The heat-ramp method to study regulated cell death in a pathogenic yeast <i>Cryptococcus neoformans</i>

Human fungal pathogens cause a significant public health burden. While no reliable surveilence data are available, estimations suggest that 1 billion infections and over 2 million deaths are attributable to fungal infections annually worldwide. This drove the World Health Organization to generate a priority list of fungal pathogens for reearch, which includes the yeast <i>Cryptococcus neoformans</i> in a top critical priority. With the rise of drug-resistance and emerging fungal pathogens, new conceptual strategies for antifungal therapies are needed in addition to existing antibiotic development pipelines to meet clinical needs. Intrinsic cell death pathways encoded by pathogenic fungi are largely unstudied but could be leveraged for antifungal therapy analogous to anti-cancer therapeutics that activate apoptosis or other cell death mechanisms. Thus far, molecularly defined fungal cell death mechanisms are best characterized for only a few, predominantly model filamentous species. To extend these studies to pathogenic yeast, here we describe and demonstrate a tunable heat-ramp stimulus that when applied to small volumes of yeast cell suspensions reveals a protracted cell death process in the pathogenic yeast <i>Cryptococcus neoformans</i>. This low cost protocol induces robust and reproducible phenotypes to study gene-dependent mechanisms in laboratory strains and clinical isolates.