Facultative Sterol Uptake in an Ergosterol-Deficient Clinical Isolate of Candida glabrata Harboring a Missense Mutation in ERG11 and Exhibiting Cross-Resistance to Azoles and Amphotericin B

Abstract

We identified a clinical isolate of Candida glabrata (CG156) exhibiting flocculent growth and cross-resistance to fluconazole (FLC), voriconazole (VRC), and amphotericin B (AMB), with MICs of >256, >256, and 32 μg ml ⁻¹ , respectively. Sterol analysis using gas chromatography-mass spectrometry (GC-MS) revealed that CG156 was a sterol 14α-demethylase (Erg11p) mutant, wherein 14α-methylated intermediates (lanosterol was >80% of the total) were the only detectable sterols. ERG11 sequencing indicated that CG156 harbored a single-amino-acid substitution (G315D) which nullified the function of native Erg11p. In heterologous expression studies using a doxycycline-regulatable Saccharomyces cerevisiae erg11 strain, wild-type C. glabrata Erg11p fully complemented the function of S. cerevisiae sterol 14α-demethylase, restoring growth and ergosterol synthesis in recombinant yeast; mutated CG156 Erg11p did not. CG156 was culturable using sterol-free, glucose-containing yeast minimal medium ( _glc YM). However, when grown on sterol-supplemented _glc YM (with ergosta 7,22-dienol, ergosterol, cholestanol, cholesterol, Δ ⁷ -cholestenol, or desmosterol), CG156 cultures exhibited shorter lag phases, reached higher cell densities, and showed alterations in cellular sterol composition. Unlike comparator isolates (harboring wild-type ERG11 ) that became less sensitive to FLC and VRC when cultured on sterol-supplemented _glc YM, facultative sterol uptake by CG156 did not affect its azole-resistant phenotype. Conversely, CG156 grown using _glc YM with ergosterol (or with ergosta 7,22-dienol) showed increased sensitivity to AMB; CG156 grown using _glc YM with cholesterol (or with cholestanol) became more resistant (MICs of 2 and >64 μg AMB ml ⁻¹ , respectively). Our results provide insights into the consequences of sterol uptake and metabolism on growth and antifungal resistance in C. glabrata .