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Silkworm model for Francisella novicida infection
Shib Shankar Saha1, Jin Suzuki2, Akihiko Uda3
1The United Graduate School of Veterinary Science, Yamaguchi University, 1677-1, Yoshida, Yamaguchi 753-8515, Japan; Department of Pathology and Parasitology, Patuakhali Science and Technology University, Babugonj, Barisal 8210, Bangladesh.
Abstract:
Understanding the virulence and pathogenesis of human pathogens using insect models is an increasingly popular method. Francisella novicida, which is virulent in mice but non-pathogenic to immunocompetent humans, is widely used as an ideal candidate for Francisella research. In this study, we developed a silkworm (Bombyx mori) infection model for F. novicida by inoculating the hemocoels of silkworms with F. novicida. We found that silkworms died within 3-7 days of F. novicida infection. However, the deletion mutant of DotU, the core part of type VI secretion systems, failed to kill silkworm. In whole silkworm bodies, the bacterial load of the DotU deletion mutant was significantly less than that of the wild-type strain. Approximately 10-fold increase in bacterial load was recorded in hemolymph and subcutaneous tissues compared with that in the silk gland, Malpighian tubule, and reproductive organs. The CFU count of the DotU deletion mutant in all organs was similar results to the whole body CFU count. Confocal microscopy further confirmed the arrested growth of the mutant strain within hemocytes. The intracellular growth of F. novicida strains was also analyzed using the silkworm ovary-derived cell line BmN4. In BmN4, both CFU count assay and confocal microscopy revealed extensive growth of the wild-type strain compared with that of the mutant strain. Francisella DotU has already been proven as a virulence factor in mammals, and it was also found to be an essential virulence factor in our silkworm infection model. Therefore, this silkworm infection model is suitable for identifying new virulence factors of Francisella.
Insights
A novel silkworm infection model effectively demonstrated Francisella novicida pathogenesis. The DotU protein was confirmed as a crucial virulence factor, essential for bacterial survival and host mortality in this insect model.
Area of Science:
- Microbiology
- Infectious Diseases
- Insect Pathology
Background:
- Insect models are valuable for studying human pathogen virulence and pathogenesis.
- Francisella novicida, a mouse-virulent but human-non-pathogenic bacterium, serves as a key research model.
- Understanding Francisella virulence factors is crucial for developing effective countermeasures.
Purpose of the Study:
- To establish and validate a silkworm (Bombyx mori) infection model for Francisella novicida.
- To investigate the role of the DotU protein in Francisella novicida pathogenesis using the silkworm model.
- To assess the suitability of the silkworm model for identifying novel bacterial virulence factors.
Main Methods:
- Inoculation of silkworm hemocoels with wild-type and DotU deletion mutant strains of Francisella novicida.
- Monitoring silkworm survival rates and quantifying bacterial loads in whole bodies and specific organs.
- Utilizing confocal microscopy to assess bacterial growth within silkworm hemocytes and the BmN4 cell line.
Main Results:
- Francisella novicida infection caused silkworm mortality within 3-7 days.
- A DotU deletion mutant significantly reduced bacterial load and failed to induce mortality, indicating DotU's essential role.
- Bacterial growth was significantly impaired in the DotU mutant within silkworm hemocytes and the BmN4 cell line.
- Bacterial load distribution showed variations across different silkworm tissues.
Conclusions:
- The silkworm (Bombyx mori) serves as a viable and effective infection model for studying Francisella novicida pathogenesis.
- The DotU protein is a critical virulence factor for Francisella novicida in the silkworm model, mirroring its role in mammalian systems.
- This silkworm infection model is a promising tool for the discovery and characterization of new bacterial virulence factors.
