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A silkworm infection model to investigate Vibrio vulnificus virulence genes
Mai Yamamoto1, Takashige Kashimoto2, Yukihiro Yoshimura1
1Department of Nutritional Science, Okayama Prefectural University, Soja, Okayama 719‑1197, Japan.
Abstract:
The halophilic marine bacterium, Vibrio vulnificus, occasionally causes fatal septicemia in immunocompromised patients. Mice are commonly used as experimental animals to investigate the virulence of V. vulnificus, however, a large number of mice are generally required for bioassays. The present study examined whether the invertebrate species, silkworms, can be used instead of mice to investigate V. vulnificus virulence. When the silkworms were inoculated with 1.2x107 colony forming units of V. vulnificus OPU1‑Rf, a virulent strain of V. vulnificus, all injected silkworms died within 48 h, however, those injected with culture filtrate or diluent did not. This silkworm infection model was then used to isolate attenuated V. vulnificus mutants from 1,016 transposon‑inserted mutants. Consequently, a harmless mutant, SW998, was isolated. In this strain, the transposon was inserted into the rtxA gene, which is a known V. vulnificus virulence gene. In conclusion, the present study demonstrated that silkworms are useful animals for investigating the virulence of V. vulnificus.
Insights
Silkworms offer a viable alternative to mice for studying Vibrio vulnificus virulence. This research successfully utilized silkworms to identify an attenuated V. vulnificus mutant, demonstrating their utility in virulence studies.
Area of Science:
- Microbiology
- Infectious Diseases
- Animal Models
Background:
- Vibrio vulnificus is a halophilic marine bacterium causing severe infections, particularly in immunocompromised individuals.
- Traditional mouse models for V. vulnificus virulence studies require significant animal numbers.
- There is a need for alternative, potentially more efficient, animal models for V. vulnificus research.
Purpose of the Study:
- To evaluate the efficacy of silkworms (invertebrates) as an alternative animal model for investigating V. vulnificus virulence.
- To establish a silkworm infection model for V. vulnificus.
- To use this model to isolate attenuated V. vulnificus mutants.
Main Methods:
- Silkworms were inoculated with a virulent strain of V. vulnificus (OPU1-Rf).
- Survival rates and time to death were monitored.
- The silkworm model was employed to screen 1,016 transposon-mutagenized V. vulnificus strains.
- Mutants were analyzed for virulence attenuation.
Main Results:
- Inoculation with virulent V. vulnificus (1.2x10^7 CFU) resulted in 100% mortality in silkworms within 48 hours.
- Silkworms injected with culture filtrate or diluent showed no mortality.
- A single attenuated mutant, SW998, was successfully isolated using the silkworm model.
- The transposon in SW998 was found to be inserted in the rtxA gene, a known virulence factor.
Conclusions:
- Silkworms serve as a practical and effective animal model for studying V. vulnificus virulence.
- This invertebrate model facilitates the isolation of attenuated V. vulnificus strains.
- The rtxA gene is confirmed as a critical virulence factor in V. vulnificus using the silkworm model.
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