2023 Fiscal Year Research-status Report
The mechanism by which Bordetella bronchiseptica avoids predation by amoebae
| Project/Area Number |
23K14520
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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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| Keywords | Bordetella / Acanthamoeba / Survival / BvgAS / Coculture / Extrahost |
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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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| Causes of Carryover |
Previously, we had planned to perform both positive and negative screening simultaneously to identify bacterial genes required for survival in amoebae. However, we decided to conduct the positive screening first because our laboratory had prepared and stocked multiple mutant strains. This allowed us to carry out the screening immediately, and achieved good results with this approach. It also enabled us to minimize our expenses as we did not have to allocate a budget for the mutant construction. Additionally, positive screening did not require any high-end reagents or tools; instead, we utilized resources already available in our laboratory. For publication, we received a publication discount by submitting our results to the American Society for Microbiology (ASM), of which we are active members. Next year, we want to continue this research by performing negative screening using Tn-Seq, which requires specific reagents, tools, and analyses, and plan to present these results at scientific conferences. As a result, we anticipate an increase in our costs. Therefore, it would be beneficial if we could transfer our remaining budget from last year to this year's budget allocation.
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Research Products
(10 results)
