Mechanisms of drug resistance in Candida albicans

DESCRIPTION (provided by applicant): A major cause of resistance to azole antifungal drugs in Candida albicans is overproduction of the plasma membrane drug efflux pumps Cdr1p, Cdr2p and/or Mdr1p, but little is known about these pumps' physical and catalytic properties. It also is not known if mutations in these pumps' structural genes (CDR1, CDR2 and MDR1) cause azole resistance in clinical C. albicans strains. This project will use genetic, cell biological and biochemical approaches to pursue two specific aims. Aim 1 is to determine if Cdr1p, Cdr2p and Mdr1p actively transport azoles across the plasma membrane and to define these proteins' properties. CDR1, CDR2 or MDR1 will be expressed in a conditional C. albicans sec4 mutant, and secretory vesicles from these cells will be tested for the ability to transport [3H]-fluconazole across their membranes. The vesicles will also be used to examine each recombinant protein's physical properties, catalytic constants, substrate specificities and inhibitor profile. Aim 2 is to determine if mutations in the CDR1, CDR2 and/or MDR1 coding sequences cause azole resistance in C. albicans. Azole-susceptible and azole-resistant C. albicans strains from different groups of patients will be tested for [3H]-fluconazole accumulation and for CDR1, CDR2 and MDR1 overexpression. The CDR1, CDR2 and MDR1 ORFs from [3H]-fluconazole-underaccumulating strains that do not overexpress CDR1, CDR2 or MDR1 will be cloned and expressed in a C. albicans cdr1 cdr2 mdr1 null mutant. Any ORF that confers higher levels of azole resistance in the cdr1 cdr2 mdr1 mutant than does its wild-type counterpart will be sequenced, its protein product will be analyzed using isolated C. albicans secretory vesicles, and its effects on azole resistance will be verified in wild-type C. albicans transformants. The longterm goal of these studies is to develop new strategies and approaches for overcoming and/or preventing the emerging problem of azole resistance in C. albicans.