WO2001086287A1 - Method of screening compound having antimicrobial activity on pathogenic microorganism infecting organism having acquired immune mechanism by using organism having natural immune mechanism alone, and method of evaluating the antimicrobial activity by using organism having natural immune mechanism alone - Google Patents

Method of screening compound having antimicrobial activity on pathogenic microorganism infecting organism having acquired immune mechanism by using organism having natural immune mechanism alone, and method of evaluating the antimicrobial activity by using organism having natural immune mechanism alone Download PDF

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WO2001086287A1
WO2001086287A1 PCT/JP2001/003945 JP0103945W WO0186287A1 WO 2001086287 A1 WO2001086287 A1 WO 2001086287A1 JP 0103945 W JP0103945 W JP 0103945W WO 0186287 A1 WO0186287 A1 WO 0186287A1
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organism
immune mechanism
pathogenic microorganism
test sample
silkworm
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PCT/JP2001/003945
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French (fr)
Japanese (ja)
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Kazuhisa Sekimizu
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Sekimizu, Nobukazu
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Priority to JP2001583180A priority Critical patent/JPWO2001086287A1/en
Priority to AU2001256708A priority patent/AU2001256708A1/en
Publication of WO2001086287A1 publication Critical patent/WO2001086287A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5085—Supracellular entities, e.g. tissue, organisms of invertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/21—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Pseudomonadaceae (F)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/305—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/43504—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates
    • G01N2333/43552—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from insects
    • G01N2333/43578—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from insects from silkworm

Definitions

  • This study aims to screen for compounds that have antimicrobial activity against JSi microorganisms by using organisms that have only the innate immunity mechanism as well as to infect organisms that have an acquired immunity mechanism.
  • the present invention relates to a method for evaluating antibacterial activity against microorganisms using a living organism having only a natural immune mechanism.
  • an infection model using an organism having only the innate immunity mechanism could be a model for microbial infection of an organism having the adaptive immunity mechanism. Therefore, it was also unclear whether an infection model using an organism having only the innate immunity mechanism could be used in screening an antibacterial agent for treating a microbial infection in an organism having an adaptive immunity mechanism. .
  • the present invention has been made in view of such circumstances, and an object thereof is to provide a model of microbial infection in which an organism having only the natural immunization mechanism is used as an Icheon-based organism, and to provide a microbial infection in an organism having an acquired immune mechanism.
  • the present invention also provides a method for screening a compound exhibiting a pharmacological activity against a pathogenic microorganism that infects an organism having an acquired immune mechanism, and a method for evaluating the antibacterial activity, using these infection models.
  • Target In a preferred embodiment of the present investigation, as the infection model, it squats to insects.
  • an organism that can be infected by a gram-positive pathogenic microorganism is used as the infection model.
  • the present invention also aims at reducing the amount of experiment space and experiment space in screening antibacterial agents and evaluating antibacterial activity.
  • the present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, silkworms (arthropods, mandibles) whose generations have been changed quickly, can be easily bred in a laboratory, and genetic analysis is progressing Submonas, winged insects, belonging to the order Lepidoptera). Bombyx larvae have large larvae, so injection of pathogens and drugs is relatively small compared to small organisms such as C. e7e /? Extremely easy
  • the present inventors have attempted to develop an infection model of a pathogenic microorganism using silkworm larvae.
  • the present inventors examined whether silkworm larvae are useful as antibacterial agent evaluation systems in individuals.
  • injection of live Staphylococcus aureus or Pseudomonas aeruginosa bacteria into the blood of the silkworm larva killed most of the larvae in a short period of time.
  • the autoclaved Staphylococcus aureus was injected, no death of the silkworm larva was observed.
  • E. coli was injected, most of the silkworm larvae survived even 5 days after the injection.
  • Staphylococcus aureus After injection of Staphylococcus aureus, blood and tissues of silkworm larvae were collected over time, and it was confirmed that Staphylococcus aureus was growing. Immunostaining using anti- & aureus antibodies suggested that S. aureus was growing in the mid-gut epithelium. These results indicate that injection of B. aureus or Pseudomonas aeruginosa into the silkworm larvae kills the silkworm larvae.
  • the present invention provides a world in which the efficacy of an antibacterial agent against pathogenic microbial infections in organisms having only the innate immunity mechanism and the efficacy of antimicrobial agents against pathogenic microbial infections in organisms having an acquired immune mechanism have been demonstrated. This is the first example. Therefore, the present invention is also the first in the world to succeed in developing a system for evaluating anti-bacterial activity against pathogenic microorganisms that infect organisms having an acquired immune system by using organisms that possess only the innate immune system. Is also an example.
  • the present invention relates to a screening of a compound having an antibacterial activity against pathogenic microorganisms infecting an organism having an adaptive immunity mechanism using an organism having only an innate immune mechanism, and a review of the antibacterial activity iilli. Is
  • test sample improves the infectious symptoms of an organism having only the natural immunological mechanism or the degree of survival as compared to the case where no test sample is administered (control) Deciding whether or not
  • the creature belonging to the insects is a larva, the method described in (5)
  • the pathogenic microorganism that infects the animal having an immune mechanism is selected from the group consisting of 1 ⁇ 2 Staphylococcus aureus, Pseudomonas aeruginosa, Cholera and pathogenic Escherichia coli. From (1)
  • the present invention provides a method for screening a compound having anti-microbial activity against an organism having an acquired immune mechanism by using an organism having innate immunity and a method for naturally immunizing the antibacterial activity.
  • the present invention provides a method for evaluation using living organisms.
  • a pathogenic microorganism and a test substance are administered to an organism that has only the natural immune system (-r. (A)).
  • ⁇ ⁇ Natural immune system Means an immune host defense mechanism (innate immunity mechanism) that does not depend on an adaptive immunity (acquired immunity) mechanism.
  • Vertebrate animals have an adaptive immunity mechanism that protects the body by using molecules such as antibodies that specifically recognize invaders against invading pathogens, while invertebrates and plants have such an adaptive immunity mechanism. do not do.
  • the “organism having only the innate immunity mechanism” in the present invention is, in other words, an invertebrate and a plant having no acquired immunity mechanism.
  • the organism to which the pathogenic microorganism is administered is not particularly limited as long as it has only the innate immunity mechanism, but an organism belonging to insects is a preferred example.
  • the term "insects" refers to a net of the arthropod phylum, and is composed of four subclasses of bryozoan, coleopteran, wingless biecta, and lepidoptera Means a leash.
  • the organism that succumbs to the worms used in the present invention is not particularly limited.
  • Larvae are preferred for convenience of handling. Larvae include, but are not limited to, larvae of the order Lepidoptera (including geese) and Coleoptera (including the beetle).
  • the larvae are preferably large.
  • the term "large larva” refers to a larva with a body length of 1 cm or more.
  • organisms other than bizoids include arthropods other than insects such as spiders and scorpions, slaughtered animals such as slugs and rodents, annelids such as earthworms, starfish, and ephemerides such as starfish.
  • arthropods other than insects such as spiders and scorpions
  • slaughtered animals such as slugs and rodents
  • annelids such as earthworms, starfish, and ephemerides such as starfish.
  • Examples include cutaneous animals, nematodes of nematodes and roundworms, nematodes such as hydra, sea anemones and jellyfish, and all plants such as rice and radish. These organisms can also be used for the present invention.
  • Organisms that possess only the innate immune system include those infected by gram bacteria, those infected by gram positive bacteria, and those infected by both.
  • a therapeutic agent for human infections caused by Gram-positive bacteria such as Staphylococcus aureus
  • those infected by Gram-positive bacteria must be used.
  • Icheon will be.
  • Silkworm used in this example Larvae are infected not only by Gram-negative bacteria but also by Gram-positive bacteria, Staphylococcus aureus, and can be suitably used for the development of these antibacterial agents.
  • pathogenic microorganism means a microorganism capable of infecting a host and causing a disease.
  • a pathogenic microorganism that infects at least one kind of organism having an acquired immune mechanism is used. From the viewpoint of the development of antibacterial agents against microbial infections in humans, it is preferable to use pathogenic microorganisms that infect humans.
  • Pathogenic microorganisms include both gram-negative and gram-positive bacteria.
  • Gram-negative bacteria that can be ffl suitable for this development include, for example, Pseudomonas aeruginosa, cholera, and pathogenic Escherichia coli (0-157).
  • Gram-positive bacteria include, for example, Staphylococcus aureus However, it is not limited to these.
  • test sample to be administered to the host organism is not particularly limited, and a desired sample to be evaluated for antibacterial activity is used.
  • the test samples e.g., cell extracts, the cell culture;, products of fermenting microorganisms, marine organism extracts, plant extracts, ⁇ made or crude proteins, peptides, non-peptide compounds, synthetic low molecular compound And natural compounds, but are not limited thereto.
  • the pathogenic microorganism and the test sample can be administered to the host by, for example, intraperitoneal administration, injection into the blood, addition to feed (feed), injection into the intestine, and the like.
  • Pathogenic microorganisms The dose of the test sample to the host varies depending on the pathogenic microorganism, the host, and the type of the test sample. In general, the pathogenic microorganism is administered as a dilution from the highest cultivable bacterial solution to about 1 / 10,000 dilution. In the case where a larva of an organism belonging to an insect is used as a host, for example, about 0.05 ml of a bacterial solution may be injected into the blood width from the leg.
  • test sample determine the river ⁇ minimum that kills the host ⁇ , and administer a smaller amount.
  • pathogenic microorganism fc 'main and the type of the test sample, and the like.
  • the pathogenic microorganisms and the test subject are examined for the sensitivity or survival of organisms exhibiting the innate immune mechanism to which the sample was administered (see (b)).
  • Infectious symptoms to be detected include, for example, 1) an increase in the number of pathogenic microorganisms in the host individual, 2) a decrease in the weight of the host or inhibition of the increase in the weight of the host, 3) a decrease in the amount of antibacterial substances in the blood of the host, and 4) a host. ⁇ deficiency of immune function, ⁇ reduction of various enzyme activities in host body fluids and internal organs.
  • the host is a larva of an insect, for example, it may be detected that the larva does not molt to an older larva or does not become a pupa or an adult.
  • the degree of survival of the host may be detected in addition to the above infection symptoms.
  • the degree of survival includes, for example, survival rate and survival period.
  • the infectious symptom of the organism having the immune system is improved or survived, as compared with the case where the test sample is not administered (control).
  • Select the compound that improves the degree step (c)).
  • the test sample improves the infectious disease state of the organism having the innate immune mechanism as compared with the case where the test sample is not administered (control).
  • the test sample modifies the infectious symptoms of the host organism or improves the degree of survival as compared to the control
  • the test sample is: f It can be determined that the antimicrobial activity against the pathogenic microorganism administered to the main organism is high, while the test sample injected does not improve the infection symptoms of the host organism or survive compared to the control. If the concentration is not increased, it can be determined that the test sample has no antibacterial activity against the pathogenic microorganisms administered to the host organism. Samples determined to have antimicrobial activity are good candidates for antimicrobial activity against pathogenic microorganisms injected into the main organism. Simple explanation of the figure
  • FIG. 1 is a diagram showing the survival rate of silkworm larvae injected with S. aureus, P. aeruginosa, and E. coli.
  • Staphylococcus aureus 4220, Smith, MSSA, MRSA
  • Pseudomon as aeruginosa S24
  • Escherichia coli K12-3, W3110, NIHJ
  • 0.05 ml 3 ⁇ 10 7 cells
  • FIG. 2 is a diagram showing the growth of Staphylococcus aureus in a silkworm larva.
  • Staphylo coccus aureus (MSSA) culture broth was diluted 10-fold with 0.6% NaCl, and 0.05 ml (3 x 10 7 cells) of the diluted culture was injected into the silkworm larvae.
  • And tissue (B) were collected, suspended in 0.6% NaCl, applied to a mannitol medium, cultured at 37 ° C overnight, and the number of colonies that appeared was counted.
  • Figure 3 is a micrograph showing the presence of Staphylococcus aureus in the midgut of the silkworm larva. 0.9% NaCl (A) or yellow globule i (MSSA) (3 x 10 7 cell s) (B) was injected into the silkworm larva, and after 40 hours, a paraffin-wrapped woven section of the paraffin of the submucosa was prepared. Contact with a S. aureus antibody was performed by the photoantibody method. The left side of the figure is the body surface and the right side is the inside of the intestine.
  • A NaCl
  • MSSA yellow globule i
  • FIG. 4 is a graph showing the effects of various antibacterial substances on infection death of silkworm larvae by Staphylococcus aureus.
  • Staphylococcus aureus (3 ⁇ 10 7 cells / 0.05 ml) was injected into 10 young silkworms and further injected with the antibiotic ft (0.2 mg / 0.05 ml), and thereafter the number of surviving animals was counted temporarily.
  • Antibiotics i were ampicillin, oxasillin, and vancomycin.
  • the green erythrocyte W RN4220 that was collected was distributed by Kawahira Tenten University Hiramatsu.
  • Staphylococcus aureus MSSA and MRSA clinical isolates from Kyushu University Hospital were used (Akimitsu, N., et al. 1999. Antimicrob Agents Chemother 43: 3042-3043.).
  • Yellow budu bulb [Smith strain and large intestine i NIHJ strain were isolated from Research Institute for Fine Materials Chemistry, Dr. Hamada]. Recitation (S24), Dai I WW3110 shares and K12-3 shares studied A laboratory stock was used.
  • Staphylococcus aureus was cultivated and confirmed on agar medium in Mannite salt medium (Eiken Chemical Co., Ltd.), Pseudomonas aeruginosa in NAC medium (Eiken Chemical Co., Ltd.), and Escherichia coli on D0C medium (Eiken Chemical Co., Ltd.). . These inverted single colonies were used overnight in LB liquid medium.
  • fertilized eggs of silkworm larvae were purchased from Japan Sericulture Industry Co., Ltd. and reared with artificial feed (silk mate: Japan Sericulture Industry Co., Ltd.) at room temperature.
  • Example 1 Infection and death of silkworm larvae by Staphylococcus aureus and Pseudomonas aeruginosa
  • Staphylococcus aureus and Pseudomonas aeruginosa are the causative agents of F1 opportunistic infection in humans.
  • the present inventors examined whether these bacteria could infect silkworm larvae by infection.
  • 0.05 ml of a bacterial solution or an antibacterial substance solution of Staphylococcus aureus and Pseudomonas aeru was injected into the abdominal leg of the fifth-instar silkworm larvae, and the bleeding was stopped for 10 seconds by finger pressure.
  • the injection needle used was 27G X 3/4 (Termo Corporation), and the injection cylinder was 1 ml (Termo Corporation). The number of surviving individuals over time after the injection was examined (FIG. 1).
  • the tail limb of the silkworm larva injected with the bacteria is cut, the body fluid is collected, diluted with 0.6% NaCl, spread on Mannit medium (Eiken Chemical Co., Ltd.), and cultured at 37 ° C overnight. The number of colonies that appeared was counted, and the number of bacteria in the silkworm body fluid was calculated.
  • the silkworm larvae were laparotomized on a paper tube, the body fluid was removed, suspended in 0.9% NaCl, spread on Mannitol medium, and the number of colonies that appeared was counted. The number of bacteria in the tissue was calculated.
  • the volume of the fluid and the tissue of the silkworm larva were calculated as 1.5 ml and 1 ml, respectively.
  • Figure 3 is a histological image of the silkworm larvae's midgut cut perpendicular to the body axis 40 hours after injection of S. aureus.
  • the silkworm larvae injected with Staphylococcus aureus clear fluorescence was observed in the midgut epithelium. This fluorescence was not observed in larvae that were not injected with Staphylococcus aureus ( Figure 3A) or without primary antibody (data not shown).
  • the present inventors investigated whether the death of Bombyx mori larva infection by Staphylococcus aureus could be suppressed by antibiotics. After injection of the clinical isolate of B. aureus into silkworm larvae, ampicillin, oxacillin and vancomycin were injected, and the number of surviving silkworm larvae was counted over time.
  • IC5 () for infected silkworm larvae 5 ⁇ 10 6 S. aureus were injected into the silkworm larva blood, and then 0.05 ml of various concentrations of antimicrobial solutions were injected. Four days after the injection, the concentration of the antibacterial substance at which the number of surviving individuals was half was determined. The IC 5 (1 value was calculated assuming that the body fluid of the silkworm larva was 1.5 ml.
  • LD5 () for the silkworm larva was injected with 0.05 ml of various concentrations of antibacterial solutions to 5 larvae, and one day after the injection The concentration at which half of the larvae die in the eye.
  • the infection model of the present invention can be used as a model for infection of various pathogens to organisms having an acquired immune mechanism, including humans, and is useful for screening antibacterial agents for infectious diseases caused by these pathogens.
  • the infectious model of the present invention can be expected to contribute to the efficient development of antibacterial agents that can be used for human clinical application by using the infectious model as a pre-stage of pathogenic microorganism infection experiments using mammals.
  • the silkworm infection model is effective for the development of antibacterial activity against opportunistic infections caused by Staphylococcus aureus, for example, because it is infected by gram-positive pathogenic bacteria.
  • the use of the infection model of the present invention greatly reduces acquisition costs, breeding costs, and experimental space per individual in drug screening, unlike the conventional case of using a mammal. It becomes possible. For example, (filtering the 1 air fill evening one, and 2 to keep the temperature and humidity constant, is necessary) 1000 mice SPF environment space required for breeding in is an approximately 25 m 2 In addition, backup facilities such as a cage washing room and an autoclave room are required.
  • the silkworm infection model of the present invention is extremely easy to inject pathogens and drugs compared to small organisms such as C.e7e /] s, and can be said to be suitable for evaluating antibacterial drugs against pathogens.
  • the infection model of the present invention is also effective for elucidating the innate immunity mechanism against pathogen infection at the molecular level using genetic techniques.

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Abstract

It is examined whether or not silkworm larvae having a natural immune mechanism alone are useful as a system for evaluating an antimicrobial agent. It is found out that silkworm larvae die from infection by the injection of Micrococcus flavus or Pseudomonas aeruginosa. Effects of antimicrobial agents on silkworm larvae surprisingly agree with the clinical efficacies of these drugs on humans, i.e., an organism having an acquired immune mechanism.

Description

明細 Ϊ 獲得免疫機構を有する生物に感染する病原微生物に対し抗菌活性を有する 化合物を自然免疫機構のみを有する生物を利用してスクリーニングする方法、 および該抗菌活性を自然免疫機構のみを有する生物を利用して評価する方法 技術分野  Description: A method for screening a compound having an antibacterial activity against a pathogenic microorganism infecting an organism having an adaptive immunity mechanism using an organism having only an innate immunity mechanism, and utilizing an organism having only the innate immunity mechanism for the antibacterial activity Technical field
本究明は、 獲 ^免疫機構を冇する生物に感染する病 JSi微生物に対し抗菌活性を 有する化合物を自然免疫機構のみを有する生物を利川してスクリーニングする方 法および獲得免疫機構を有する生物に感染する病 I 微生物に対する抗菌活性を自 然免疫機構のみを有する生物を利用して評価する方法に関する。 背景技術  This study aims to screen for compounds that have antimicrobial activity against JSi microorganisms by using organisms that have only the innate immunity mechanism as well as to infect organisms that have an acquired immunity mechanism. The present invention relates to a method for evaluating antibacterial activity against microorganisms using a living organism having only a natural immune mechanism. Background art
微生物感染症に対する新薬問 においては、 カビ、 放射菌、 海洋生物などを含 む各種生物资源から抽出精製された化合物、 —機化学的方法により合成された化 学物質、 あるいは造伝子工学的手法に基づいて^られた蛋白 （リコンビナント ドラッグ） について、 それらの薬品としての杭 |'効¾や安全性を励物実験により 確認することが必要不可欠である。 従来、 突験川の動物としては、 主にマウス、 希にサルゃブ夕などの哺乳動物が利用されてきた。  In the case of new drugs for microbial infectious diseases, compounds extracted and purified from various biological sources including molds, radioactive bacteria, marine organisms, etc., — chemical substances synthesized by mechanochemical methods, or genetic engineering techniques It is indispensable to confirm the efficacy and safety of the proteins (recombinant drugs) based on the stakes | Heretofore, animals of the Sudokugawa River have been mainly mammals such as mice, and rarely Salvage.
しかしながら、 これら突験用の哺乳動物の飼育に っては、 SPF (specific pa thogen-free；特定病 βΐ休除去 ) と呼ばれる; められた細菌及びウィルスの感染 がな I、条件を満たす必要がある。 それに加えて、 SPF勅物による病原微生物の 染実験を行なうには、 施設や運川に多大の経 を^する。 さらに、 医薬品 究に 多数の哺乳動物を川いることは、 倫理的に 1 题があるとの指摘もなされている。 そこで、 微生物感¾ に対する新薬 ^発において、 これら喊乳勁物に代 しう る実験動物の探索の必^ H :がある。 ところで、 生物の長い進化の歴史の中で、 病原体による侵入と宿主との間の攻 防は、 様々な免疫機構を導いてきた。 ヒトを含む脊椎動物は抗体という、 侵入者 を特異的に認識する分子による 「獲得免疫機構」 を確立した。 しかしながら、 脊 椎動物が現れる前に既に無脊椎動物は、 抗体によらない 「自然免疫機構」 を持つ ていた。 無脊椎動物である昆虫は、 この地球上で最も繁栄していると考えられる 生物の一つであるが、 「自然免疫機構」 により外来者の侵入を防いでいる。 However, when rearing these abrupt mammals, it is called SPF (specific pa thogen-free); it is necessary that the bacteria and viruses that have been infected meet the conditions. is there. In addition, experiments to stain pathogenic microorganisms with the SPF Imperative require significant facilities and canals. Furthermore, it has been pointed out that there is an ethical implication of having a large number of mammals in pharmaceutical research. Therefore, in the development of new drugs against microbial sensation, there is a need to search for experimental animals that can substitute for these drugs. By the way, in the long history of evolution of living things, invasion by pathogens and defense between the host have led to various immune mechanisms. Vertebrate animals, including humans, have established an "acquired immune system" through antibodies, molecules that specifically recognize invaders. However, before the appearance of vertebrates, invertebrates already had an "innate immune system" that did not rely on antibodies. Invertebrates, insects, are considered one of the most prosperous on the planet, but their “natural immunity” keeps out invaders.
近年の研究により、 その分子レベルでの機構は、 ヒトのそれと共通しているこ とが明らかになりつつある。 例えば、 細菌感染に対する応^において、 哺乳動物 と昆虫は、 防御遺伝子を発現させるための 通に保 されたシグナル絰路を持つ ている。 すなわち、 哺乳動物においては、 病原体の体内への侵入により、 免疫細 胞において TOLL like receptorを介して、 NF- Bの発現誘導が引き起こされる (Medzhitov, R. et al. 1997. Nature 388: 394-397.) 。 一方、 Drosophira me a/jo asie尸の成虫においても、 TOLL like receptorである dToll と 18- wheeler を介して NF- Λ:Βのホモログである Dif と Relishが誘導され、 抗閣応答系が活性 化される（Lemaitre, B. et al. 1996. Cell 86: 973-983·、 Bernal, A., and D. A. Kimbell. 2000. Proc. Natl. Acad. Sci. USA 97: 6019-6024·)。  Recent studies have revealed that its molecular mechanism is similar to that of humans. For example, in responding to bacterial infection, mammals and insects have signal pathways through which defense genes can be expressed. In other words, in mammals, the invasion of the pathogen into the body causes induction of NF-B expression in immune cells via TOLL like receptor (Medzhitov, R. et al. 1997.Nature 388: 394- 397.). On the other hand, in Drosophira me a / jo asie society adults, Dif and Relish, homologs of NF-II: Β, are induced via dToll, a TOLL-like receptor, and 18-wheeler, activating the anti-cancer response system. (Lemaitre, B. et al. 1996. Cell 86: 973-983 ·, Bernal, A., and DA Kimbell. 2000. Proc. Natl. Acad. Sci. USA 97: 6019-6024 ·).
また、 最近になり、 咄乳動物以外の動物を利川した病原微生物の感染モデルに つき、 いくつかの報告がなされた。 例えば、 Caenorhabditis elegans (Tan, M. W. et al. 1999. Proc. Natl. Acad. Sci. USA. 96: 715-20.、 Tan, M, W.,et al. 1 999. Proc. Natl. Acad. Sci. USA 96: 2408-13.、 Mahajan-Miklos, S. et al., 199 9. Cell 96: 47-56.)、 Arabidopsis thai i ana (Reuber, T. L. et al. 1998. PI ant J. 16: 473- 85. )を川いた緑 菌感染の解析、 Dictyostelium discoideum^ 用いた Legionella p?e"M¾D?i 感染の解析（Solomon, J. M. et al. 2000. Infe ct Immun 68: 2939—47.)、 Caenorhabditis elegans (Aballay, A. et al. 2000. Curr Biol 10: 1539-42. ), yeast (Scherer, C. A. et al. 2000. Mol Microbi ol . 37: 1133- 45. )を用いた■a wje a P ; ™感染の研究例が報告され ている。 Also, recently, several reports have been made on models of infection with pathogenic microorganisms that used Icheon in animals other than infants. For example, Caenorhabditis elegans (Tan, MW et al. 1999. Proc. Natl. Acad. Sci. USA. 96: 715-20., Tan, M, W., et al. 1 999. Proc. Natl. Acad. Sci. USA 96: 2408-13., Mahajan-Miklos, S. et al., 199 9. Cell 96: 47-56.), Arabidopsis thai iana (Reuber, TL et al. 1998. PI ant J. 16: 473-85.)), Analysis of Legionella p? E "M¾D? I infection using Dictyostelium discoideum ^ (Solomon, JM et al. 2000. Infect Immun 68: 2939-47.), Caenorhabditis elegans (Aballay, A. et al. 2000.Curr Biol 10: 1539-42.), Yeast (Scherer, CA et al. 2000.Mol Microbi ol. 37: 1133-45.), a study of 研究 a wje a P; ™ infection has been reported.
しかしながら、 これら自然免疫機構のみを有する生物を利用した感染モデルが、 獲得免疫機構を有する生物の微生物感染のモデルとなり得るかは全く不明であつ た。 従って、 また、 獲得免疫機構を有する生物の微生物感染症の治療のための抗 菌剤のスクリーニングにおいて、 これら自然免疫機構のみを有する生物を利用し た感染モデルを利用できるか否かも不明であった。  However, it was completely unclear whether an infection model using an organism having only the innate immunity mechanism could be a model for microbial infection of an organism having the adaptive immunity mechanism. Therefore, it was also unclear whether an infection model using an organism having only the innate immunity mechanism could be used in screening an antibacterial agent for treating a microbial infection in an organism having an adaptive immunity mechanism. .
特に、 グラム陽性の病原性細菌に関しては、 これまで哺乳動物以外の動物個体 を用いた感染モデルの報告例すらないのが現状である。 グラム 性細菌の中でも 黄色ブドウ球菌は、 ヒトに日和見感染症の原因菌であり、 近年、 多剂耐性能を持 つ RSAが出現し、 臨床上問题となっている （Speller, DC et al .， Lancet 350 : 323-325. ) 。 このため、 黄色ブドウ球菌に対する抗菌剂の開発が強く望まれて いる。 発明の開示  In particular, with regard to gram-positive pathogenic bacteria, there is no report of an infection model using an animal other than a mammal so far. Among gram bacteria, Staphylococcus aureus is the causative agent of opportunistic infections in humans. In recent years, the emergence of highly resistant RSA has caused clinical problems (Speller, DC et al., Lancet 350: 323-325.). Therefore, development of antibacterial agent against Staphylococcus aureus is strongly desired. Disclosure of the invention
本発明は、 このような状況に鑑みてなされたものであり、 その目的は、 自然免 疫機構のみを有する生物を利川した微生物感染のモデルであって、 獲得免疫機構 を有する生物における微生物感染症の抗菌剂開発に有川なモデルを提供すること にある。 本発明は、 これら感染モデルを利用して、 獲得免疫機構を有する生物に 感染する病原微生物に対し杭閣活性を冇する化合物をスクリーニングする方法お よび該抗菌活性の評価方法を捉供することをも 的とする。 本究明の好ましい態 様において、 該感染モデルとして、 昆虫類に屈する it物を利川する。 また、 本発 明の他の好ましい態様において、 該感染モデルとしてグラム陽性の病原微生物に 感染され得る生物を利用する。 本 ¾明は、 また、 抗菌剤のスクリーニングおよび 抗菌活性の評価において、 実験絰 や実験スペースを節減することをも目的とす る。 本発明者らは、 上記課題を解決すべく鋭意検討を行なった結果、 世代交代が早 く、 研究室で容易に飼育でき、 遺伝学的解析が進んでいるカイコ （節足動物門、 大顎亜門、 有翅昆虫類、 チョウ目に属する） に着 した。 カイコは幼虫が大型で あるため、 C. e7e /? s (線形動物門、 双腺網、 桿線虫亜門、 カンセンチユウ目に 属する） などの小型の生物と比較して病原体や薬物の注射が極めて容易であるThe present invention has been made in view of such circumstances, and an object thereof is to provide a model of microbial infection in which an organism having only the natural immunization mechanism is used as an Icheon-based organism, and to provide a microbial infection in an organism having an acquired immune mechanism. To provide Arikawa's model for antimicrobial development. The present invention also provides a method for screening a compound exhibiting a pharmacological activity against a pathogenic microorganism that infects an organism having an acquired immune mechanism, and a method for evaluating the antibacterial activity, using these infection models. Target. In a preferred embodiment of the present investigation, as the infection model, it squats to insects. In another preferred embodiment of the present invention, an organism that can be infected by a gram-positive pathogenic microorganism is used as the infection model. The present invention also aims at reducing the amount of experiment space and experiment space in screening antibacterial agents and evaluating antibacterial activity. The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, silkworms (arthropods, mandibles) whose generations have been changed quickly, can be easily bred in a laboratory, and genetic analysis is progressing Submonas, winged insects, belonging to the order Lepidoptera). Bombyx larvae have large larvae, so injection of pathogens and drugs is relatively small compared to small organisms such as C. e7e /? Extremely easy
(Okada, E. et al . 1997. J. Seric . Sci . Jpn. 66 : 116-122. ) 。 このため、 病原 体に対する抗菌薬の評価に極めて適していると考えられる。 また、 カイコ幼虫を 使用することは、 廉価であり、 倫理上の問題もなく、 哺乳！ J物と比較して有用性 が高い。 (Okada, E. et al. 1997. J. Seric. Sci. Jpn. 66: 116-122.). Therefore, it is considered to be extremely suitable for evaluating antimicrobial agents against pathogens. Also, using silkworm larvae is inexpensive, has no ethical problems, and sucks! It is more useful than the J product.
そこで、 本発明者らは、 カイコ幼虫を利用した病原微生物の感染モデルの開発 を試みた。 まず、 本発明者らは、 個体における抗菌剤評価系として、 カイコ幼虫 が有用であるかについて検討した。 その結果、 カイコ幼虫の血液中へ黄色ブドウ 球菌あるいは緑膿菌の生菌を注射すると、 短期 ^で大部分の力ィコ幼虫が死亡し た。 一方、 ォ一トクレーブ処理した黄色ブドゥ球菌を注射した場合には、 カイコ 幼虫の死亡は認められなかった。 大腸菌を注射した場合では、 注射後 5日目にお いても大部分のカイコ幼虫が生存した。 黄色ブドウ球菌を注射した後、 経時的に カイコ幼虫の血液及び組織を採取した結架、 黄色ブドゥ球菌が増殖していること が確認された。 抗 & aureus抗体を用いた免疫染色により、 中腸上皮において 黄色ブドウ球菌が増殖していることが示唆された。 これらの結果は、 カイコ幼虫 への黄色ブドゥ球菌あるいは綠膿菌の注射により、 カイコ幼虫が感染死すること を示している。  Therefore, the present inventors have attempted to develop an infection model of a pathogenic microorganism using silkworm larvae. First, the present inventors examined whether silkworm larvae are useful as antibacterial agent evaluation systems in individuals. As a result, injection of live Staphylococcus aureus or Pseudomonas aeruginosa bacteria into the blood of the silkworm larva killed most of the larvae in a short period of time. On the other hand, when the autoclaved Staphylococcus aureus was injected, no death of the silkworm larva was observed. When E. coli was injected, most of the silkworm larvae survived even 5 days after the injection. After injection of Staphylococcus aureus, blood and tissues of silkworm larvae were collected over time, and it was confirmed that Staphylococcus aureus was growing. Immunostaining using anti- & aureus antibodies suggested that S. aureus was growing in the mid-gut epithelium. These results indicate that injection of B. aureus or Pseudomonas aeruginosa into the silkworm larvae kills the silkworm larvae.
さらに、 メチシリン感受性 ί½色ブドウ球 li (MSSA) によるカイコ幼虫の感染死 は、 アンピシリン、 ォキサシリン、 バンコマイシンで抑えられるのに対し、 メチ シリン耐性黄色ブドゥ球菌 (MRSA) によるカイコ幼虫の感染死は、 アンピシリン、 ォキサシリンでは抑えられず、 バンコマイシンで抑えられた。 また、 MSSAによ るカイコ幼虫の感¾死は、 ；Π ¾剂である EtOH、 塩化ベンザルコニゥム、 ポビド ンョードでは抑えることができなかった。 驚くべきことに、 この結果は、 ヒトの 臨床におけるこれら薬剤の有効性と一致している。 従って、 カイコ幼虫を用いた 系は動物個体に対する感染モデルとして新規抗菌剤のスクリーニングや評価に極 めて有効であると考えられる。 本発明は、 自然免疫機構のみを有する生物におけ る病原微生物感染に対する抗菌剤の有効性と、 獲得免疫機構を有する生物におけ る病原微生物感染に対する抗菌剤の有効性の一致が示された世界で初めての例で ある。 従って、 また、 本発明は、 自然免疫機構のみを冇する生物を利用して、 獲 得免疫機構を有する生物に感染する病原微生物に対する抗 ¾活性の評価系を開発 することに成功した世界で初めての例でもある。 Furthermore, the death of silkworm larvae caused by methicillin-sensitive green staphylococcus li (MSSA) is suppressed by ampicillin, oxacillin, and vancomycin, whereas the death of silkworm larvae caused by methicillin-resistant Staphylococcus aureus (MRSA) is reduced by ampicillin. It was not suppressed by oxacillin, but was suppressed by vancomycin. The death of silkworm larvae by MSSA was caused by: EtOH, benzalkonium chloride, povid I couldn't suppress it in Nohdo. Surprisingly, this result is consistent with the efficacy of these agents in human clinical practice. Therefore, a system using silkworm larvae is considered to be extremely effective in screening and evaluating new antibacterial agents as a model for infection of animal individuals. The present invention provides a world in which the efficacy of an antibacterial agent against pathogenic microbial infections in organisms having only the innate immunity mechanism and the efficacy of antimicrobial agents against pathogenic microbial infections in organisms having an acquired immune mechanism have been demonstrated. This is the first example. Therefore, the present invention is also the first in the world to succeed in developing a system for evaluating anti-bacterial activity against pathogenic microorganisms that infect organisms having an acquired immune system by using organisms that possess only the innate immune system. Is also an example.
即ち、 本発明は、 自然免疫機構のみを有する生物を利用した、 獲得免疫機構を 有する生物に感染する病原微生物に対し抗菌活性を有する化合物のスクリーニン グおよび該抗菌活性の評 iilliに関し、 より詳しくは、  That is, the present invention relates to a screening of a compound having an antibacterial activity against pathogenic microorganisms infecting an organism having an adaptive immunity mechanism using an organism having only an innate immune mechanism, and a review of the antibacterial activity iilli. Is
( 1 ) 獲得免疫機構を有する生物に感染する病原微生物に対し抗菌活性を有す る化合物をスクリーニングする方法であって、  (1) A method for screening a compound having antibacterial activity against a pathogenic microorganism that infects an organism having an adaptive immune mechanism,
( a ) 自然免疫機構のみを有する生物に該病原微生物および被検試料を投与する 工程、  (a) administering the pathogenic microorganism and a test sample to an organism having only an innate immune mechanism,
( b ) 該自然免疫機構のみを有する生物の感染症状または生存の程度を検出する 工程、 および  (b) detecting an infection symptom or a degree of survival of an organism having only the innate immunity mechanism, and
( c ) 被検試料を投与しない場合 （対照） と比較して、 該自然免疫機構のみを有 する生物の感染症状を改善する、 または生存の程度を向上させる化合物を選択す る工程、 を含む方法、  (c) a step of selecting a compound that improves the infectious symptoms of an organism having only the innate immune mechanism or improves the degree of survival as compared to the case where no test sample is administered (control). Method,
( 2 ) 獲得免疫機構を ¾する生物に感染する病原微生物に対する、 被検試料の 抗菌活性を評価する方法であって、  (2) A method for evaluating the antibacterial activity of a test sample against a pathogenic microorganism that infects an organism having an acquired immune mechanism,
( a ) 自然免疫機構のみを右する生物に病原微生物および被検試料を投与するェ fe、 (b) 該自然免疫機構のみを有する生物の感染症状または生存の程度を検出する 工程、 および (a) administer pathogenic microorganisms and test samples to organisms that have only an innate immune system, (b) detecting an infection symptom or a degree of survival of an organism having only the innate immunity mechanism, and
(c) 被検試料を投与しない場合 （対照） と比較して、 該被検試料が、 該自然免 疫機構のみを有する生物の感染症状を改善するか否か、 または生存の程度を向上 させるか否かを判定する工程、 を含む方法、  (c) whether or not the test sample improves the infectious symptoms of an organism having only the natural immunological mechanism or the degree of survival as compared to the case where no test sample is administered (control) Deciding whether or not
(3) 獲得免疫機構を有する生物が哺乳動物である、 （ 1) または （2) に記 載の方法、  (3) The method according to (1) or (2), wherein the organism having the acquired immune mechanism is a mammal,
(4) 哺乳動物がヒトである、 （3) に記載の方法、  (4) The method according to (3), wherein the mammal is a human,
(5) 自然免疫機構のみを する生物が昆虫類に屈する生物である、 （ 1 ) カ ら （4) のいずれかに記載の方法、  (5) The method according to any one of (1) to (4), wherein the organism having only the innate immune mechanism is an organism that yields to insects.
(6) 昆虫類に属する生物が幼虫である、 （5) に記叔の方法、  (6) The creature belonging to the insects is a larva, the method described in (5)
(7) 幼虫が大型である、 （6) に記載の方法、  (7) The method according to (6), wherein the larva is large.
(8) 毘虫類に屈する生物がカイコである、 （5) から （7) のいずれかに記 載の方法、  (8) The method described in any one of (5) to (7), wherein the organism that succumbs to the worms is the silkworm.
(9) 然免疫機構のみをおする生物がグラム陽性の病原微生物により感染さ れるものである、 （ 1) から （7) のいずれかに,] ΰ載の方法、  (9) An organism that has only the immune system is infected by a gram-positive pathogenic microorganism, according to any one of (1) to (7),]
( 10) 狻 ^免疫機構を有する尘物に感染する病原微生物が j½色ブドウ球菌、 緑濃菌、 コレラ菌および病原性大腸菌からなる胙より選択される、 （ 1) から (10) The pathogenic microorganism that infects the animal having an immune mechanism is selected from the group consisting of ½ Staphylococcus aureus, Pseudomonas aeruginosa, Cholera and pathogenic Escherichia coli. From (1)
(9) のいずれかに記載の方法、 および The method according to any of (9), and
( 1 1) ( 1) から （ 10) のいずれかに記战の方法により同定される抗菌沾 性を冇する化 物、 を提供するものである。  (11) A compound having antibacterial properties, which is identified by the method described in any one of (1) to (10).
本¾明は、 獲得免疫機構を /する生物に感¾する^ ό ^微生物に対し抗¾¾性を 有する化合物を自然免疫を有する生物を利川してスクリーニングする方法および 該抗菌活性を 然免疫を冇する生物を利用して評価する方法を提供する。  The present invention provides a method for screening a compound having anti-microbial activity against an organism having an acquired immune mechanism by using an organism having innate immunity and a method for naturally immunizing the antibacterial activity. The present invention provides a method for evaluation using living organisms.
本発叨の方法においては、 まず、 ΙΊ然免疫機構のみをィ ίする生物に病原微生物 および被検^料を投与する (― r. (a) ) 。 木允 njjにおいて 「Γι然免疫機構」 と は、 獲得免疫 （後天性免疫） 機構によらない免疫的生体防御機構 （先天性免疫機 構） を意味する。 脊椎動物は、 病原体の侵入に対し抗体などの侵入者を特異的に 認識する分子を利用して生体を防御する獲得免疫機構を有するが、 無脊椎動物や 植物はこのような獲得免疫機構を有しない。 本発明における 「自然免疫機構のみ を有する生物」 とは、 換言すれば、 獲得免疫機構を有しない無脊椎動物および植 物である。 In the method of the present invention, first, a pathogenic microorganism and a test substance are administered to an organism that has only the natural immune system (-r. (A)). In Kijyun njj, “然 ι Natural immune system” Means an immune host defense mechanism (innate immunity mechanism) that does not depend on an adaptive immunity (acquired immunity) mechanism. Vertebrate animals have an adaptive immunity mechanism that protects the body by using molecules such as antibodies that specifically recognize invaders against invading pathogens, while invertebrates and plants have such an adaptive immunity mechanism. do not do. The “organism having only the innate immunity mechanism” in the present invention is, in other words, an invertebrate and a plant having no acquired immunity mechanism.
本発明において病原微生物を投与する生物としては、 自然免疫機構のみを有す る生物であれば特に制限はないが、 昆虫類に属する生物が好適な一例である。 本 発明において 「昆虫類」 とは、 節足動物門大颚亚門の一網であって、 力マァシム シ類、 トビムシ類、 無翅毘虫類および冇翅毘虫類の 4亜綱からなる綱を意味する。 本発明に用いる毘虫類に屈する生物としては、 特に制限はない。 取り扱いの便宜 性から幼虫であることが好ましい。 幼虫としては、 例えば、 鱗翅目 （ガゃチョウ を含む） 及び甲虫目 （力ブトムシを含む） の幼虫が挙げられるが、 これらに制限 されない。 病原微生物ゃ被検試料の投与のしゃすさの観点から、 幼虫は大型のも のであることが好ましい。 本発叨において 「大型の幼虫」 とは、 体長が l cm以 上である幼虫を指す。 毘虫類以外の生物としては、 例えば、 クモ、 サソリ等の昆 虫類以外の節足動物、 ナメクジ、 力夕ツムリ等の軟休勋物、 ミミズ等の環形動物、 ヒトデ、 ゥ二等のキヨク皮動物、 ギヨゥ虫、 回虫 の線形動物、 ヒドラ、 イソギ ンチヤク、 クラゲ等の腔腸動物、 イネ、 ダイコン等のすべての植物が挙げられ、 これら生物も本発叨に用いることが考えられる。  In the present invention, the organism to which the pathogenic microorganism is administered is not particularly limited as long as it has only the innate immunity mechanism, but an organism belonging to insects is a preferred example. In the present invention, the term "insects" refers to a net of the arthropod phylum, and is composed of four subclasses of bryozoan, coleopteran, wingless biecta, and lepidoptera Means a leash. The organism that succumbs to the worms used in the present invention is not particularly limited. Larvae are preferred for convenience of handling. Larvae include, but are not limited to, larvae of the order Lepidoptera (including geese) and Coleoptera (including the beetle). Pathogenic microorganisms. From the viewpoint of the ease of administration of the test sample, the larvae are preferably large. In this development, the term "large larva" refers to a larva with a body length of 1 cm or more. Examples of organisms other than bizoids include arthropods other than insects such as spiders and scorpions, slaughtered animals such as slugs and rodents, annelids such as earthworms, starfish, and ephemerides such as starfish. Examples include cutaneous animals, nematodes of nematodes and roundworms, nematodes such as hydra, sea anemones and jellyfish, and all plants such as rice and radish. These organisms can also be used for the present invention.
自然免疫機構のみを仃する生物には、 グラム 性細菌により感染されるもの、 グラム陽性細菌により感染されるもの、 およびそれら双方により感染されるもの が含まれる。 現在、 黄色ブドウ球菌などのグラム陽性菌によるヒトの感染淀に対 する治療薬の^ ¾が望まれているが、 このような治療薬の開発においては、 グラ ム陽性細菌により感染されるものが利川される。 本 施例において用いたカイコ 幼虫は、 グラム陰性細菌のみならず、 グラム陽性細菌である黄色ブドウ球菌によ り感染されるため、 これら抗菌剤の開発に好適に用いることができる。 Organisms that possess only the innate immune system include those infected by gram bacteria, those infected by gram positive bacteria, and those infected by both. At present, there is a demand for a therapeutic agent for human infections caused by Gram-positive bacteria such as Staphylococcus aureus, but in the development of such therapeutic agents, those infected by Gram-positive bacteria must be used. Icheon will be. Silkworm used in this example Larvae are infected not only by Gram-negative bacteria but also by Gram-positive bacteria, Staphylococcus aureus, and can be suitably used for the development of these antibacterial agents.
本発明において 「病原微生物」 とは、 宿主に感染して病気を引き起こす能力を 有する微生物を意味する。 本発明においては、 獲得免疫機構を有する生物の少な くとも 1種に感染する病原微生物を用いる。 ヒ卜における微生物感染症に対する 抗菌剤の開発の点からは、 病原微生物としては、 ヒトに感染するものを用いるこ とが好ましい。 病原微生物には、 グラム陰性細菌およびグラム陽性細菌の双方が 含まれる。 本発叨に適 ffl可能なグラム陰性細菌としては、 例えば、 緑膿菌、 コレ ラ菌、 病原性大腸菌 （0-157)を、 グラム陽性細菌としては、 例えば、 黄色ブドウ 球菌を挙げることができるが、 これらに制限されるものではない。  In the present invention, "pathogenic microorganism" means a microorganism capable of infecting a host and causing a disease. In the present invention, a pathogenic microorganism that infects at least one kind of organism having an acquired immune mechanism is used. From the viewpoint of the development of antibacterial agents against microbial infections in humans, it is preferable to use pathogenic microorganisms that infect humans. Pathogenic microorganisms include both gram-negative and gram-positive bacteria. Gram-negative bacteria that can be ffl suitable for this development include, for example, Pseudomonas aeruginosa, cholera, and pathogenic Escherichia coli (0-157). Gram-positive bacteria include, for example, Staphylococcus aureus However, it is not limited to these.
宿主生物に投与される被検試料としては特に制限はなく、 抗菌活性の評価を行 ないたい所望の試料が用いられる。 被検試料としては、 例えば、 細胞抽出物、 細 胞培養上 ;、 発酵微生物産生物、 海洋生物抽出物、 植物抽出物、 ^製若しくは粗 精製蛋白質、 ペプチド、 非ペプチド性化合物、 合成低分子化合物、 天然化合物な どが挙げられるが、 これらに制限されるものではない。 The test sample to be administered to the host organism is not particularly limited, and a desired sample to be evaluated for antibacterial activity is used. The test samples, e.g., cell extracts, the cell culture;, products of fermenting microorganisms, marine organism extracts, plant extracts, ^ made or crude proteins, peptides, non-peptide compounds, synthetic low molecular compound And natural compounds, but are not limited thereto.
病原微生物および被検試料の宿主への投与は、 例えば、 腹腔内投与、 血液中へ の注射、 飼料 （エサ） への添加、 腸内への注入などの方法で行なうことができる。 病原微生物ゃ被検試料の宿主への投与量は、 病原微生物、 宿主及び被検試料の 種類などにより変動する。 一般的には、 病原微生物は、 培養可能な最高密度の菌 液からその 10000分の 1 くらいまで稀釈液を投与する。 昆虫類に属する生物の幼 虫を宿主に用いる場 rには、 例えば、 菌液 0.05ml程度を脚部から血液巾に注射 すればよい。 被検試料は、 川いる ί主を殺傷する最小] ¾を求め、 それ以下の量を 投与する。 当業者であれば、 病原微生物、 fc'主及び被検試料の種類などに応じて、 適切な投与量を選択することが可能であろう。  The pathogenic microorganism and the test sample can be administered to the host by, for example, intraperitoneal administration, injection into the blood, addition to feed (feed), injection into the intestine, and the like. Pathogenic microorganisms. The dose of the test sample to the host varies depending on the pathogenic microorganism, the host, and the type of the test sample. In general, the pathogenic microorganism is administered as a dilution from the highest cultivable bacterial solution to about 1 / 10,000 dilution. In the case where a larva of an organism belonging to an insect is used as a host, for example, about 0.05 ml of a bacterial solution may be injected into the blood width from the leg. For the test sample, determine the river {minimum that kills the host}, and administer a smaller amount. Those skilled in the art will be able to select an appropriate dose depending on the pathogenic microorganism, fc 'main and the type of the test sample, and the like.
本発明においては、 次いで、 病原微生物および被検,试料が投与された自然免疫 機構を冇する生物の感¾ 状または生存の^度を検 M;する ( に程（b ) ) 。 検出する感染症状としては、 例えば、 ①宿主個体内における病原微生物の数の 増加、 ②宿主の体重の減少あるいは宿主の体重の増加の阻害、 ③宿主の血液中の 抗菌物質量の低下、 ④宿主の免疫機能の不全、 ⑤宿主の体液及び体内臓器中の 種々の酵素活性の低下などが挙げられる。 宿主が昆虫の幼虫であれば、 例えば、 高齢幼虫へと脱皮しない、 あるいは蛹や成虫とならないことなどを検出してもよ い。 本発明においては、 また、 上記感染症状以外に、 宿主の生存の程度を検出し てもよい。 生存の程度としては、 例えば、 生存率や生存期問が挙げられる。 Next, in the present invention, the pathogenic microorganisms and the test subject are examined for the sensitivity or survival of organisms exhibiting the innate immune mechanism to which the sample was administered (see (b)). Infectious symptoms to be detected include, for example, 1) an increase in the number of pathogenic microorganisms in the host individual, 2) a decrease in the weight of the host or inhibition of the increase in the weight of the host, 3) a decrease in the amount of antibacterial substances in the blood of the host, and 4) a host.免疫 deficiency of immune function, 低下 reduction of various enzyme activities in host body fluids and internal organs. If the host is a larva of an insect, for example, it may be detected that the larva does not molt to an older larva or does not become a pupa or an adult. In the present invention, the degree of survival of the host may be detected in addition to the above infection symptoms. The degree of survival includes, for example, survival rate and survival period.
本発明の抗菌活性を有する化合物のスクリーニングにおいては、 次いで、 被検 試料を投与しない場合 （対照） と比較して、 該 [^然免疫機構を冇する生物の感染 症状を改善する、 または生存の程度を向上させる化合物を選択する （工程（ c ) ) 。 一方、 本究明の抗菌活性の評価方法においては、 次いで、 被検試料を投^しない 場合 （対照） と比較して、 該被検試料が、 該自然免疫機構を有する生物の感染症 状を改善するか否か、 または生存の程度を向上させるか否かを判定する （工程 In the screening of the compound having the antibacterial activity of the present invention, the infectious symptom of the organism having the immune system is improved or survived, as compared with the case where the test sample is not administered (control). Select the compound that improves the degree (step (c)). On the other hand, in the antibacterial activity evaluation method of the present invention, the test sample improves the infectious disease state of the organism having the innate immune mechanism as compared with the case where the test sample is not administered (control). To determine whether or not to improve the degree of survival (process
( C ) ) 。 (C)).
投与した被検試料が、 対照と比較して、 ¾主生物の感染症状を改 する、 また は生存の程度を向上させる場合には、 該被検試料は、 ？f主生物に投与した病原微 生物に対し抗菌活性を冇すると判定することができ、 一方、 投^した被検試料が、 対照と比較して、 宿主生物の感染症状を改善しない、 または生存の ¾度を^上さ せない場合には、 該被検試料は、 宿主生物に投与した病原微生物に対し抗菌活性 を有しないと判定することができる。 抗菌活性を冇すると判定された試料は、 主生物に投^した病原微生物に対する抗菌剂の打力な候補となる。 図而の簡 i lな説明  If the administered test sample modifies the infectious symptoms of the host organism or improves the degree of survival as compared to the control, the test sample is: f It can be determined that the antimicrobial activity against the pathogenic microorganism administered to the main organism is high, while the test sample injected does not improve the infection symptoms of the host organism or survive compared to the control. If the concentration is not increased, it can be determined that the test sample has no antibacterial activity against the pathogenic microorganisms administered to the host organism. Samples determined to have antimicrobial activity are good candidates for antimicrobial activity against pathogenic microorganisms injected into the main organism. Simple explanation of the figure
図 1は、 黄色ブドウ球 、 緑膿菌、 大腸菌を注射したカイコ幼虫の生存率を示 す図である。 Staphylococcus aureus ( 4220， Smith, MSSA, MRSA)ヽ Pseudomon as aeruginosa ( S24) 、 Escherichia coli ( K12-3, W3110, NIHJ )の一晚 ^液 を 0.6% NaClで 10倍に希釈して、 その 0.05 ml (3 x 107 cells) を 10匹の力 ィコ 5令幼虫血液中に注射した。 生存個体数を絰時的にカウントした。 FIG. 1 is a diagram showing the survival rate of silkworm larvae injected with S. aureus, P. aeruginosa, and E. coli. Staphylococcus aureus (4220, Smith, MSSA, MRSA) 晚 solution of Pseudomon as aeruginosa (S24), Escherichia coli (K12-3, W3110, NIHJ) Was diluted 10-fold with 0.6% NaCl, and 0.05 ml (3 × 10 7 cells) thereof was injected into the blood of 10 mice of the 5th instar larva. The number of surviving animals was counted temporarily.
図 2は、 カイコ幼虫体内での黄色ブドゥ球菌の増殖を示す図である。 Staphylo coccus aureus (MSSA)のー晚培養液を 0.6% NaClで 10倍に希釈して、 その 0.05 ml (3 X 107 cells) をカイコ幼虫に注射し、 経時的にカイコ幼虫の体液 （A) 、 及び、 組織 （B) を回収し、 0.6% NaCl中に懸濁し、 マンニット培地に塗布し、 3 7°Cで一晩培養後、 現れたコロニー数をカウントした。 FIG. 2 is a diagram showing the growth of Staphylococcus aureus in a silkworm larva. Staphylo coccus aureus (MSSA) culture broth was diluted 10-fold with 0.6% NaCl, and 0.05 ml (3 x 10 7 cells) of the diluted culture was injected into the silkworm larvae. , And tissue (B) were collected, suspended in 0.6% NaCl, applied to a mannitol medium, cultured at 37 ° C overnight, and the number of colonies that appeared was counted.
図 3は、 カイコ幼虫の中腸における Staphylococcus aureusの 在を示す顕微 鏡写 ¾である。 0.9% NaCl (A) あるいは、 黄色ブドウ球 i(MSSA) (3 x 107 cell s) (B) をカイコ幼虫に注射し、 40時問後に屮腸のバラフィン包 ¾紐織切片を 作成し、 黄色ブドウ球菌抗体による問接' 光抗体法を行った。 図の左側が体表側、 右側が腸の内部側である。 Figure 3 is a micrograph showing the presence of Staphylococcus aureus in the midgut of the silkworm larva. 0.9% NaCl (A) or yellow globule i (MSSA) (3 x 10 7 cell s) (B) was injected into the silkworm larva, and after 40 hours, a paraffin-wrapped woven section of the paraffin of the submucosa was prepared. Contact with a S. aureus antibody was performed by the photoantibody method. The left side of the figure is the body surface and the right side is the inside of the intestine.
図 4は、 Staphylococcus aureusによるカイコ幼虫の感染死に対する種々の抗 生物質の効果を示す図である。 Staphylococcus aureus (3 x 107 cells/0.05 m 1) をカイコ幼 l lO匹に注射し、 さらに抗生物 ft (0.2 mg/0.05 ml) を注射し、 その後絰時的に生存個体数をカウントした。 抗生物 iとしてアンピシリン、 ォキ サシリン、 バンコマイシンを川いた。 A、 MSSA B、 MRSA。 発明を実施するための最良の形態 FIG. 4 is a graph showing the effects of various antibacterial substances on infection death of silkworm larvae by Staphylococcus aureus. Staphylococcus aureus (3 × 10 7 cells / 0.05 ml) was injected into 10 young silkworms and further injected with the antibiotic ft (0.2 mg / 0.05 ml), and thereafter the number of surviving animals was counted temporarily. Antibiotics i were ampicillin, oxasillin, and vancomycin. A, MSSA B, MRSA. BEST MODE FOR CARRYING OUT THE INVENTION
以下、 本¾明を突施例により、 さらに詳細に説明する力 水 は以下の実施 例に制限されるものではない。  Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.
なお、 本実施例において川いた ¾色ブドウ球 W RN4220は川 11天' 大学平松 ^士 より分与された。 色ブドウ球菌 MSSA、 MRSAは九州大学付属病院における臨床 分離株を用いた (Akimitsu, N.， et al. 1999. Antimicrob Agents Chemother 4 3: 3042-3043. )。 黄色ブドゥ球 [ Smith株、 大腸 i NIHJ株は微^物化学研究所 浜田^士より分- された。 誦 (S24) 、 大 I WW3110株及び K12-3株は研究 室保存株を用いた。 黄色ブドウ球菌はマンニッ ト食塩培地 （栄研化学株式会社） 、 緑膿菌は NAC培地 （栄研化学株式会社） 、 大腸菌は D0C培地 （栄研化学株式会 社） の寒天培地上で確認培養した。 これらの翻のシングルコロニーを LB液体培 地中で一晩培養して使用した。 In addition, in this example, the green erythrocyte W RN4220 that was collected was distributed by Kawahira Tenten University Hiramatsu. For Staphylococcus aureus MSSA and MRSA, clinical isolates from Kyushu University Hospital were used (Akimitsu, N., et al. 1999. Antimicrob Agents Chemother 43: 3042-3043.). Yellow budu bulb [Smith strain and large intestine i NIHJ strain were isolated from Research Institute for Fine Materials Chemistry, Dr. Hamada]. Recitation (S24), Dai I WW3110 shares and K12-3 shares studied A laboratory stock was used. Staphylococcus aureus was cultivated and confirmed on agar medium in Mannite salt medium (Eiken Chemical Co., Ltd.), Pseudomonas aeruginosa in NAC medium (Eiken Chemical Co., Ltd.), and Escherichia coli on D0C medium (Eiken Chemical Co., Ltd.). . These inverted single colonies were used overnight in LB liquid medium.
また、 カイコ幼虫の受精卵を日本養蚕工業株式会社より購入し、 室温で人工飼 料 （シルクメイ ト ： 日本養蚕工業株式会社） を与えて飼育した。  In addition, fertilized eggs of silkworm larvae were purchased from Japan Sericulture Industry Co., Ltd. and reared with artificial feed (silk mate: Japan Sericulture Industry Co., Ltd.) at room temperature.
[実施例 1 ] 黄色ブドゥ球菌及び緑膿菌によるカイコ幼虫の感染死  [Example 1] Infection and death of silkworm larvae by Staphylococcus aureus and Pseudomonas aeruginosa
黄色ブドウ球菌及び緑膿菌はヒ卜の F1和見感染^の原因菌である。 本発明者ら は、 これらの細菌がカイコ幼虫を感染死させるか否かを検討した。  Staphylococcus aureus and Pseudomonas aeruginosa are the causative agents of F1 opportunistic infection in humans. The present inventors examined whether these bacteria could infect silkworm larvae by infection.
5令カイコ幼虫の Ί—-腹脚部に Staphylococcus aureus及び Pseudomonas aeru の菌液または抗菌物質溶液を 0. 05 ml注射し、 指圧により 10秒 ^止血し た。 注射針は 27G X 3/4 (テルモ株式会社) 、 注射筒は l ml (テルモ株式会社) を使用した。 注射後の経時的な生存個体数を調べた （図 1 )。  0.05 ml of a bacterial solution or an antibacterial substance solution of Staphylococcus aureus and Pseudomonas aeru was injected into the abdominal leg of the fifth-instar silkworm larvae, and the bleeding was stopped for 10 seconds by finger pressure. The injection needle used was 27G X 3/4 (Termo Corporation), and the injection cylinder was 1 ml (Termo Corporation). The number of surviving individuals over time after the injection was examined (FIG. 1).
菌を含まない培地の希釈液を注射した場合は、 全ての個休が 5日以上生存して いた。 これに対し、 3 X 107個の Staphylococcus 尸 eusを注射した場合、 4つ の株 (RN4220, Smith MSSA MRSA) のいずれにおいても注射後 2日以内にカイ コ幼虫の 90%以上が死亡した （図 1 ) 。 黄色ブドウ球菌を注射したカイコ幼虫は、 注射後、 次第に餌を食べなくなり、 動きが緩愷になり、 2曰 Πに表皮が簿ぃ褐色 を呈し、 死亡した。 一方、 ォ一トクレーブ処理を行った黄色ブドウ球菌 （3 X 10 8 cel ls) を注射した場合のカイコ幼虫の生存率は、 注射 5日後においても、 8 0%以上であった (データは示さず) o 20系統の臨床分離 MRSA (Akimitsu, N . , e t al . 1999. Antimicrob Agents Chemother 43 : 3042-3043. ) について調べた結 果では、 MSSAに比べ特に! II性の い株は見出されなかったが、 MRSA問で感染死 を引き起こす必要な細胞数には違いが兄られた （デ一夕は示さず） 。 さらに、 注 射する黄色ブドウ球蘭 (MSSA) の閑数を、 3 X 10,; cel lsとした場合には、 注射 5 H後に全ての 1休が死 したが、 3 X 10;Ί ce l l sでは、 注射 5 [ |後の生 は 50%であった （デ一夕は示さず） 。 一方、 3 X 107個の ¾ei/cto/K>/?as aeruginosa (S24) を注射した場合、 1日後に全ての個体が死亡した （図 1 ) 。 緑膿菌を注 射したカイコ幼虫も、 注射後、 餌を食べなくなり、 表皮が黒色を呈して死亡した。 一方、 3 X 107個の fed? / c? a coli を注射した場合、 検討した 3つの株 （NIH J、 K12- 3、 W3110) いずれにおいても注射 5日後の生存率は 90%以上であり、 力 ィコ幼虫に対して病原性を示さなかった。 All injections survived for more than 5 days when injected with a dilution of the culture medium without bacteria. In contrast, when 3 × 10 7 Staphylococcus s.eus were injected, more than 90% of the silkworm larvae died within 2 days after injection in any of the four strains (RN4220, Smith MSSA MRSA) ( Figure 1 ) . After injection, the larvae of the silkworm, which had been injected with Staphylococcus aureus, gradually stopped eating and became slow to move, and died due to the appearance of a dark brown epidermis. On the other hand, the survival rate of silkworm larvae when injected with autoclaved S. aureus (3 × 10 8 cels) was still more than 80% even 5 days after injection (data not shown). o In clinical examination of 20 strains of clinically isolated MRSA (Akimitsu, N., et al. 1999. Antimicrob Agents Chemother 43: 3042-3043.) However, there was a difference in the number of cells required to cause infection death in MRSA (data not shown). Furthermore, if the number of yellow staphylococcal orchids (MSSA) to be injected is 3 × 10 ,; cel ls, all the 1-day rests died 5 H after injection, but 3 × 10 ; Ί ce lls Then, injection 5 [| 50% (not shown). On the other hand, when 3 × 10 7 ¾ei / cto / K> /? As aeruginosa (S24) were injected, all the animals died one day later (FIG. 1). The silkworm larva injected with Pseudomonas aeruginosa also stopped eating food after the injection and died with a black epidermis. On the other hand, when 3 × 10 7 fed? / C? A coli were injected, the survival rate 5 days after injection was 90% or more for all three strains (NIH J, K12-3, and W3110). However, it did not show any pathogenicity against the larvae of the larvae.
次ぎに、 注射した黄色ブドウ球菌がカイコ幼虫体内で増殖しているか否かを検 討した。 カイコ幼虫体内の菌数の測定を以下のように行なった。  Next, it was examined whether the injected Staphylococcus aureus was growing in the silkworm larvae. The number of bacteria in the silkworm larva was measured as follows.
菌を注射したカイコ幼虫の尾脚を切断し、 体液を採取後、 0.6% NaClで希釈し、 マンニット培地 （栄研化学株式会社） 上に塗布して、 37°Cにて一晩培養後、 現れ たコロニー数をカウントし、 カイコ体液中の菌数を計算した。 組織中の菌につい ては、 カイコ幼虫をべ一パー夕オル上で開腹し、 体液を除去後、 0.9% NaClに懸 濁した後、 マンニット培地上に塗布し、 現れたコロニー数をカウントし、 組織中 の菌数を計算した。 カイコ幼虫の体液及び、 組織の容積をそれそれ 1.5 ml及び 1 mlとして計算した。  The tail limb of the silkworm larva injected with the bacteria is cut, the body fluid is collected, diluted with 0.6% NaCl, spread on Mannit medium (Eiken Chemical Co., Ltd.), and cultured at 37 ° C overnight. The number of colonies that appeared was counted, and the number of bacteria in the silkworm body fluid was calculated. For the bacteria in the tissues, the silkworm larvae were laparotomized on a paper tube, the body fluid was removed, suspended in 0.9% NaCl, spread on Mannitol medium, and the number of colonies that appeared was counted. The number of bacteria in the tissue was calculated. The volume of the fluid and the tissue of the silkworm larva were calculated as 1.5 ml and 1 ml, respectively.
その結果、 力ィコ幼虫組織、 体液のどちらにおいても、 注射後菌数の顕著な増 加が認められ（図 2 )、 注射後 2日目にはカイコ体内の総菌数は 1 X 10x個に到達 した。 As a result, a marked increase in the number of bacteria after injection was observed in both the larval tissue and the body fluid (Fig. 2), and the total number of bacteria in the silkworm body was 1 x 10 x 2 days after the injection. Reached.
次に、 カイコ幼虫体内組織における、 黄色ブドウ球菌の増殖部位をパラフィン 組織切片に対する ίϊί光抗体法を用いて調べた。 カイコ幼虫をカルノア液 （EtOH ： chloroform ： CH 00H = 6 : 3 : 1)で室温にて 10時^固定した。 その後、 パ ラフィンに包埋し、 10〃mの厚さで切片を作成した。 脱バラフィンを行った組織 切片に対し、 1% BSA (Sigma社） でブロッキング操作を施した後、 黄色ブドウ球 菌モノクローナル抗体 （15702 QED Bioscience Inc.) を反応させた。 その後、 サンプルを PBSで 3回洗い、 光標識された 2次抗体 （FluoroLinl^Cy'1^ label led goat ant i -mouse IgG (H+L ) , Amersham社） を反応させた。 さらにサンプル を PBSで 3回洗い、 蛍光顕微鏡 （ォリンパス BH2) で観察した。 Next, the growth sites of Staphylococcus aureus in the body tissues of the silkworm larva were examined using a fluorescent antibody method to paraffin tissue sections. The silkworm larva was fixed with Carnoy's solution (EtOH: chloroform: CH00H = 6: 3: 1) at room temperature for 10 hours. After that, the sections were embedded in paraffin and sectioned to a thickness of 10 mm. The deparaffinized tissue section was subjected to a blocking operation with 1% BSA (Sigma), and then reacted with a Staphylococcus aureus monoclonal antibody (15702 QED Bioscience Inc.). The samples were then washed three times with PBS and photolabeled secondary antibody (FluoroLinl ^ Cy ' 1 ^ label led goat anti-mouse IgG (H + L), Amersham). The samples were further washed three times with PBS and observed under a fluorescence microscope (Olympus BH2).
図 3は、 黄色ブドゥ球菌を注射後 40時間後のカイコ幼虫の中腸を体軸に対し て垂直に切断した組織像である。 黄色ブドウ球菌を注射したカイコ幼虫では中腸 上皮に明瞭な蛍光が観察された。 この蛍光は黄色ブドウ球菌を注射しなかった力 ィコ幼虫 （図 3A) 、 あるいは 1次抗体を用いなかった場合 （データは示さず） は認められなかった。 以上の結果は、 カイコ幼虫の黄色ブドウ球菌による死が、 黄色ブドゥ球菌のカイコ幼虫体液中での増殖、 及び中腸組織への侵入と増殖によ る感染死であることを示唆する。  Figure 3 is a histological image of the silkworm larvae's midgut cut perpendicular to the body axis 40 hours after injection of S. aureus. In the silkworm larvae injected with Staphylococcus aureus, clear fluorescence was observed in the midgut epithelium. This fluorescence was not observed in larvae that were not injected with Staphylococcus aureus (Figure 3A) or without primary antibody (data not shown). These results suggest that the death of the silkworm larvae by Staphylococcus aureus is the growth of Staphylococcus aureus in the bodily fluid of the silkworm larvae and the death of infection by invasion and growth into the midgut tissue.
[実施例 2 ] 黄色ブドゥ球菌によるカイコ幼虫の感染死に対する抗生物質及び 消毒剤の効果  [Example 2] Effects of antibiotics and disinfectants on infection death of silkworm larvae caused by Staphylococcus aureus
さらに本発明者らは、 カイコ幼虫の黄色ブドウ球菌による感染死が抗生物質で 抑えられるか否かを調べた。 黄色ブドゥ球菌臨床分離株をカイコ幼虫に注射した 後、 アンピシリン、 ォキサシリン、 バンコマイシンを注射し、 経時的にカイコ幼 虫の生存個体数をカウン卜した。  Furthermore, the present inventors investigated whether the death of Bombyx mori larva infection by Staphylococcus aureus could be suppressed by antibiotics. After injection of the clinical isolate of B. aureus into silkworm larvae, ampicillin, oxacillin and vancomycin were injected, and the number of surviving silkworm larvae was counted over time.
MSSAを単独注射した場合、 2日後にはすべてのカイコ幼虫が死亡した。 この時、 アンピシリン （200 g/body)、 ォキサシリン （200〃g/body )、 バンコマイシン (200/zg/body) を注射した場合、 注射 4日目においても生存率は 90%以上であ つた （図 4A) 。 すなわち、 これら 3種の抗生物質はいずれも MSSAによるカイコ 幼虫の感染死を抑えた。  All silkworm larvae died two days later when MSSA was injected alone. At this time, when ampicillin (200 g / body), oxacillin (200 mg / body), and vancomycin (200 / zg / body) were injected, the survival rate was more than 90% even on the fourth day of injection (Fig. 4A). In other words, all three of these antibiotics suppressed MSSA larval infection and death.
MRSA準独を注射した場合、 1口後には 90%のカイコ幼虫が死亡した。 この時、 アンピシリン （200〃g/body )、 ォキサシリン （200 ig/body) を注射した場合で も、 注射 2日目にすべてのカイコ幼虫が死亡した。 これに対してバンコマイシン ( 200 g/body) を注射した場合には、 注射 4曰目においても生存率は 80%以上 であった （図 4B )。 よって、 MRSAによるカイコ幼虫の感染死はアンピシリン、 ォ キサシリンにより抑えられないが、 バンコマイシンにより抑えられることが判明 した。 When injected with MRSA quasi-German, 90% of the silkworm larvae died after one mouthful. At this time, all the silkworm larvae died on the second day of the injection, even if ampicillin (200 μg / body) or oxacillin (200 ig / body) was injected. On the other hand, when vancomycin (200 g / body) was injected, the survival rate was more than 80% even in the case of injection 4 (Fig. 4B). Therefore, infection death of silkworm larvae by MRSA is It was found that it was not suppressed by xacillin, but was suppressed by vancomycin.
さらに、 種々の消毒剤によって MSSA、 MRSAによるカイコ幼虫の死が抑えられ るか否かを検討した。 まず最初に、 各種消毒剤の寒天培地における黄色ブドウ球 菌に対する MIC値とカイコ幼虫に対する LD5。値を比較した （表 1 ) 。 Furthermore, we examined whether various disinfectants can suppress the death of silkworm larvae by MSSA and MRSA. First, LD 5 for MIC values and silkworm larvae against Staphylococcus sphere bacteria in the agar medium Disinfectants. The values were compared (Table 1).
表 1  table 1
Figure imgf000015_0001
Figure imgf000015_0001
N D '測定していない その結 ¾、 EtOH及びポビドンョードは MICよりも LDaiの値が低く治疮効采は 期待できないことが判叨した。 一方、 塩化べンザルコニゥムの MIC値は LD i値よ りも小さい値を示したが、 LD5I1に近い の塩化ベンザルコニゥムによっても MSSA によるカイコ幼虫の死は抑えられなかった。 すなわち、 黄色ブドウ球菌による力 ィコ幼虫の感染死に対して、 消毒剤による治療は有効でないことが判明した。 なお、 この実験に用いたアンピシリン （萬有製薬株式会社）、 ォキサシリン （S igma) 、 バンコマイシン （塩！!] ί¾製薬株式会社） 、 塩化ベンザルコニゥム （古 Π1 製薬株式会社） 、 ポビドンョ一ド （明治製菜株式会社） はすべて、 0.6% NaClで 希釈して川いた。 それそれの杭 物 fTの ¾色ブドウ球 wに対する MICは、 種々の 濃度の抗菌物 を含む LB10寒天培地上に蘭の一晩培 ¾液 ( 1 X 109 cel ls/ml) を 10:i分の 1に希釈して、 その 1〃1をェ一ゼで広げ、 37°Cで 72時問培養して求 めた (Akimitsu, N. , et al . 1999. Antimicrob Agents Chemother. 43 : 3042-30 3. ) 。 感染したカイコ幼虫に対する IC5()を求める場合には、 5 X 106乗個の黄色 ブドウ球菌をカイコ幼虫血液中に注射後、 さらに種々の濃度の抗菌物質溶液を 0. 05 ml注射した。 注射 4日後に生存個体数が半数となる抗菌物質の濃度を求めた。 カイコ幼虫の体液を 1.5 ml として IC5(1値を計算した。 カイコ幼虫に対する LD5() は種々の濃度の抗菌物質の溶液 0. 05 mlを 5匹の幼虫に注射し、 注射後 1日目に 半数の幼虫が死亡する濃度とした。 産業上の利用の可能性 As a result, it was found that EtOH and povidone were lower in LD ai value than MIC and could not be expected to cure. Meanwhile, MIC values of the base chloride Nzarukoniumu showed a LD i values by remote small value, the death of the silkworm larva by MSSA was not suppressed by chloride Benzarukoniumu near the LD 5I1. In other words, it was found that treatment with a disinfectant was not effective for the death of infection of D. larvae by Staphylococcus aureus. The ampicillin (Banyu Pharmaceutical Co., Ltd.), oxacillin (Sigma), vancomycin (Salt !!) Pharmaceutical Co., Ltd., benzalkonium chloride (Old Pharmaceutical Co., Ltd.), and povidonide (Meiji Co., Ltd.) used in this experiment All of them were diluted with 0.6% NaCl. The MIC of each pile fT against green globule w was determined by transferring an orchid overnight culture (1 × 10 9 cel ls / ml) on LB10 agar medium containing various concentrations of antibacterial substances at 10 : i. After diluting to 1/1, spreading the 1〃1 with a protease and culturing at 37 ° C for 72 hours (Akimitsu, N., et al. 1999. Antimicrob Agents Chemother. 43: 3042 -30 3.) To determine IC5 () for infected silkworm larvae, 5 × 10 6 S. aureus were injected into the silkworm larva blood, and then 0.05 ml of various concentrations of antimicrobial solutions were injected. Four days after the injection, the concentration of the antibacterial substance at which the number of surviving individuals was half was determined. The IC 5 (1 value was calculated assuming that the body fluid of the silkworm larva was 1.5 ml. LD5 () for the silkworm larva was injected with 0.05 ml of various concentrations of antibacterial solutions to 5 larvae, and one day after the injection The concentration at which half of the larvae die in the eye.
本発明により、 自然免疫機構のみを冇する生物を利川した、 獲得免疫機構を有 する生物に対する病原微生物感染のモデルが提供された。 本発明の感染モデルを 利用した抗菌剤の効果は、 獲得免疫機構を有する生物におけるこれらの薬剤の有 効性に対応することが期待される。 従って、 本発明の感染モデルは、 ヒトを含む 獲得免疫機構を有する生物に対する様々な病原体の感染モデルとなり得、 これら 病原体による感染症の抗菌剤のスクリーニングに有用である。 本発明の感染モデ ルは、 哺乳動物を用いた病原微生物の感染実験の前段階として用いることにより、 ヒ卜の臨床応用に可能な抗菌剤の開発の効率化に寄与することが期待できる。 特 にカイコの感染モデルは、 グラム陽性の病原性細菌により感染されることから、 例えば、 黄色ブドゥ球菌による日和見感染症に対する抗菌剂の開発に有効である。 また、 本発明の感染モデルを利用すれば、 薬剤のスクリーニングにおいて、 従 来の哺乳勤物を利 fflする場合と異なり、 一個体当りの入手費用、 飼育費用、 およ び実験スペースを大幅に節減することが可能となる。 例えば、 マウス 1000匹を SPF環境 （①空気をフィル夕一で濾過すること、 および②温度及び湿度を一定に 保つこと、 が必要） で飼育するのに必要なスペースは、 およそ 25 m2であり、 こ れに加えてケージ洗浄室、 オートクレーブ室などのバックアツプ設備が必要とな る。 一方、 カイコの幼虫では、 1 にインキュベーターを設； すれば、 1000〜10 000匹 (齢数により ¾なる) の飼 が可能であり、 度を一 （例えば、 30。C ) に保つこと以外に、 ケージ洗浄室、 オートクレープ室などの特別なバックアップ 設備は不要である （匹数、 必要な面積数などは目安の概数で諸条件により変化す る） 。 According to the present invention, a model of infection of pathogenic microorganisms to an organism having an adaptive immunity mechanism, which has an organism having only an innate immunity mechanism, has been provided. The effects of antibacterial agents using the infection model of the present invention are expected to correspond to the efficacy of these agents in organisms having an acquired immune mechanism. Therefore, the infection model of the present invention can be used as a model for infection of various pathogens to organisms having an acquired immune mechanism, including humans, and is useful for screening antibacterial agents for infectious diseases caused by these pathogens. The infectious model of the present invention can be expected to contribute to the efficient development of antibacterial agents that can be used for human clinical application by using the infectious model as a pre-stage of pathogenic microorganism infection experiments using mammals. In particular, the silkworm infection model is effective for the development of antibacterial activity against opportunistic infections caused by Staphylococcus aureus, for example, because it is infected by gram-positive pathogenic bacteria. In addition, the use of the infection model of the present invention greatly reduces acquisition costs, breeding costs, and experimental space per individual in drug screening, unlike the conventional case of using a mammal. It becomes possible. For example, (filtering the ① air fill evening one, and ② to keep the temperature and humidity constant, is necessary) 1000 mice SPF environment space required for breeding in is an approximately 25 m 2 In addition, backup facilities such as a cage washing room and an autoclave room are required. On the other hand, for the silkworm larvae, if an incubator is set at 1, it is possible to raise 1000 to 10000 animals (depending on the age), and it is possible to keep them at a single degree (for example, 30.C). No special backup equipment such as a cage washing room and an autoclave room is required other than keeping the number of animals (the number of animals, the required number of areas, etc. is an approximate number and varies depending on various conditions).
また、 本発明のカイコ感染モデルは、 C. e7e /] sなどの小型の生物と比較し て病原体や薬物の注射が極めて容易であり、 病原体に対する抗菌薬の評価に適し ていると言える。  In addition, the silkworm infection model of the present invention is extremely easy to inject pathogens and drugs compared to small organisms such as C.e7e /] s, and can be said to be suitable for evaluating antibacterial drugs against pathogens.
カイコを含めた無脊椎動物や植物には、 抗体を介した獲得免疫機構は存在しな いが、 ヒトと共通した自然免疫機構が存在する。 本発明の感染モデルは、 病原体 の感染に対する自然免疫機構を遺伝学的手法を用いて分子レベルで解明するため にも有効である。  Invertebrates and plants, including silkworms, do not have an antibody-mediated adaptive immunity mechanism, but do have an innate immunity mechanism common to humans. The infection model of the present invention is also effective for elucidating the innate immunity mechanism against pathogen infection at the molecular level using genetic techniques.

Claims

請求の範囲 The scope of the claims
1. 獲得免疫機構を有する生物に感染する病原微生物に対し抗菌活性を有する 化合物をスクリーニングする方法であって、 1. A method for screening a compound having antibacterial activity against a pathogenic microorganism that infects an organism having an adaptive immune mechanism,
(a) 自然免疫機構のみを有する生物に該病原微生物および被検試料を投与する 工程、  (a) administering the pathogenic microorganism and a test sample to an organism having only an innate immune mechanism,
(b) 該自然免疫機構のみを有する生物の感染症状または生存の程度を検出する 工程、 および  (b) detecting an infection symptom or a degree of survival of an organism having only the innate immunity mechanism, and
(c) 被検試料を投与しない場合 （対照） と比較して、 該自然免疫機構のみを冇 する生物の感染症状を改蕃する、 または生存の程度を向上させる化合物を選択す る工程、 を含む方法。  (c) a step of selecting a compound that ameliorates the infectious symptoms of an organism having only the innate immunity mechanism or improves the degree of survival as compared to the case where the test sample is not administered (control). Including methods.
2. 獲得免疫機構を有する生物に感染する病原微生物に対する、 被検試料の抗 菌活性を評価する方法であって、  2. A method for evaluating the antibacterial activity of a test sample against a pathogenic microorganism that infects an organism having an acquired immune mechanism,
(a) 自然免疫機構のみを有する生物に病原微生物および被検試料を投与するェ 程、  (a) administering the pathogenic microorganism and the test sample to an organism having only the innate immune mechanism,
(b) 該自然免疫機構のみを有する生物の感染症状または生存の程度を検出する 工程、 および  (b) detecting an infection symptom or a degree of survival of an organism having only the innate immunity mechanism, and
(c) 被検試料を投与しない場合 （対照） と比較して、 該被検試料が、 該自然免 疫機構のみを有する生物の感染症状を改善するか否か、 または生存の程度を向上 させるか否かを判定する工程、 を含む方法。  (c) whether or not the test sample improves the infectious symptoms of an organism having only the natural immunological mechanism or the degree of survival as compared to the case where no test sample is administered (control) Deciding whether or not.
3. 狻捋免疫機構を有する生物が哺乳動物である、 詰求 ¾11または 2に記載の 方法。  3. The method according to claim 11 or 2, wherein the organism having an immune mechanism is a mammal.
4. 哺乳動物がヒトである、 詰求項 3に記載の方法。  4. The method of claim 3, wherein the mammal is a human.
5. 自然免疫機構のみを有する生物が昆虫類に属する生物である、 請求項 1か ら 4のいずれかに記載の方法。  5. The method according to any one of claims 1 to 4, wherein the organism having only the innate immunity mechanism is an organism belonging to insects.
6. 昆虫類に屈する生物が幼虫である、 詰求项 5に記^の方法。 6. The larva is the creature that yields to insects.
7 . 幼虫が大型である、 請求項 6に記載の方法。 7. The method of claim 6, wherein the larva is large.
8 . 昆虫類に属する生物がカイコである、 請求項 5から 7のいずれかに記載の 方法。  8. The method according to claim 5, wherein the organism belonging to insects is a silkworm.
9 . 自然免疫機構のみを有する生物がグラム陽性の病原微生物により感染され るものである、 請求項 1から 7のいずれかに記載の方法。  9. The method according to any one of claims 1 to 7, wherein the organism having only the innate immune mechanism is infected by a gram-positive pathogenic microorganism.
1 0 . 獲得免疫機構を有する生物に感染する病原微生物が黄色ブドウ球菌、 緑濃 菌、 コレラ菌および病原性大腸菌からなる群より選択される、 請求項 1から 9の いずれかに記載の方法。  10. The method according to any one of claims 1 to 9, wherein the pathogenic microorganism that infects an organism having an adaptive immune mechanism is selected from the group consisting of Staphylococcus aureus, Pseudomonas aeruginosa, Vibrio cholerae, and pathogenic Escherichia coli.
1 1 . 請求項 1から 1 0のいずれかに記載の方法により同定される抗菌活性を有 する化合物。  11. A compound having an antibacterial activity identified by the method according to any one of claims 1 to 10.
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