Lipid flippase in echinocandin drug resistance in Cryptococcus neoformans - Abstract
Cryptococcosis is a deadly fungal disease that accounts for over 15% HIV/AIDS related deaths. Treatment
options for cryptococcosis are limited. Currently available antifungal drugs are either highly toxic (polyenes) or
exert a fungistatic effect (triazoles), necessitating long treatment regimens and leaving open the avenue for
emergence of drug resistance. The third major antifungal drug class, the echinocandins, show low toxicity and
are fungicidal against several other prevalent fungal pathogens. However, Cryptococci are resistant to
echinocandins and the mechanisms of this resistance remain unknown. We recently reported that loss of lipid
flippase, the enzyme responsible for maintaining the asymmetry of membrane lipid bilayers and normal
intracellular vesicle trafficking, sensitizes C. neoformans to caspofungin, a drug of the echinocandin class, as
well as to several triazoles. We also found that lipid flippase was essential for virulence in a murine model of
cryptococcosis and sensitized C. neoformans to killing by macrophages, suggesting that it may be a novel
antifungal drug target. In this project, we propose to decipher the mechanism of lipid flippase in cryptococcal
echinocandin resistance and to conduct proof-of-principle studies inhibiting flippase function in C. neoformans.
In the first Aim, we will test two related, non-mutually exclusive hypotheses regarding the role of lipid flippase in
drug resistance: (1) that loss of lipid flippase changes membrane structure, e.g. PS distribution on membrane,
to promote the interaction of caspofungin with its target β-1,3-D-glucan synthase (Fks1), and (2) that in the
absence of lipid flippase certain drug resistance pathways, such as calcineurin pathway are compromised,
disrupting cellular calcium homeostasis and inducing killing by drugs. We will test these hypotheses by employing
a host of cellular, molecular, biochemical, and genetic approaches. In the second Aim, we propose to develop
an antibody Fab fragment against the extracellular loop of lipid flippase, which is essential for flippase function,
and to test its ability to sensitize C. neoformans to antifungal drugs and to killing by macrophages. The region of
lipid flippase 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. The success of
this study will lead to a better understanding of lipid flippase mediated drug resistance in C. neoformans, which
could help expand the use of echinocandin drugs against Cryptococci and other resistant fungal pathogens.
Furthermore, generation of flippase inhibitory antibodies will provide a valuable research tool and may lead to
future development of novel combination therapy approaches. Finally, successful development of antibody-
based inhibitors could open a new avenue of research and drug development against other membrane proteins
in fungi and bacteria.
