Cargando…

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 (CaLDM) Y132H I471T by the Darlington strain strongly incr...

Descripción completa

Detalles Bibliográficos
Autores principales: Graham, Danyon O., Wilson, Rajni K., Ruma, Yasmeen N., Keniya, Mikhail V., Tyndall, Joel D. A., Monk, Brian C.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8621857/
https://www.ncbi.nlm.nih.gov/pubmed/34829185
http://dx.doi.org/10.3390/jof7110897
_version_ 1784605555974733824
author Graham, Danyon O.
Wilson, Rajni K.
Ruma, Yasmeen N.
Keniya, Mikhail V.
Tyndall, Joel D. A.
Monk, Brian C.
author_facet Graham, Danyon O.
Wilson, Rajni K.
Ruma, Yasmeen N.
Keniya, Mikhail V.
Tyndall, Joel D. A.
Monk, Brian C.
author_sort Graham, Danyon O.
collection PubMed
description 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 (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 Saccharomyces cerevisiae LDM6×His (ScLDM6×His) to elucidate how differential susceptibility to azole drugs is conferred by LDM of the C. albicans 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 PDR5 locus of an azole hypersensitive strain of S. cerevisiae. 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 Online
Article
Text
id pubmed-8621857
institution National Center for Biotechnology Information
language English
publishDate 2021
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-86218572021-11-27 Structural Insights into the Azole Resistance of the Candida albicans Darlington Strain Using Saccharomyces cerevisiae Lanosterol 14α-Demethylase as a Surrogate Graham, Danyon O. Wilson, Rajni K. Ruma, Yasmeen N. Keniya, Mikhail V. Tyndall, Joel D. A. Monk, Brian C. J Fungi (Basel) Article 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 (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 Saccharomyces cerevisiae LDM6×His (ScLDM6×His) to elucidate how differential susceptibility to azole drugs is conferred by LDM of the C. albicans 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 PDR5 locus of an azole hypersensitive strain of S. cerevisiae. 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. MDPI 2021-10-24 /pmc/articles/PMC8621857/ /pubmed/34829185 http://dx.doi.org/10.3390/jof7110897 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Graham, Danyon O.
Wilson, Rajni K.
Ruma, Yasmeen N.
Keniya, Mikhail V.
Tyndall, Joel D. A.
Monk, Brian C.
Structural Insights into the Azole Resistance of the Candida albicans Darlington Strain Using Saccharomyces cerevisiae Lanosterol 14α-Demethylase as a Surrogate
title Structural Insights into the Azole Resistance of the Candida albicans Darlington Strain Using Saccharomyces cerevisiae Lanosterol 14α-Demethylase as a Surrogate
title_full Structural Insights into the Azole Resistance of the Candida albicans Darlington Strain Using Saccharomyces cerevisiae Lanosterol 14α-Demethylase as a Surrogate
title_fullStr Structural Insights into the Azole Resistance of the Candida albicans Darlington Strain Using Saccharomyces cerevisiae Lanosterol 14α-Demethylase as a Surrogate
title_full_unstemmed Structural Insights into the Azole Resistance of the Candida albicans Darlington Strain Using Saccharomyces cerevisiae Lanosterol 14α-Demethylase as a Surrogate
title_short Structural Insights into the Azole Resistance of the Candida albicans Darlington Strain Using Saccharomyces cerevisiae Lanosterol 14α-Demethylase as a Surrogate
title_sort structural insights into the azole resistance of the candida albicans darlington strain using saccharomyces cerevisiae lanosterol 14α-demethylase as a surrogate
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8621857/
https://www.ncbi.nlm.nih.gov/pubmed/34829185
http://dx.doi.org/10.3390/jof7110897
work_keys_str_mv AT grahamdanyono structuralinsightsintotheazoleresistanceofthecandidaalbicansdarlingtonstrainusingsaccharomycescerevisiaelanosterol14ademethylaseasasurrogate
AT wilsonrajnik structuralinsightsintotheazoleresistanceofthecandidaalbicansdarlingtonstrainusingsaccharomycescerevisiaelanosterol14ademethylaseasasurrogate
AT rumayasmeenn structuralinsightsintotheazoleresistanceofthecandidaalbicansdarlingtonstrainusingsaccharomycescerevisiaelanosterol14ademethylaseasasurrogate
AT keniyamikhailv structuralinsightsintotheazoleresistanceofthecandidaalbicansdarlingtonstrainusingsaccharomycescerevisiaelanosterol14ademethylaseasasurrogate
AT tyndalljoelda structuralinsightsintotheazoleresistanceofthecandidaalbicansdarlingtonstrainusingsaccharomycescerevisiaelanosterol14ademethylaseasasurrogate
AT monkbrianc structuralinsightsintotheazoleresistanceofthecandidaalbicansdarlingtonstrainusingsaccharomycescerevisiaelanosterol14ademethylaseasasurrogate