Search

The facile inoculum delivery and handling of the insect Galleriamellonellamake it a desirable model for the study of fungal pathogenesis. Here we present methods to study fungal virulence, filamentation and fungal cell associates with insect hemocytes using Candidaalbicansand Cryptococcusneoformansto illustrate the use of this model. The two types of fungi cause distinct infections thus we compare and contrast the infection characteristics observed in G. mellonella. The protocols presented herein can be adapted to the study of other fungal pathogens using G. mellonellaas an infection model.

ABSTRACTEvaluation of Cryptococcus neoformansvirulence in a number of nonmammalian hosts suggests that C. neoformansis a nonspecific pathogen. We used the killing of Galleria mellonella(the greater wax moth) caterpillar by C. neoformansto develop an invertebrate host model system that can be used to study cryptococcal virulence, host immune responses to infection, and the effects of antifungal compounds. All varieties of C. neoformanskilled G. mellonella. After injection into the insect hemocoel, C. neoformansproliferated and, despite successful phagocytosis by host hemocytes, killed caterpillars both at 37°C and 30°C. The rate and extent of killing depended on the cryptococcal strain and the number of fungal cells injected. The sequenced C. neoformansclinical strain H99 was the most virulent of the strains tested and killed caterpillars with inocula as low as 20 CFU/caterpillar. Several C. neoformansgenes previously shown to be involved in mammalian virulence (CAP59, GPA1, RAS1, and PKA1) also played a role in G. mellonellakilling. Combination antifungal therapy (amphotericin B plus flucytosine) administered before or after inoculation was more effective than monotherapy in prolonging survival and in decreasing the tissue burden of cryptococci in the hemocoel. The G. mellonella-C. neoformanspathogenicity model may be a substitute for mammalian models of infection with C. neoformansand may facilitate the in vivo study of fungal virulence and efficacy of antifungal therapies.