Glutathione metabolism modulates redox homeostasis and virulence in the pathogenic fungus Cryptococcus neoformans

Pathogens must overcome the hostile conditions of their hosts to survive, proliferate, and cause disease. The fungal pathogen Cryptococcus neoformans is particularly adept at mitigating challenges in the host environment and has developed an arsenal of defense mechanisms to evade oxidative and nitrosative agents released by phagocytic cells during infection. Among these mechanisms, melanin production is crucially linked to both fungal virulence and defense against harmful free radicals that facilitate host innate immunity and clearance of invading pathogens. Redox-active thiols such as glutathione (GSH) are also key for the oxidative stress response of fungi and comprise systems that depend on enzymatic and non-enzymatic mechanisms to enable pathogens to evade host immunity and cause disease. However, the connection between redox regulation and elaboration of major virulence factors in C. neoformans remains unclear. In this thesis, I employed genetic and metabolomic approaches to demonstrate that the redox metabolite GSH is inextricably linked to redox-active processes that facilitate melanin and titan cell production. Furthermore, I demonstrated that genetic perturbations in GSH biosynthesis result in global metabolic changes that ultimately affect survival in macrophages and virulence in a murine model of cryptococcosis. Genetically encoded redox sensors were designed to detect these metabolic changes and quantify fluctuations in the oxidation status of intracellular GSH pools in response to exogenous oxidizing/reducing agents. I also designed and species-optimized a CRISPR-based gene interference platform (CRISPRi) to manipulate and characterize pathways involved in general redox regulation in C. neoformans, and targeted the ADE2 and LAC1 genes for transcriptional repression as a proof of concept. Upon further optimization, these new genetic tools will enable quantification and manipulation of redox pathways to improve our understanding of redox-mediated regulation of fungal virulence. Together, the work presented in this thesis identifies key mechanistic insights into the redox-dependent factors drive fungal survival, growth, and virulence, and highlights the importance of GSH-mediated redox regulation for pathogen adaptation to the host environment, suggesting new avenues for antifungal drug development.