Fungal pathogens cause a tremendous health burden worldwide and are associated with over 1.5 million deaths per year globally, eclipsing or equaling malaria, TB, or HIV. The last several decades have seen a shift in the epidemiology of Candida infections, with a decreased incidence of pan-sensitive C. albicans and a concomitant increase in non-albicans Candida species, such as C. glabrata and C. parapsilosis, which either have reduced intrinsic sensitivity to commonly used antifungals or can rapidly acquire drug-resistant mutations. Echinocandins are inhibitors of b-glucan synthase (GS) that are cidal in Candida and well tolerated by patients and are thus used as first-line agents for invasive Candida infections, especially in light of increasing insensitivity to azoles in species such as C. glabrata. However, resistance to echinocandins is also increasing, and in C. glabrata it is the highest among major Candida pathogens (including C. auris). Clinical echinocandin resistance (ECHR) is associated with mutations in genes encoding GS, FKS1 and FKS2, and C. glabrata can rapidly evolve ECHR mutations both in vitro and during patient treatment. However, the cellular mechanisms underpinning this rapid emergence of resistance are not well understood. Previously, we described a universal paradigm for fungi, in which drug exposure by a cidal agent effectively kills susceptible cells but leaves a sub-population of viable non- growing cells that are drug-tolerant. Ultimately, some drug-tolerant cells escape drug action by forming heritable drug resistant mutations. We have identified the gastrointestinal (GI) tract and macrophages as important in-host niches of echinocandin-tolerant C. glabrata, in which ECHR mutants form. We have also shown that the evolution of resistance to the echinocandin caspofungin in the GI tract is more complex than was previously appreciated, involving formation of mutations in FEN1, a sphingolipid biosynthesis gene. Importantly, we also identified fen1 mutations in multiple clinical isolates, where they contribute to reduced caspofungin susceptibility. Finally, we discovered that reactive oxygen species (ROS) are formed in C. glabrata cells during echinocandin treatment. However, genes involved in ROS detoxification are downregulated, and deletion of several such genes greatly induces the formation of ECHR mutations, suggesting that ROS induction is a programmed C. glabrata response that may promote the emergence of resistance. In this grant we propose to use transposon insertion screening to comprehensively identify C. glabrata vulnerabilities specific to the host niches where echinocandin tolerance and resistance occur (Aim 1), to comprehensively define the clinically relevant mutational routes to tolerance and resistance against multiple echinocandins in the gut (Aim 2), and to use accurate multidisciplinary complementary approaches, including electron paramagnetic/spin resonance (EPR/ESR) and in vivo ROS reporters, to clearl...