ATP Binding Cassette (ABC) Transporters in Fungal Drug Tolerance

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Members of the Multi-drug Resistance Protein (MRP) family of ATP Binding Cassette (ABC) transporters contribute to drug tolerance in major fungal pathogens, including Candida species, in 2 main ways: 1) they detoxify the cell of cytotoxic molecules such as antifungal drugs and electrophiles/oxidants by sequestering them in the vacuole, and 2) they cause complex morphological changes such as hyphal extension and biofilm formation linked to drug tolerance. Because of their role in these survival mechanisms, MRP family members are often required for infection and are tightly regulated by the cell. The overall objective of this proposal is to bridge gaps in our understanding of how these transporters contribute to the increasing threat of drug resistance in fungal pathogens. Mechanistic models that explain this resistance are especially lacking, limiting antifungal treatment efficacy. The long-term goal is to understand how anti-fungal treatments fail. To this end, we specifically focus on early stage anti-stress responses through enzymatic, structural, and cellular investigations of two prominent vacuolar MRP transporters: Ycf1 and Mlt1. Our rationale is that these insights will provide a foundation for exploiting unique aspects of transporter architecture in order to generate more effective therapies that overcome drug tolerance in fungal infections. Our preliminary work identifies key features of MRP family transporter regulation during fungal stress responses. These are complex substrate binding sites and the regulation of different states of an intrinsically disordered region called the Regulatory-domain (R- domain) by phosphorylation. Our central hypothesis is that MRP transporters are governed by domain insertions outside of the conserved transporter fold that regulate multi-segmented substrate binding sites. Our specific aims testing this hypothesis in MPR transporters are to define: (1) the regulatory architectures adopted across transport cycles, (2) the functional and structural roles of R-domain phosphorylation on catalytic regulation, and (3) the molecular basis of substrate selection and lipid transport in membrane homeostasis. This application uses an innovative multidisciplinary approach that applies advances in membrane protein biochemistry and electron cryo-microscopy to structurally and biophysically undercharacterized fungal integral membrane proteins. In light of the growing threat of Candida infections, the significance of our proposal is twofold. First, we will establish a mechanistic framework for understanding the molecular basis of therapeutic failure driven by important Candida virulence factors and their yeast homologs. Second, we will establish a foundation useful for developing allosteric therapeutics against drug-tolerance linked to fungal MRP family transporters, with general applicability to all MRP transporters.