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

ABSTRACT: 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.

SUBMITTER: Graham DO

PROVIDER: S-EPMC8621857 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The cytochrome P-450 lanosterol 14alpha-demethylase (CYP51A1) of yeasts is involved in an important step in the biosynthesis of ergosterol. Since CYP51A1 is the target of azole antifungal agents, this enzyme is potentially prone to alterations leading to resistance to these agents. Among them, a decrease in the affinity of CYP51A1 for these agents is possible. We showed in a group of Candida albicans isolates from AIDS patients that multidrug efflux transporters were playing an important role in the resistance of C. albicans to azole antifungal agents, but without excluding the involvement of other factors (D. Sanglard, K. Kuchler, F. Ischer, J.-L. Pagani, M. Monod, and J. Bille, Antimicrob. Agents Chemother. 39:2378-2386, 1995). We therefore analyzed in closer detail changes in the affinity of CYP51A1 for azole antifungal agents. A strategy consisting of functional expression in Saccharomyces cerevisiae of the C. albicans CYP51A1 genes of sequential clinical isolates from patients was designed. This selection, which was coupled with a test of susceptibility to the azole derivatives fluconazole, ketoconazole, and itraconazole, enabled the detection of mutations in different cloned CYP51A1 genes, whose products are potentially affected in their affinity for azole derivatives. This selection enabled the detection of five different mutations in the cloned CYP51A1 genes which correlated with the occurrence of azole resistance in clinical C. albicans isolates. These mutations were as follows: replacement of the glycine at position 129 with alanine (G129A), Y132H, S405F, G464S, and R467K. While the S405F mutation was found as a single amino acid substitution in a CYP51A1 gene from an azole-resistant yeast, other mutations were found simultaneously in individual CYP51A1 genes, i.e., R467K with G464S, S405F with Y132H, G129A with G464S, and R467K with G464S and Y132H. Site-directed mutagenesis of a wild-type CYP51A1 gene was performed to estimate the effect of each of these mutations on resistance to azole derivatives. Each single mutation, with the exception of G129A, had a measurable effect on the affinity of the target enzyme for specific azole derivatives. We speculate that these specific mutations could combine with the effect of multidrug efflux transporters in the clinical isolates and contribute to different patterns and stepwise increases in resistance to azole derivatives.

Project description:Azole resistant fungal infections remain a health problem for the immune compromised. Current therapies are limited due to rises in new resistance mechanisms. Therefore, it is important to identify new drug targets for drug discovery and novel therapeutics. Arv1 (are1 are2 required for viability 1) function is highly conserved between multiple pathogenic fungal species. Candida albicans (C. albicans) cells lacking CaArv1 are azole hypersusceptible and lack virulence. Saccharomyces cerevisiae (S. cerevisiae) Scarv1 cells are also azole hypersusceptible, a phenotype reversed by expression of CaArv1, indicating conservation in the molecular mechanism for azole susceptibility. To define the relationship between Arv1 function and azole susceptibility, we undertook a structure/function analysis of ScArv1. We identified several conserved amino acids within the ScArv1 homology domain (ScAhd) required for maintaining normal azole susceptibility. Erg11 lanosterol 14-α-demethylase is the rate-limiting enzyme in sterol biosynthesis and is the direct target of azole antifungals, so we used our ScArv1 mutants in order to explore the relationship between ScArv1 and ScErg11. Specific ScArv1 mutants ectopically expressed from a low copy plasmid were unable to restore normal azole susceptibility to Scarv1 cells and had reduced Erg11 protein levels. Erg11 protein stability depended on its ability to form a heterodimeric complex with Arv1. Complex formation was required for maintaining normal azole susceptibility. Scarv1 cells expressing orthologous CaArv1 mutants also had reduced CaErg11 levels, were unable to form a CaArv1-CaErg11 complex, and were azole hypersusceptible. Scarv1 cells expressing CaArv1 mutants unable to interact with CaErg11 could not sustain proper levels of the azole resistant CaErg11Y132F F145L protein. Caarv1/Caarv1 cells expressing CaArv1 mutants unable to interact with CaErg11 were found to lack virulence using a disseminated candidiasis mouse model. Expressing CaErg11Y132F F145L did not reverse the lack of virulence. We hypothesize that the role of Arv1 in Erg11-dependent azole resistance is to stabilize Erg11 protein level. Arv1 inhibition may represent an avenue for treating azole resistance.

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

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