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 ß-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 resistant fungal pathogens. Our successful
development of antibody-based inhibitors will establish a new avenue of research and drug development against
other membrane proteins in fungi and bacteria.