The process of autophagy is conserved among all eukaryotes from yeast to humans, and is mainly responsible for bulk degradation of cellular contents and nutrient recycling during starvation. Autophagy has been suggested to play a role in the pathogenesis of the opportunistic human fungal pathogen Cryptococcus neoformans, potentially through a contribution to export virulence factors. The purpose of the study is to define the roles of different C. neoformans autophagy genes in nutritional aspects of amino acid homeostasis, in the secretion of virulence factors, and in virulence in mice.
We examined the functions of four ATG genes representing steps in induction (ATG1), nucleation of the phagophore (ATG9), and expansion of the phagophore (ATG7, ATG8) by generating single gene deletion mutants and their respective complemented strains. Phenotypic characterization of each atgΔ mutant were carried out including in vitro production of melanin and polysaccharide capsule, the ability to grow at body temperature (37ºC), and intracellular replication in murine macrophage-like cells J774A.1. In addition, a mouse inhalation model of cryptococcosis was used to evaluate the effect of each ATG gene on virulence of C. neoformans.
In this study, we showed that deletion of each of the ATG1, ATG7, ATG8, and ATG9 genes in C. neoformans leads to autophagy-related phenotypes including impaired amino acid homeostasis under nitrogen starvation. In addition, the atgΔ mutants were hypersensitive to inhibition of the ubiquitin-proteasome system, a finding consistent with a role in amino acid homeostasis. Although each atgΔ mutant was not markedly impaired in virulence factor production in vitro, we found that ATG7, ATG8, and ATG9 contribute to C. neoformans virulence in a murine inhalation model of cryptococcosis. Interestingly, these mutants displayed significant differences in disease development. A more detailed investigation of virulence for the atg8Δ mutant revealed that this strain stimulated an exaggerated host immune response, which in turn contributed to disease progression.
Our results suggest that different ATG genes are involved in different non-autophagic functions, and contribute to C. neoformans virulence beyond their core functions in autophagy.


ATG genes influence the virulence of Cryptococcus neoformans through contributions beyond core autophagy functions