Metabolic determinants of carbon dioxide tolerance in the pathogenic fungi, Cryptococcus neoformans

Project Summary/Abstract Invasive fungal diseases account for 1.5 million deaths annually. Cryptococcus neoformans contributes to much of this burden with a mortality rate that nears 70% and contributes to more than 180,000 deaths globally. The high mortality rate is largely attributed to the lack of effective anti-cryptococcal therapies. Our long-term goal is to understand the underlying mechanisms that drive Cryptococcus virulence and to exploit these mechanisms for rational drug development. Virulence requires Cryptococcus to overcome many stressors imposed in the human host. We have recently identified the ability to tolerate host carbon dioxide as a novel virulence trait. The objective of this proposal is to specifically identify the mechanisms that allow for tolerance of host carbon dioxide as a requirement for Cryptococcus virulence. A reverse genetic screen of kinases uncovered multiple effectors of the protein kinase Tor as requirements for carbon dioxide tolerance. Tor is a known to regulate metabolic processes across eukaryotes but is not well characterized in Cryptococcus. We performed metabolic profiling under carbon dioxide stress and indeed uncovered a strong metabolic shift. This proposal sets out to define the metabolic profile induced by carbon dioxide across diverse clinical and environmental isolates. We also identified that general membrane stress and specific inhibition of sphingolipid biosynthesis potentiate carbon dioxide sen- sitivity. Tor pathways are known to regulate acetyl-CoA metabolism and sphingolipid biosynthesis but are not well characterized in Cryptococcus. Combining our investigation of these carbon dioxide response pathways with virulence studies will help us uncover the underlying regulatory machinery and response kinetics required for achieving carbon dioxide tolerance as a requirement for virulence. In this pursuit, we will achieve a more direct understanding of the specific requirements for carbon dioxide tolerance. Our combination of genetics, biochemical, and cellular approaches will guide us toward the underlying regulatory machinery and direct effec- tors that dictate carbon dioxide tolerance as a requirement for virulence. The proposed experimental approaches combined with professional development activities will provide a strong foundation for the applicant to launch an independent scientific career.