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

Abstract

A strain of the opportunistic pathogenic yeast Candida lusitaniae was genetically modified for use as a cellular model for assessing by allele replacement the impact of lanosterol C14α-demethylase ERG11 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 the Candida 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 the ERG11 locus by the reconstitution of a functional auxotrophic marker in the 3' intergenic region of ERG11 Homologous and heterologous ERG11 alleles are expressed from the resident ERG11 promoter of C. lusitaniae, allowing accurate comparison of the phenotypic change in azole susceptibility. As a proof of concept, we successfully expressed in C. lusitaniae different ERG11 alleles, either bearing or not bearing mutations retrieved from a clinical context, from two phylogenetically distant yeasts, C. albicans and Kluyveromyces marxianusCandida lusitaniae constitutes a high-fidelity expression system, giving specific Erg11p-dependent fluconazole MICs very close to those observed with the ERG11 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 in Candida species, and the equivalent position K151 in K. marxianus, plays a critical role in fluconazole resistance.

Keywords: Candida; Candida lusitaniae; ERG11 mutation; Kluyveromyces; fluconazole resistance; heterologous expression.

FIG 1

Maximum likelihood tree of pathogenic filamentous fungi and pathogenic and nonpathogenic yeasts using a Clustal Omega alignment of amino acid sequences of lanosterol 14-alpha-demethylase. Bootstrap values: thick branch, 90 to 100%; medium branch, 70 to 89%; thin branch, lower than 70%. Asterisks indicate species that are not considered to be pathogenic. Arrows show the position of the yeast species used in this study.

FIG 2

Molecular modifications sequentially achieved to obtain the recipient strain E11ura3Δ5′. (A) Transformation of the 6936 ura3Δ strain with a DNA cassette bearing the genetic marker URA3 (white box) flanked by DNA sequences homologous to the 3′-UTR region of the ERG11 locus (gray boxes). Crossing lines symbolize recombination events that allow the incoming linear DNA cassette to be integrated into the recipient chromosome to give the E11URA3 strain. (B) Transformation of the E11URA3 strain with a DNA cassette bearing a nonfunctional URA3 truncated of its promoter and of the NH2 part of the ORF (white box), resulting after recombination in the strain E11ura3Δ5′. (C) Southern blot analysis of the different strains used and constructed in this study after digestion of the genomic DNA with EcoRV and hybridization with URA3 probes. DNA fragment size is given in kilobase pairs. WT, wild-type strain 6936; EV, EcoRV site; ATG, initiation codon; TAA, stop codon; UTR, untranslated regulatory region; IG, intergenic region.

FIG 3

Strategy used to replace the resident ERG11 allele of C. lusitaniae by any other ERG11 allele bearing or not bearing a mutation. The E11ura3Δ5′ recipient strain was transformed by a DNA cassette made of the promoter region of the C. lusitaniae ERG11 gene (gray box, 5′-UTR), the ORF, and the 3′-UTR region of any new ERG11 allele (black box) and the URA3 complementary information (white box), allowing reconstitution and expression of a functional URA3 allele downstream of ERG11. *, mutation; abbreviations are defined in the legend to Fig. 2.

FIG 4

Etest determination of fluconazole susceptibility after 24 h of incubation on RPMI medium. (A) C. albicans SC5314. (B) C. lusitaniae E11ura3Δ5′ strain expressing the ERG11 allele A of C. albicans SC5314.

FIG 5

Normalized expression levels of different ERG11 alleles in the C. lusitaniae E11ura3Δ5′ strain grown in RPMI medium. (A) C. albicans ERG11 allele expression in fold variation relative to the expression level of the reference SC5314 WT ERG11 allele A in C. lusitaniae. (B) K. marxianus ERG11 allele expression in fold variation relative to the expression level of the reference K. marxianus PAZ clinical resistant ERG11 allele in C. lusitaniae. (C) C. lusitaniae ERG11 allele expression in fold variation relative to the expression level of the reference 6936 WT ERG11 allele in C. lusitaniae. Expression of reference alleles (described in Table 1) in the C. lusitaniae E11ura3Δ5′ strain was set to 1. ERG11 allele expression levels were quantified and normalized relative to the housekeeping gene ACT1. Values are presented with SEM (standard errors of the means). *, P < 0.05 compared to the respective reference ERG11 allele.