Pathogenic diversity of Cryptococcus in Galleria mellonella extends beyond classical virulence factors

Pathogenic determinants in the <i>Cryptococcus</i> genus have been extensively studied, often using standard laboratory isolates. Here, we analyzed the virulence of ten <i>Cryptococcus</i> isolates from diverse sources, species, and genotypes. These isolates exhibited marked differences in their ability to colonize and kill the invertebrate host <i>Galleria mellonella</i>, as well as in their interactions with hemocytes. Capsule formation also varied widely among isolates, with no clear correlation between virulence in <i>G. mellonella</i> and source of isolation, species, fungal burden, capsule size, or interaction with larval hemocytes. To further investigate the basis of this pathogenic diversity, we selected two hypervirulent isolates (<i>C. deuterogattii</i> and <i>C. neoformans</i>) and two hypovirulent isolates (<i>C. gattii</i> and <i>C. neoformans</i>) for in-depth analysis. Differences in the induction of antimicrobial peptides during infection did not account for the observed variation in virulence. Genomic analysis of capsule-related genes, scanning electron microscopy of capsule morphology in vitro and in vivo, and nuclear magnetic resonance of the major capsular polysaccharide all revealed high variability among isolates, but none of these features correlated with virulence. Proteomic profiling of cellular extracts showed that virulent strains were enriched in proteins associated with oxidative processes. Supplementation with an antioxidant during infection increased the virulence of hypovirulent isolates in <i>G. mellonella</i>. These results reveal a high pathogenic diversity in <i>Cryptococcus</i> that goes beyond its classical virulence factors.