title: 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.
authors.affiliation: Institute of Life Science and College of Medicine, Swansea University, Swansea, Wales, United Kingdom.
authors.forename: Claire M
authors.initials: CM
authors.lastname: Hull
authors.forename: Josie E
authors.initials: JE
authors.lastname: Parker
authors.forename: Oliver
authors.initials: O
authors.lastname: Bader
authors.forename: Michael
authors.initials: M
authors.lastname: Weig
authors.forename: Uwe
authors.initials: U
authors.lastname: Gross
authors.forename: Andrew G S
authors.initials: AG
authors.lastname: Warrilow
authors.forename: Diane E
authors.initials: DE
authors.lastname: Kelly
authors.forename: Steven L
authors.initials: SL
authors.lastname: Kelly
mesh_terms: D000666:Amphotericin B / Q000494:pharmacology*; D000935:Antifungal Agents / Q000494:pharmacology*; D001393:Azoles / Q000494:pharmacology*; D001483:Base Sequence; D001692:Biological Transport; D041221:Candida glabrata / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D026141:Drug Resistance, Multiple, Fungal / Q000235:genetics*; D004875:Ergosterol / Q000378:metabolism; D015725:Fluconazole / Q000494:pharmacology; D005656:Fungal Proteins / Q000235:genetics; D006801:Humans; D008826:Microbial Sensitivity Tests; D020125:Mutation, Missense; D011743:Pyrimidines / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D017422:Sequence Analysis, DNA; D058886:Sterol 14-Demethylase / Q000235:genetics*; D013261:Sterols / Q000378:metabolism; D014230:Triazoles / Q000494:pharmacology; D065819:Voriconazole
chemical_list: D000935:Antifungal Agents; D001393:Azoles; D005656:Fungal Proteins; D011743:Pyrimidines; D013261:Sterols; D014230:Triazoles; D000666:Amphotericin B; D015725:Fluconazole; D058886:Sterol 14-Demethylase; D065819:Voriconazole; D004875:Ergosterol