Structural Insights into the Azole Resistance of the Candida albicans Darlington Strain Using Saccharomyces cerevisiae Lanosterol 14α-Demethylase as a Surrogate

Target-based azole resistance in Candida albicans involves overexpression of the ERG11 gene encoding lanosterol 14α-demethylase (LDM), and/or the presence of single or multiple mutations in this enzyme. Overexpression of Candida albicans LDM (...

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Autores principales:	Danyon O. Graham, Rajni K. Wilson, Yasmeen N. Ruma, Mikhail V. Keniya, Joel D. A. Tyndall, Brian C. Monk
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	azole resistance <i>Candida albicans</i> Darlington strain <i>Saccharomyces cerevisiae</i> CYP51 lanosterol 14α-methylase Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/2fb14a794e4d4733aafc6a6df72d6c0b
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spelling	oai:doaj.org-article:2fb14a794e4d4733aafc6a6df72d6c0b2021-11-25T18:05:32ZStructural Insights into the Azole Resistance of the <i>Candida albicans</i> Darlington Strain Using <i>Saccharomyces cerevisiae</i> Lanosterol 14α-Demethylase as a Surrogate10.3390/jof71108972309-608Xhttps://doaj.org/article/2fb14a794e4d4733aafc6a6df72d6c0b2021-10-01T00:00:00Zhttps://www.mdpi.com/2309-608X/7/11/897https://doaj.org/toc/2309-608XTarget-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.Danyon O. GrahamRajni K. WilsonYasmeen N. RumaMikhail V. KeniyaJoel D. A. TyndallBrian C. MonkMDPI AGarticleazole resistance<i>Candida albicans</i>Darlington strain<i>Saccharomyces cerevisiae</i>CYP51lanosterol 14α-methylaseBiology (General)QH301-705.5ENJournal of Fungi, Vol 7, Iss 897, p 897 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	azole resistance <i>Candida albicans</i> Darlington strain <i>Saccharomyces cerevisiae</i> CYP51 lanosterol 14α-methylase Biology (General) QH301-705.5
spellingShingle	azole resistance <i>Candida albicans</i> Darlington strain <i>Saccharomyces cerevisiae</i> CYP51 lanosterol 14α-methylase Biology (General) QH301-705.5 Danyon O. Graham Rajni K. Wilson Yasmeen N. Ruma Mikhail V. Keniya Joel D. A. Tyndall Brian C. Monk 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
description	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.
format	article
author	Danyon O. Graham Rajni K. Wilson Yasmeen N. Ruma Mikhail V. Keniya Joel D. A. Tyndall Brian C. Monk
author_facet	Danyon O. Graham Rajni K. Wilson Yasmeen N. Ruma Mikhail V. Keniya Joel D. A. Tyndall Brian C. Monk
author_sort	Danyon O. Graham
title	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
title_short	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
title_full	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
title_fullStr	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
title_full_unstemmed	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
title_sort	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
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/2fb14a794e4d4733aafc6a6df72d6c0b
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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

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