Structural Insights into the Azole Resistance of the <i>Candida albicans</i> Darlington Strain Using <i>Saccharomyces cerevisiae</i> Lanosterol 14α-Demethylase as a Surrogate
Target-based azole resistance in <i>Candida albicans</i> involves overexpression of the <i>ERG11</i> gene encoding lanosterol 14α-demethylase (LDM), and/or the presence of single or multiple mutations in this enzyme. Overexpression of <i>Candida albicans</i> LDM (...
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| Autores principales: | , , , , , |
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| Formato: | article |
| Lenguaje: | EN |
| Publicado: |
MDPI AG
2021
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| Materias: | |
| Acceso en línea: | https://doaj.org/article/2fb14a794e4d4733aafc6a6df72d6c0b |
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| Sumario: | Target-based azole resistance in <i>Candida albicans</i> involves overexpression of the <i>ERG11</i> gene encoding lanosterol 14α-demethylase (LDM), and/or the presence of single or multiple mutations in this enzyme. Overexpression of <i>Candida albicans</i> LDM (CaLDM) Y132H I471T by the Darlington strain strongly increased resistance to the short-tailed azoles fluconazole and voriconazole, and weakly increased resistance to the longer-tailed azoles VT-1161, itraconazole and posaconazole. We have used, as surrogates, structurally aligned mutations in recombinant hexahistidine-tagged full-length <i>Saccharomyces cerevisiae</i> LDM6×His (ScLDM6×His) to elucidate how differential susceptibility to azole drugs is conferred by LDM of the <i>C. albicans</i> Darlington strain. The mutations Y140H and I471T were introduced, either alone or in combination, into ScLDM6×His via overexpression of the recombinant enzyme from the <i>PDR5</i> locus of an azole hypersensitive strain of <i>S. cerevisiae</i>. Phenotypes and high-resolution X-ray crystal structures were determined for the surrogate enzymes in complex with representative short-tailed (voriconazole) and long-tailed (itraconazole) triazoles. The preferential high-level resistance to short-tailed azoles conferred by the ScLDM Y140H I471T mutant required both mutations, despite the I471T mutation conferring only a slight increase in resistance. Crystal structures did not detect changes in the position/tilt of the heme co-factor of wild-type ScLDM, I471T and Y140H single mutants, or the Y140H I471T double-mutant. The mutant threonine sidechain in the Darlington strain CaLDM perturbs the environment of the neighboring C-helix, affects the electronic environment of the heme, and may, via differences in closure of the neck of the substrate entry channel, increase preferential competition between lanosterol and short-tailed azole drugs. |
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