The evolution of fungal virulence

Abstract

Maintenance of fungal virulence in soil is enigmatic since mammalian infection is not necessary for the fungal life cycle. We sought to determine if soil amoebae may be involved in maintaining fungal virulence in the environment. Using both Acanthamoebae castellanii and Dictyostelium discoideum we demonstrated that soil amoebae can be infected by fungal pathogens. Cryptococcus neoformans, a saprophytic soil fungus, is phagocytosed by A. castellanii which results in killing of the amoebae and replication of C. neoformans. C. neoformans cells with attenuated virulence because of defects in capsule synthesis, melanin production, or phospholipase secretion were also avirulent to amoebae. Using both immunogold electron microscopy and immunofluorescence we demonstrated that C. neoformans cells secrete capsular polysaccharide into discrete vesicles. We also investigated the interaction of amoebae and the dimorphic fungi Histoplasma capsulatum, Blastomyces dermatitidis, and Sporothrix schenkii. Infection of amoebae with H. capsulatum, B. dermatitidis and S. schenckii yeast forms resulted in fungal growth and amoebae killing. The environmental conidial forms of H. capsulatum were determined to be infectious to amoebae. Passage of an avirulent strain of H. capsulatum with A. castellanii resulted in the recovery of virulence as determined by CFU and histopathology. Since there are a wide variety of soil amoebae, we investigated whether D. discoideum could also support C. neoformans growth. Phagocytosis of C. neoformans results in amoebae killing and fungal growth. Mice were infected with cryptococcal cells that had been passaged with D. discoideum and mouse survival time was drastically decreased, indicating a significant increase in cryptococcal virulence after amoebae passage. Time to melanization and capsule size was affected by amoebae passage while cryptococcal growth curves were unchanged. The relevance of our studies was evaluated by isolating both amoebae and C. neoformans from the same ecological niche in the Bronx. Combined these results indicate that amoebae are a host for pathogenic fungal studies and we propose that virulence of fungi is maintained in the environment through selection by amoebae and other soil predators.