FK506 Resistance of Saccharomyces cerevisiae Pdr5 and Candida albicans Cdr1 Involves Mutations in the Transmembrane Domains and Extracellular Loops

Abstract

The 23-membered-ring macrolide tacrolimus, a commonly used immunosuppressant, also known as FK506, is a broad-spectrum inhibitor and an efflux pump substrate of pleiotropic drug resistance (PDR) ATP-binding cassette (ABC) transporters. Little, however, is known about the molecular mechanism by which FK506 inhibits PDR transporter drug efflux. Thus, to obtain further insights we searched for FK506-resistant mutants ofSaccharomyces cerevisiaecells overexpressing either the endogenous multidrug efflux pump Pdr5 or itsCandida albicansorthologue, Cdr1. A simple but powerful screen gave 69 FK506-resistant mutants with, between them, 72 mutations in either Pdr5 or Cdr1. Twenty mutations were in just three Pdr5/Cdr1 equivalent amino acid positions, T550/T540 and T552/S542 of extracellular loop 1 (EL1) and A723/A713 of EL3. Sixty of the 72 mutations were either in the ELs or the extracellular halves of individual transmembrane spans (TMSs), while 11 mutations were found near the center of individual TMSs, mostly in predicted TMS-TMS contact points, and only two mutations were in the cytosolic nucleotide-binding domains of Pdr5. We propose that FK506 inhibits Pdr5 and Cdr1 drug efflux by slowing transporter opening and/or substrate release, and that FK506 resistance of Pdr5/Cdr1 drug efflux is achieved by modifying critical intramolecular contact points that, when mutated, enable the cotransport of FK506 with other pump substrates. This may also explain why the 35 Cdr1 mutations that caused FK506 insensitivity of fluconazole efflux differed from the 13 Cdr1 mutations that caused FK506 insensitivity of cycloheximide efflux.

An A666G mutation in transmembrane helix 5 of the yeast multidrug transporter Pdr5 increases drug efflux by enhancing cooperativity between transport sites

“…This might also result in a longer period for a substrate to interact with Pdr5 and perhaps allow increased cooperativity. This model may very well explain some of the interesting FK506 hyperreistant mutants in Cdr1 and Pdr5 (Tanabe et al , ). However, we have no concrete kinetic evidence that it explains the behavior of the A666G mutant, some observations from this study lend support to this model.…”

Candida albicans Antifungal Resistance and Tolerance in Bloodstream Infections: The Triad Yeast-Host-Antifungal

“…In humans, one of the factors that is responsible for the failure of cancer therapy are ATP-dependent drug efflux pumps, such as P-glycoprotein (P-gp) [149]. P-gp substrates such as FK506 [150][151][152] or cyclosporine A (CsA) are immunosuppressors that are able to inhibit efflux [153]. They act similarly in C. albicans, inhibiting the calcineurin-mediated azole tolerance by binding to small, abundant, conserved binding proteins called immunophilins.…”

Engineering a Cysteine-Deficient Functional Candida albicans Cdr1 Molecule Reveals a Conserved Region at the Cytosolic Apex of ABCG Transporters Important for Correct Folding and Trafficking of Cdr1

“…Isolation of CDR1PC-NT1 and -NTS12 suppressor mutations. Naturally arising ADDD-CaCDR1PC-NT1-GFP or -NTS12-GFP suppressor mutants with reduced FLC efflux pump function were selected by plating 10 6 to 10 7 logarithmic-phase cells on CSM agar plates supplemented with high concentrations of FLC (;4Â to 8Â MIC FLC ) and incubating the plates for up to 2 weeks at 30°C, as previously described (12,24). Individual colonies were tested for possible mutations by sequencing the entire CDR1 ORF.…”