Galleria mellonella as a model system to study Cryptococcus neoformans pathogenesis

Abstract

Evaluation of Cryptococcus 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 degrees C and 30 degrees 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.

FIG. 1.

Killing of G. mellonella caterpillars by C. neoformans depends on the number of cryptococcal cells inoculated. Kaplan-Meier plots of G. mellonella survival after injection of different inocula of C. neoformans strain H99 (ATCC 208821). There was no killing of caterpillars that received PBS or heat-killed cryptococcal cells of the same C. neoformans strain (10⁵ cells/larva).

FIG. 2.

Binding, phagocytosis, and nodulation of C. neoformans cells by G. mellonella hemocytes. C. neoformans fungal cells binding to (A) or phagocytosed by G. mellonella hemocytes (B and C) are shown. (D) A C. neoformans fungal cell is surrounded by layers of hemocytes in a process known as nodulation that has been described previously for G. mellonella in association with pathogens. Fungal cells (B) were stained with calcofluor white, and a phagocytosed C. neoformans is identified by a white arrowhead. (C and D) C. neoformans cells were stained with FITC before inoculation as described in Materials and Methods. Original magnification in all panels, ×100; bar, 50 μM.

FIG. 3.

C. neoformans virulence factors important for mammalian infection also enhance killing of C. elegans. Survival of G. mellonella after injection of 1.5 × 10⁴ CFU/larva of wild-type C. neoformans strain H99 or mutants with disruptions in the genes encoding CAP59 (essential for capsule formation) or the G protein-cAMP-PKA or the RAS1-controlled signal transduction cascades demonstrated hypovirulence (cap59, gpa1, ras1, and pka1). There was a significant decrease in virulence between the wild type and the mutants at both 37°C (A) and 30°C (B). P values were <0.01 for each of the mutants compared to the parental strain H99 (Table 1).

FIG. 4.

Melanin biosynthesis is involved in G. mellonella killing by C. neoformans. A mutation involving the gene that encodes laccase (an enzyme essential for melanin biosynthesis) renders C. neoformans less virulent in the G. mellonella system at 37°C than the wild type (P = 0.001) (in this experiment, caterpillars received 2.5 × 10⁴ CFU/larva).

FIG. 5.

The C. neoformans MFα1 promoter is expressed in the G. mellonella hemocoel during the proliferative stage. Fluorescent microscopy of G. mellonella hemolymph on day 3 (proliferative stage) after injection with 1.25 × 10⁴ CFU/larva of C. neoformans strain H99 expressing GFP fused to the MFα1 promoter. (A and B) Hemolymph was undiluted to provide a better appreciation of the fungal burden within the insect hemolymph. (C and D) Cells were fixed as detailed in Materials and Methods. Images in panels A and C are fluorescence images, and images in panels B and D are the corresponding confocal images. There was no fluorescence observed on day 1 or 2 of this experiment.

FIG. 6.

Antifungal drugs prolong the survival of G. mellonella caterpillars after challenge with C. neoformans. We examined the role of the most commonly used agents for C. neoformans infection by administering a single dose of amphotericin B (AMB; 1.5 mg/kg), fluconazole (FLU; 14 mg/kg), or flucytosine (5-FC; 20 mg/kg) alone or in combination 48 h after the inoculation of caterpillars with 1.2 × 10³ CFU of C. neoformans strain H99 per larva. A control group received the C. neoformans inoculum and PBS instead of antifungal drugs. Monotherapy with amphotericin B prolonged the survival of G. mellonella caterpillars (P = 0.001 compared to control). Also, there was a trend suggesting that fluconazole was effective (P = 0.072). The combination of amphotericin B plus flucytosine was significantly more effective than amphotericin B alone (P = 0.0002).