Galleria mellonella as a Model System To Study Cryptococcus neoformans Pathogenesis

Abstract

Evaluation ofCryptococcus neoformans virulence in a number of nonmammalian hosts suggests that C. neoformans is a nonspecific pathogen. We used the killing of Galleria mellonella (the greater wax moth) caterpillar by C. neoformans to 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. neoformans killed G. mellonella. After injection into the insect hemocoel, C. neoformans proliferated 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. neoformans clinical strain H99 was the most virulent of the strains tested and killed caterpillars with inocula as low as 20 CFU/caterpillar. Several C. neoformans genes previously shown to be involved in mammalian virulence (CAP59, GPA1, RAS1, and PKA1) also played a role in G. mellonella killing. 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. neoformans pathogenicity model may be a substitute for mammalian models of infection with C. neoformans and may facilitate the in vivo study of fungal virulence and efficacy of antifungal therapies.The yeast Cryptococcus neoformans is amenable to a variety of genetic manipulations, making it an excellent model fungal pathogen in which to identify and study fungal virulence factors. C. neoformans is an important human pathogen that infects individuals with compromised immune function and less commonly infects immunocompetent hosts as well (30,36). Recent studies have taken advantage of the broad host range of C. neoformans to develop facile invertebrate model systems utilizing the amoeba Acanthamoeba castellanii (44), the nematode Caenorhabditis elegans (31), the slime mold Dictyostelium discoideum (45,46), and the insect Drosophila melanogaster (3) as hosts for the study of established virulence attributes or to identify novel genes involved in virulence (32). Some of these previously developed alternative models are limited by the inability of the host system to survive at 37°C, the difficulty of administering exact fungal inocula, or the difficulty of administering antifungal chemotherapy. Moreover, wild-type D. melanogaster is extremely resistant to systemic infection by a number of fungi, including C. neoformans (3).To address these limitations, we developed a new system using caterpillars of the greater wax moth (Galleria mellonella) as an alternative model for the study of C. neoformans. We report here that various serotypes of C. neoformans proliferate inside the hemocoel and kill the caterpillar, even though the fungi undergo phagocytosis by G. mellonella hemocytes. Caterpillar killing correlates with the number of CFU of C. n...

Fungal extracellular ribotoxins as insecticidal agents

“…Results shown are the average of at least three independent assays. Assays were performed as described (Fuchs et al, 2010;Mylonakis et al, 2005;Mylonakis, 2008). A 10-μl Hamilton syringe was used to inject 8-μl aliquots of the inoculum into the hemocoel of each caterpillar via the last left proleg.…”

Pre-exposure ofGalleria mellonellalarvae to different doses ofAspergillus fumigatusconidia causes differential activation of cellular and humoral immune responses

“…There was a significant increase in the density of circulating hemocytes at 24 h in those larvae that received the initial doses of response of mammals 4 insects have been used as alternatives to mammals for the screening of microbial pathogens [13][14][15] or for evaluating the potency of antimicrobial agents. 16,17 Understanding the mechanisms employed by insects to withstand infection is critical to the successful use of them as models.…”

The development of animal infection models and antifungal efficacy assays against clinical isolates ofTrichosporon asahii,T. asteroidesandT. inkin

“…41,42 A high dose of FLC or VRC was able to improve the survival of the animals compared to the untreated control groups infected with any of the 3 Trichosporon species, while AMB was not able to reduce mortality in any of the 3 species (Fig. 4).…”