined in detail. Then, in the genome database of S. aureus N315 strain, an ORF encoding 70 amino acids not registered as ORF was found (upper right right portion of FIG. 4A). Therefore, a plasmid (pInt fudoh) having this gene was introduced into a Newman strain, and its gliding ability was examined. As a result, the Newman strain into which pInt fudoh was introduced had suppressed gliding ability (FIG. 4 (B), FIG. 4 (C) fudoh-introduced strain). The present inventor named the novel gene having a function of suppressing the sliding ability as “fudh”.
  • Primer sets (Table 2, fudhoh-F, fudhoh-R) were designed by a conventional method so that they could be annealed with a specific base sequence contained in the fudhoh gene, and synthesized with a DNA synthesizer. Producing various labeled primers such as biotin label and dinitrophenyl label by introducing an amino group into the 5 'terminal base of this primer using Aminolink II (Trademark: Applied Biosystems Japan). Can do.
  • PCR implementation The reaction was carried out using a PC-350 gene amplification apparatus manufactured by ASTEC under the following reaction conditions. DNA denaturation: 1st 94 ° C, 120 sec From the second time 94 °C, 15sec Annealing: 52 ° C, 30 sec Extension reaction 72 °C, 60sec The above reaction was performed for 25 cycles. Confirmation of the obtained PCR product was performed by agarose gel electrophoresis.
  • kits for using the method for evaluating the pathogenicity of pathogenic bacteria, and a primer for detecting a gene encoding a protein having a function of suppressing the gliding ability of pathogenic bacteria or It was found that a kit for evaluating the pathogenicity of pathogenic bacteria having at least one primer for detecting a mutation that suppresses the function of the previous protein in the gene can be actually produced.
  • Example 3 Gliding ability of Newman strain introduced with mutant fudhoh gene
  • K29Rfudhoh was introduced into a Newman strain with high gliding ability.
  • the ability to suppress the sliding ability was confirmed.
  • K29Rfudhoh did not inhibit the sliding ability of the Newman strain at all (FIGS. 6A and 6B).
  • Example 1 From these results and the results of Example 1, it was clarified that the high gliding ability of 11 strains can be explained by the mutation of the fudhoh gene in 10 strains and the absence of the fudhoh gene in 1 strain.
  • the relationship between the gliding ability of S. aureus and the pathogenicity to the host For the purpose of studying the silkworm of the NI-5 strain (parent strain MRSA strain) whose gliding ability was increased by dropping the SCCmec region, and conversely, the Newman strain (parent strain MSSA strain) whose gliding ability was suppressed by introduction of the fudh gene. Pathogenicity was examined.
  • Example 4-1 Pathogenicity of SCCmec-dropped MRSA strain against silkworms NI-5 strain or SCCmec-dropped NI-5 strain (1.9 ⁇ 10 4 each) in which control vector pND50 was introduced into 10 5-year-old silkworms per group 50 ⁇ L of cfu / overnight culture was diluted with physiological saline) and injected into the blood. The physiological saline injection group was used as a sterile control. Each silkworm was fasted and kept at 37 ° C., and the survival rate per hour after injection was monitored. The results are shown in FIG. 7A.
  • Example 4-2 Pathogenicity of fudoh-introduced MSSA strains against silkworms: Newman strain in which empty vector pInt has been introduced into 10 groups of five-year-old silkworms or Newman strains into which fudoh gene has been introduced (each 1.5 ⁇ 10 4 cfu) / The overnight culture was diluted with physiological saline) and 50 ⁇ L was injected into the blood. The physiological saline injection group was used as a sterility control. Silkworms were fasted and kept at 40 ° C., and the survival rate per hour after injection was monitored. The result is shown in FIG. 7B.
  • the newly discovered fudoh gene has a function of suppressing the ability of Staphylococcus aureus to slide, and is also involved in the high pathogenicity of Staphylococcus aureus.
  • Example 3 since S. aureus having a mutated fudoh gene has increased gliding ability, it is considered that S. aureus having a mutated fudoh gene has increased pathogenicity to the host. It is done. Performing a means for detecting the presence or absence of a single base mutation that results in substitution of the amino acid of the 29th amino acid part of fudhoh found in the present invention is similar to performing a means for detecting the presence or absence of fudoh itself. It is clear that this is a method for evaluating the high pathogenicity of pathogenic bacteria to a host. Similarly, even when a new mutation is found in fudoh, using the phenotype “sliding ability” as a clue, it is easy to detect the mutation, and the detection of the mutation The pathogenicity of pathogenic bacteria can be evaluated.
  • the present invention is not limited to the invention relating to the specific “fudhoh gene”, but extends to the invention relating to “a gene encoding a protein having a function of controlling the gliding ability of pathogenic bacteria”. It is also clear from the basic inventive properties of the invention.
  • the pathogenicity evaluation method for pathogenic bacteria of the present invention evaluates the high pathogenicity of pathogenic bacteria that could not be evaluated for the high pathogenicity depending on the genotype related to conventional toxins and drug resistance. Therefore, it is widely used in fields where new drugs and treatment methods for pathogenic bacteria are being developed. In addition, by clarifying the genes used for pathogenicity assessment and the mutations in those genes, it is possible to use any gene amplification method targeting them, so clinical trials that require rapid and accurate result determination are required. Since an evaluation kit based on the principle of a method for evaluating a pathogenic bacterium or a gene amplification method can be provided at the site, it is widely used in the clinical field.

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Abstract

La présente invention concerne un procédé d’évaluation par lequel la pathogénicité d’une bactérie pathogène peut être rapidement et sûrement évaluée par détection du génotype caractéristique de la bactérie et un kit d’évaluation pour celui-ci. La présente invention concerne un procédé d’évaluation de la pathogénicité d’une bactérie pathogène caractérisé en ce qu’il comprend la confirmation de la présence ou l’absence d’un gène qui code pour une protéine ayant une fonction de contrôle de la motilité par glissement de la bactérie pathogène ou l’occurrence ou la non-occurrence d’au moins une mutation affectant la fonction précédente de la protéine dans le gène ; un kit pour évaluer la pathogénicité d’une bactérie pathogène en utilisant le procédé d’évaluation de la pathogénicité comme décrit ci-dessus ; et un gène qui comprend l’ADN suivant (a) ou (b) : (a) un ADN comprenant la séquence de bases représentée par SEQ ID N° 1, ayant une fonction de contrôle de la motilité par glissement d’une bactérie pathogène et participant à la pathogénicité de la bactérie ; et (b) un ADN étant hybridable dans des conditions stringentes avec un ADN comprenant la séquence de bases représentée par SEQ ID N° 1.
PCT/JP2009/052647 2008-06-17 2009-02-17 Procédé d’évaluation de la pathogénicité de bactérie pathogène, kit pour évaluer la pathogénicité et gène pour évaluer la pathogénicité WO2009154016A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
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JP2008148669A (ja) * 2006-12-20 2008-07-03 Genome Soyaku Kenkyusho:Kk 感染症の予防又は治療のための薬剤及びその製造方法、評価方法及びスクリーニング方法、並びに、病原性細菌の病原性の評価方法及び感染症の検査方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008148669A (ja) * 2006-12-20 2008-07-03 Genome Soyaku Kenkyusho:Kk 感染症の予防又は治療のための薬剤及びその製造方法、評価方法及びスクリーニング方法、並びに、病原性細菌の病原性の評価方法及び感染症の検査方法

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"Dai 53 Kai Japanese Symposium on Staphylococci and Staphylococcal Infections Program · Koen Yoshishu, 2008.", vol. 10, article YOSUKE OMAE ET AL.: "Oshoku Budo Kyukin no Kasso Noryoku to Byogensei Yokuatsu ni Hataraku SCCmec Ryoiki Chu no Shinki Idenshi fudoh no Dotei", pages: 20 *
ITO, T. ET AL.: "Insights on antibiotic resistance of Staphylococcus aureus from its whole genome: genomic island SCC", DRUG RESISTANCE UPDATES, vol. 6, 2003, pages 41 - 52 *
KAITO, C. ET AL.: "Colony spreading in Staphylococcus aureus", J. BACTERIOLOGY, vol. 189, no. 6, 2007, pages 2553 - 2557 *
RIKI KAKIUCHI ET AL.: "Oshoku Budo Kyukin no Nankanten Baichi Jo deno Sliding Noryoku", JAPANESE SOCIETY OF BACTERIOLOGY 79TH SOKAI, 25 February 2006 (2006-02-25), pages 142 *
RIKI KAKIUCHI ET AL.: "SCCmec Ryoiki ni Sonzai suru Oshoku Budo Kyukin Kasso Noryoku ni Kan'yo suru Shinki Idenshi", JAPANESE SOCIETY OF BACTERIOLOGY 81TH SOKAI, 25 February 2008 (2008-02-25), pages 134 *
RIKI KAKIUCHI ET AL.: "SCCmec Ryoiki no Shinki Idenshi fudoh ga Oshoku Budo Kyukin no Kasso Noryoku to Byogensei o Seigyo Suru", SEIKAGAKU [DAI 81 KAI THE JAPANESE BIOCHEMICAL SOCIETY TAIKAI KOEN YOSHISHU, SHOROKU CD], vol. 4, 4 December 2008 (2008-12-04), pages 4P1081 *

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