Abstract Cryptococcus neoformans and its sibling species C. gattii cause Cryptococcosis, a deadly fungal disease that accounts for over 15% of HIV/AIDS related deaths. Treatment options for cryptococcosis remain limited to two drug classes that are either highly toxic (polyenes) or exert a fungistatic effect (triazoles) that necessitate long treatment regimens and can induce drug resistance. The third antifungal drug class, echinocandins, shows low toxicity and is fungicidal against some prevalent fungal pathogens. However, Cryptococcus species are resistant to echinocandins through an unknown resistance mechanism. We found that loss of Cdc50, the regulatory subunit of lipid flippase, an enzyme that maintains asymmetry of the membrane lipid bilayers and regulates intracellular vesicle trafficking, sensitizes C. neoformans to the echinocandin drug caspofungin and several triazoles. We further showed that the cdc50∆ mutant abolishes lipid flippase activity. We also found that this Cdc50-mediated echinocandin resistance requires a mechanosensitive calcium channel protein, Crm1, which modulates intracellular calcium homeostasis. Strikingly, we discovered that lipid flippase function is essential for virulence in a murine model of cryptococcosis, suggesting that lipid flippase may be a novel antifungal drug target. In this project, our goals are to determine how lipid flippase mediates cryptococcal echinocandin resistance, and to conduct proof-of-principle studies of antibody-based inhibitors targeting flippase function as novel therapeutics for Cryptococcus infections. We hypothesize that C. neoformans has a unique plasma membrane structure and that loss of lipid flippase alters that structure to promote the interaction of caspofungin with its target and compromises fungal drug resistance mechanisms. We propose three Aims to test our hypothesis. In Aim 1, we will elucidate how loss of Cdc50 changes membrane structure to promote the interaction of caspofungin with its membrane target β-1,3-D-glucan synthase (Fks1). Aim 2 will identify the downstream drug resistance pathways that are compromised by the absence of Cdc50, which disrupts intracellular calcium homeostasis and promotes cell death. In Aim 3, we will develop an antibody Fab fragment and a stable peptide against the exoplasmic loop of Cdc50, which is essential for flippase function. We will validate how inhibitors sensitize C. neoformans to antifungal drugs and macrophage killing in vitro and in vivo in animal models. The region of Cdc50 targeted by this antibody-based approach has low sequence homology to its human counterpart, and our preliminary studies showed that an antibody raised against this region is fungal- specific, reducing the chance of off-target effects. The impact of this study to elucidate the mechanisms underlying lipid flippase mediated drug resistance in C. neoformans will be developing strategies for exploiting echinocandin drugs to effectively treat Cryptococci and other ...