Quantitative evaluation of cryptococcal pathogenesis and antifungal drugs using a silkworm infection model with Cryptococcus neoformans

Abstract

Aims: To develop an in vivo system that could quantitatively evaluate the therapeutic effects of antifungal drugs using a silkworm infection model with Cryptococcus neoformans. Methods and Results: Silkworms reared at 37°C died after an injection of viable serotype A C. neoformans fungus into the haemolymph. The serotype A C. neoformans, which is known to have higher mammal pathogenicity than the serotype D, was also more virulent against the silkworm. Furthermore, the deletion mutants of genes gpa1, pka1 and cna1, which are genes known to be necessary for the pathogenesis in mammals, showed an increase in the number of fungal cells necessary to kill half of the silkworm population (LD50 value). Antifungal drugs, amphotericin B, flucytosine, fluconazole and ketoconazole, showed therapeutic effects in silkworms infected with C. neoformans. However, amphotericin B was not therapeutically effective when injected into the silkworm intestine, comparable to the fact that amphotericin B is not absorbed by the intestine in mammals. Conclusions: The silkworm–C. neoformans infection model is useful for evaluating the therapeutic effects of antifungal drugs. Significance and Impact of the Study: The silkworm infection model has various advantages for screening antifungal drug candidates. We can also elucidate the cryptococcal pathogenesis and evaluate the in vivo pharmacokinetics and toxicity of each drug.

Silkworm fungal infection model for identification of virulence genes in pathogenic fungus and screening of novel antifungal drugs

“…At 37°C, C. neoformans grows in the body of silkworms resulting in a killing effect, whereas at 27°C, the fungus does not kill silkworms. The capsule thickness and the overall size of the cell of C. neoformans increase in the silkworm hemolymph at 37°C, where pathogenicity of C. neoformans is exhibited, whereas apparent morphological changes are not observed at 27°C (12). The capsule has been shown to be necessary for pathogenicity of C. neoformans against mammals.…”

“…We found that strains deficient in the can, gpa1, or pka1 genes, which are required for virulence in C. neoformans against mammals, also showed low pathogenicity against silkworms (12). The product of the can gene is considered to contribute to the pathogenicity in mammals via the calcineurin signaling pathway (22).…”

“…Heat treatment (121°C, 15 min) of the fungi eliminates the killing ability of C. albicans or C. neoformans (12,14). Killing of the silkworms by C. neoformans infection is greatly influenced by temperatures.…”

“…Silkworms die within a few days after injection with Aspergillus fumigatus, Candida albicans, Candida tropicalis, Candida glabrata, or Cryptococcus neoformans (7,(10)(11)(12)(13). Heat treatment (121°C, 15 min) of the fungi eliminates the killing ability of C. albicans or C. neoformans (12,14).…”

Histological Observation and Expression Patterns of antimicrobial peptides during Fungal Infection in Musca domestica (Diptera: Muscidae) Larvae

“…These methods model the natural infectious process of pathogenic invasion into the epidermis and intestinal tract, which can then cause local infection that may gradually develop into systemic infection (Onfelt et al 2001;Nayduch et al 2013;Wu et al 2014). The injury method mainly used needles to deliver pathogens by piercing the somatic layer or directly injects pathogens into the insect coelom to cause acute infection and rapidly activate the immune response (Matsumoto et al 2012;Singh et al 2014). However, the inevitable mechanical injury caused by this method may affect the experimental results.…”