Functional Expression of Recombinant Candida auris Proteins in Saccharomyces cerevisiae Enables Azole Susceptibility Evaluation and Drug Discovery

Abstract

Candida auris infections are difficult to treat due to acquired drug resistance against one or multiple antifungal drug classes. The most prominent resistance mechanisms in C. auris are overexpression and point mutations in Erg11, and the overexpression of efflux pump genes CDR1 and MDR1. We report the establishment of a novel platform for molecular analysis and drug screening based on acquired azole-resistance mechanisms found in C. auris. Constitutive functional overexpression of wild-type C. auris Erg11, Erg11 with amino acid substitutions Y132F or K143R and the recombinant efflux pumps Cdr1 and Mdr1 has been achieved in Saccharomyces cerevisiae. Phenotypes were evaluated for standard azoles and the tetrazole VT-1161. Overexpression of CauErg11 Y132F, CauErg11 K143R, and CauMdr1 conferred resistance exclusively to the short-tailed azoles Fluconazole and Voriconazole. Strains overexpressing the Cdr1 protein were pan-azole resistant. While CauErg11 Y132F increased VT-1161 resistance, K143R had no impact. Type II binding spectra showed tight azole binding to the affinity-purified recombinant CauErg11 protein. The Nile Red assay confirmed the efflux functions of CauMdr1 and CauCdr1, which were specifically inhibited by MCC1189 and Beauvericin, respectively. CauCdr1 exhibited ATPase activity that was inhibited by Oligomycin. The S. cerevisiae overexpression platform enables evaluation of the interaction of existing and novel azole drugs with their primary target CauErg11 and their susceptibility to drug efflux.

Overcoming Drug Resistance in a Clinical C. albicans Strain Using Photoactivated Curcumin as an Adjuvant

“…Fluorescent microscopy utilizing Nile Red revealed that it is removed from yeast cells by specialized efflux pumps [ 14 ]. Using Nile Red, which was reported as a substrate for Cdr1p, Cdr2p, and CaMDR1p [ 30 ], it was determined if the fluconazole-resistant C. albicans strain was capable of removing this compound [ 14 , 31 ]. Candida ATCC strains that have been treated with Nile Red were able to continue retaining this dye, whereas fluconazole-resistant cells exhibited only a slight fluorescence.…”

“…The effect of small molecule inhibitors on a glucose-dependent efflux was previously studied with the help of Nile Red as a substrate for multidrug transporters overexpressed in S. cerevisiae [ 31 ]. In the current investigation, the evaluation of the activity of glucose-dependent efflux pumps showed the ability of photoactivated curcumin to inhibit pump-mediated Nile Red efflux.…”

Clorgyline Analogs Synergize with Azoles against Drug Efflux in Candida auris

“…The Nile Red efflux pump assay was performed as described previously [ 25 , 77 , 82 ]. Cells (grown to OD 600nm = 2) starved for 30 min at 30 °C with 2-DOG (5mM final concentration) were loaded with Nile Red (7.5 µM final concentration).…”

“…Instances of the CauErg11 K143R mutation have occasionally been found in clade II isolates [ 24 ]. Recombinant Sacharomyces cerevisiae strains expressing CauErg11 Y132 and CauErg11 K143R showed elevated minimum inhibitory concentration (MIC) values for FLC and Voriconazole (VRC) but not POS when compared to wild-type CauErg11 [ 25 ]. Gain-of-function (GOF) mutations in the transcriptional factor Upc2 up-regulate ergosterol biosynthesis genes in C. albicans , leading to Erg11 overexpression and FLC resistance [ 26 ].…”

“…We used agarose diffusion assays to screen the in vitro activity of POS, VRC, and Isavuconazole (ISA) alone and in combination with CLO and a range of CLO analogs and then characterized two hits as inhibitors of C. auris drug efflux. Recombinant S. cerevisiae strains overexpressing CauMdr1 (Y2765) and CauCdr1 (Y2766) [ 25 ] were used to determine how the hits interfere with individual mechanisms of drug efflux pump-mediated azole resistance prior to checkerboard assays with representative C. auris clade I and clade II clinical isolates.…”