| Project/Area Number |
23K14520
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| Research Category |
Grant-in-Aid for Early-Career Scientists
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| Allocation Type | Multi-year Fund |
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| Review Section |
Basic Section 49050:Bacteriology-related
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| Research Institution | Osaka University |
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Principal Investigator |
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| Project Period (FY) |
2023-04-01 – 2025-03-31
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| Project Status |
Granted (Fiscal Year 2023)
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| Budget Amount *help |
¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000)
Fiscal Year 2024: ¥2,340,000 (Direct Cost: ¥1,800,000、Indirect Cost: ¥540,000)
Fiscal Year 2023: ¥2,340,000 (Direct Cost: ¥1,800,000、Indirect Cost: ¥540,000)
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| Keywords | Bordetella / Acanthamoeba / Survival / BvgAS / Coculture / Extrahost / 気管支敗血症菌 / 環境アメーバ |
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| Outline of Research at the Start |
気管支敗血症菌(Bb)は広く哺乳動物に呼吸器感染を起こす病原細菌である。本菌は宿主動物間で感染伝播するほか、環境中で土壌アメー バ(Acanthamoeba castellanii, Ac; Dictyostelium discoideum, Dd)内などで生息し、そこから宿主に感染伝播する可能性が指摘されてきた。もしBbが環境中から宿主感染をするならば、宿主間感染の場合とは別の感染症防除法を考察する必要がある。そこで本研究では、Bbが土壌アメーバの貪食を回避して生息するメカニズムを詳細に解析して、環境からの感染伝播の可能性を議論する材料を提供する。
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| Outline of Annual Research Achievements |
B. bronchiseptica exhibits distinct Bvg+ and Bvg－ phenotypes. Our experimental results have indicated that the growth of a mutant strain locked in the Bvg+ phase in coculture with A. castellanii was significantly reduced compared to the wild type or a Bvg－ phase-locked strain. Since Bvg－ phase-specific factors are less understood, we focused on screening genes expressed in the Bvg+ phase, including known virulence genes, that are involved in intra-amoeba survival. To this end, we used a series of Bvg+ phase-locked strain-derived mutants deficient in multiple Bvg+ phase-specific virulence factors and subjected them to a coculture assay. Our findings indicated that some multiple mutants deficient in filamentous hemagglutinin (FHA) and/or fimbriae (FIM), the major adhesins produced by B. bronchiseptica in the Bvg+ phase, survived similarly to the wild type and the Bvg－ phase-locked mutant. Independently, ΔfhaB, ΔfimBCD, and ΔfhaB/ΔfimBCD derived from the Bvg+ phase-locked mutant survived well in coculture with A. castellanii, similar to the Bvg－ phase-locked mutant. The numbers of intracellular Bvg+ phase-locked ΔfhaB and ΔfimBCD strains recovered from A. castellanii were similar to those of the wild type and the Bvg－ phase-locked mutant. Furthermore, microscopic analyses demonstrated that the Bvg+ phase-locked ΔfhaB and/or ΔfimBCD strains enter contractile vacuoles of A. castellanii and evade its predation, similar to the wild type. These results indicate that FHA and FIM encoded by fhaB and fimBCD, respectively, are targeted for predation by A. castellanii.
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| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
Our positive screening strategy using multiple-mutant strains that led to the identification of FHA and FIM, two Bvg+ phase-specific adhesins targeted by A. castellanii predation, has been published as a research article in Microbiology Spectrum. We were able to proceed smoothly because our laboratory had prepared and stocked the multiple-mutant strains, which allowed us to start screening immediately. Additionally, the results of our positive screening were also straightforward, as shown directly by the increase in total CFU of the mutant strains after the coculture assays, which made analysis easier. Overall, this progress enables us to move on to the next phase of our research, which involves screening for bacterial factors specifically expressed in the Bvg－ phase that may also support B. bronchiseptica survival in A. castellanii.
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| Strategy for Future Research Activity |
There are two possible explanations for why the Bvg+ phase-locked mutant, but not the Bvg－ phase-locked mutant, served as prey for the amoebae: factors specifically expressed in the Bvg－ phase protect the bacteria from predation, or Bvg+ phase-specific factors are recognized by the amoebae for digestion. While we have identified Bvg+ phase-specific factors that may be targeted by amoeba predation, we still believe that Bvg－ phase-specific factors play a role in the survival of B. bronchiseptica in A. castellanii, based on the fact that B. bronchiseptica switches to Bvg－ phase at temperatures where the bacteria normally encounter these amoebae. Next year, we will attempt to screen these genes using Transposon Sequencing (Tn-Seq). To achieve this, we have constructed a genome-wide library consisting of approximately ~40,000 transposon-inserted B. bronchiseptica mutants. In addition, we will also optimize the coculture assay protocol used for the negative screening procedure. If promising candidate genes are found through Tn-Seq, we will perform further experiments to characterize these genes.
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