| Abstrakt: |
ABSTRACTA strain of the opportunistic pathogenic yeast Candida lusitaniaewas genetically modified for use as a cellular model for assessing by allele replacement the impact of lanosterol C14α-demethylase ERG11mutations on azole resistance. Candida lusitaniaewas chosen because it is susceptible to azole antifungals, it belongs to the CTG clade of yeast, which includes most of the Candidaspecies pathogenic for humans, and it is haploid and easily amenable to genetic transformation and molecular modeling. In this work, allelic replacement is targeted at the ERG11locus by the reconstitution of a functional auxotrophic marker in the 3′ intergenic region of ERG11. Homologous and heterologous ERG11alleles are expressed from the resident ERG11promoter of C. lusitaniae, allowing accurate comparison of the phenotypic change in azole susceptibility. As a proof of concept, we successfully expressed in C. lusitaniaedifferent ERG11alleles, either bearing or not bearing mutations retrieved from a clinical context, from two phylogenetically distant yeasts, C. albicansand Kluyveromyces marxianus. Candida lusitaniaeconstitutes a high-fidelity expression system, giving specific Erg11p-dependent fluconazole MICs very close to those observed with the ERG11donor 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 in Candidaspecies, and the equivalent position K151 in K. marxianus, plays a critical role in fluconazole resistance. |
