A novel silkworm infection model with fluorescence imaging using transgenic Trichosporon asahii expressing eGFP

Abstract

Trichosporon asahii is a pathogenic fungus that causes deep mycosis in patients with neutropenia. Establishing an experimental animal model for quantitatively evaluating pathogenicity and developing a genetic recombination technology will help to elucidate the infection mechanism of T. asahii and promote the development of antifungal drugs. Here we established a silkworm infection model with a transgenic T. asahii strain expressing eGFP. Injecting T. asahii into silkworms eventually killed the silkworms. Moreover, the administration of antifungal agents, such as amphotericin B, fluconazole, and voriconazole, prolonged the survival time of silkworms infected with T. asahii. A transgenic T. asahii strain expressing eGFP was obtained using a gene recombination method with Agrobacterium tumefaciens. The T. asahii strain expressing eGFP showed hyphal formation in the silkworm hemolymph. Both hyphal growth and the inhibition of hyphal growth by the administration of antifungal agents were quantitatively estimated by monitoring fluorescence. Our findings suggest that a silkworm infection model using T. asahii expressing eGFP is useful for evaluating both the pathogenicity of T. asahii and the efficacy of antifungal drugs. Trichosporon asahii is a basidiomycetous yeast widely distributed in the environment 1-4 , and commonly isolated from human blood, sputum, skin, feces, and urine 5-8. T. asahii is a pathogenic fungus that causes severe deep mycosis in patients with neutropenia 8-10. Whereas the mortality rate of deep mycosis caused by Candida albicans is approximately 40%, that caused by T. asahii is approximately 80%, and the prognosis is poor 11. T. asahii is resistant to echinocandin antifungal drugs and causes severe infections in patients treated with micafungin 12. Moreover, strains resistant to amphotericin B and azole antifungal drugs such as fluconazole have been isolated from patients 13,14. Therefore, T. asahii has become a serious clinical problem as a pathogenic fungus that causes systemic infection in immunocompromised hosts 8. T. asahii forms hyphae, which are branching filamentous structures 14. In pathogenic fungi that form hyphae such as Candida albicans, hyphal formation is crucial for the host epithelial cell damage and biofilm formation that are involved in infections 15. As with C. albicans, T. asahii makes treatment difficult by forming a biofilm on devices such as catheters 14,16. Hyphal growth of T. asahii in blood vessels causes necrotic thrombi, and may contribute to infection 17. The molecular mechanisms of infection caused by T. asahii, however, remain unclear. Establishing a simple animal experimental system for systemic infection with hyphal formation of T. asahii and developing genetic recombination technology in T. asahii will contribute to elucidate the infection mechanism. The silkworm, an invertebrate, is a useful experimental animal for evaluating the pathogenicity of pathogenic microorganisms and the therapeutic effects of antibacterial, antifungal, and antiviral drugs 18-22. Silkw...

Direct Visualization of Fungal Burden in Filamentous Fungus-Infected Silkworms

“…Traditionally, studying fungal burden in infected silkworms was limited to the usage of transgenic fungal mutants expressing fluorescent proteins [ 19 , 23 ], or to the application of time-consuming histological staining procedures [ 24 , 25 ]. To circumvent these limitations, we established a simple staining protocol using a common fluorescent dye, CW, to stain the cell wall of fungal hyphae in freshly extracted organs of A. fumigatus -infected silkworms, as demonstrated in Figure 1 .…”

“…In the case of C. neoformans, 37 °C had been necessary for this pathogen to establish infection in the silkworm, and monitoring of survival had been stopped before 70h [55]. In the case of T. asahii, the survival of silkworms at 37 °C was monitored for less than 55 h [19]. Unlike these two previous studies, when choosing the incubation temperature, we aimed to find a balance between minimizing thermal stress on silkworms and the rapid growth of A. fumigatus in vivo.…”

“…(3) Larger injection volume (up to 50 µL), which enables accurate dose titrations; (4) Feasibility of both intra-hemolymph and intra-midgut injections, which mimic intravenous and oral administrations in mammals, respectively; (5) Comparable therapeutic effects, pharmacokinetic parameters, and toxicity of antimicrobial drugs in silkworms and mammals. Silkworm infection models have been established for various human fungal pathogens [14,19], including yeasts and filamentous fungi, such as Candida species, Cryptococcus neoformans, Trichosporon asahii, Aspergillus fumigatus, Rhizopus oryzae, and several dermatophytes. A silkworm infection model of Candida albicans has been applied for screening of null mutants to identify virulence-related genes [20].…”

“…Silkworm infection models have been established for various human fungal pathogens [ 14 , 19 ], including yeasts and filamentous fungi, such as Candida species, Cryptococcus neoformans , Trichosporon asahii , Aspergillus fumigatus , Rhizopus oryzae , and several dermatophytes. A silkworm infection model of Candida albicans has been applied for screening of null mutants to identify virulence-related genes [ 20 ].…”

“…Although silkworm infection models have been established for different fungal pathogens, microscopical visualization of fungal burden in silkworms so far relies on recombinant fungal strains that express fluorescent proteins [ 19 , 23 ] or on classical histological staining procedures such as Grocott-Gomori’s methenamine silver stain or periodic acid-Schiff stain in tissues, which are time-consuming due to fixation, sectioning, dehydration, and staining steps [ 24 , 25 ].…”