Genetically Encoded Sensors for Monitoring Intracellular Redox Health of the Pathogenic Fungus <i>Cryptococcus neoformans</i>

Redox sensing and regulation are critical to both the survival and virulence strategies used by the pathogenic fungus <i>Cryptococcus neoformans</i> to evade host immunity and establish infection. However, the precise genetic and biochemical mechanisms driving these redox regulation systems in the context of fungal virulence are unclear. To address this limitation, we designed genetically encoded redox sensors optimized for expression in <i>C. neoformans</i> and linked these sensors to cryptococcal redox proteins for real-time monitoring of intracellular redox status. Using these sensors, we established several fluorescence-based techniques for monitoring dose-responsive changes in the intracellular oxidation status of <i>C. neoformans</i> under stress. Specifically, we demonstrated sensor responsiveness to nontoxic doses of peroxide stress and during different stages of cell growth, and we verified sensor responsiveness in a mutant with known sensitivity to oxidative stress. This approach provides a framework for developing and deploying biosensors in pathogenic fungi and in basidiomycetes─a group of microorganisms with relatively few sophisticated genetic tools for molecular and synthetic biology. Overall, our sensors enable real-time insights into the key redox mechanisms driving growth and survival of a globally important pathogen and pave the way for tool development in other fungi.