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A strain of the opportunistic pathogenic yeastCandida lusitaniae was genetically modified for use as a cellular model for assessing by allele replacement the impact of lanosterol C14α-demethylaseERG11 mutations on azole resistance.Candida lusitaniae was chosen because it is susceptible to azole antifungals, it belongs to the CTG clade of yeast, which includes most of theCandida species pathogenic for humans, and it is haploid and easily amenable to genetic transformation and molecular modeling. In this work, allelic replacement is targeted at theERG11 locus by the reconstitution of a functional auxotrophic marker in the 3′ intergenic region ofERG11 . Homologous and heterologousERG11 alleles are expressed from the residentERG11 promoter ofC. lusitaniae , allowing accurate comparison of the phenotypic change in azole susceptibility. As a proof of concept, we successfully expressed inC. lusitaniae differentERG11 alleles, either bearing or not bearing mutations retrieved from a clinical context, from two phylogenetically distant yeasts,C. albicans andKluyveromyces marxianus .Candida lusitaniae constitutes a high-fidelity expression system, giving specific Erg11p-dependent fluconazole MICs very close to those observed with theERG11 donor strain. This work led us to characterize the phenotypic effect of two kinds of mutation: mutation conferring decreased fluconazole susceptibility in a species-specific manner and mutation conferring fluconazole resistance in several yeast species. In particular, a missense mutation affecting amino acid K143 of Erg11p inCandida species, and the equivalent position K151 inK. marxianus , plays a critical role in fluconazole resistance.

A CTG Clade Candida Yeast Genetically Engineered for the Genotype-Phenotype Characterization of Azole Antifungal Resistance in Human-Pathogenic Yeasts