title: Genetic dissection of azole resistance mechanisms in Candida albicans and their validation in a mouse model of disseminated infection.
abstract: Principal mechanisms of resistance to azole antifungals include the upregulation of multidrug transporters and the modification of the target enzyme, a cytochrome P450 (Erg11) involved in the 14alpha-demethylation of ergosterol. These mechanisms are often combined in azole-resistant Candida albicans isolates recovered from patients. However, the precise contributions of individual mechanisms to C. albicans resistance to specific azoles have been difficult to establish because of the technical difficulties in the genetic manipulation of this diploid species. Recent advances have made genetic manipulations easier, and we therefore undertook the genetic dissection of resistance mechanisms in an azole-resistant clinical isolate. This isolate (DSY296) upregulates the multidrug transporter genes CDR1 and CDR2 and has acquired a G464S substitution in both ERG11 alleles. In DSY296, inactivation of TAC1, a transcription factor containing a gain-of-function mutation, followed by sequential replacement of ERG11 mutant alleles with wild-type alleles, restored azole susceptibility to the levels measured for a parent azole-susceptible isolate (DSY294). These sequential genetic manipulations not only demonstrated that these two resistance mechanisms were those responsible for the development of resistance in DSY296 but also indicated that the quantitative level of resistance as measured in vitro by MIC determinations was a function of the number of genetic resistance mechanisms operating in any strain. The engineered strains were also tested for their responses to fluconazole treatment in a novel 3-day model of invasive C. albicans infection of mice. Fifty percent effective doses (ED(50)s) of fluconazole were highest for DSY296 and decreased proportionally with the sequential removal of each resistance mechanism. However, while the fold differences in ED(50) were proportional to the fold differences in MICs, their magnitude was lower than that measured in vitro and depended on the specific resistance mechanism operating.
authors.affiliation: Institute of Microbiology, University of Lausanne and University Hospital Center, CH-1011 Lausanne, Switzerland.
authors.forename: Donna M
authors.initials: DM
authors.lastname: MacCallum
authors.forename: Alix
authors.initials: A
authors.lastname: Coste
authors.forename: Françoise
authors.initials: F
authors.lastname: Ischer
authors.forename: Mette D
authors.initials: MD
authors.lastname: Jacobsen
authors.forename: Frank C
authors.initials: FC
authors.lastname: Odds
authors.forename: Dominique
authors.initials: D
authors.lastname: Sanglard
mesh_terms: D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology*; D001483:Base Sequence; D002176:Candida albicans / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification / Q000472:pathogenicity; D002177:Candidiasis / Q000188:drug therapy* / Q000382:microbiology*; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D004195:Disease Models, Animal; D025141:Drug Resistance, Fungal / Q000235:genetics*; D005260:Female; D015725:Fluconazole / Q000494:pharmacology*; D005656:Fungal Proteins / Q000235:genetics; D005800:Genes, Fungal; D005818:Genetic Engineering; D006801:Humans; D066298:In Vitro Techniques; D026901:Membrane Transport Proteins / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008826:Microbial Sensitivity Tests; D009154:Mutation
chemical_list: D000935:Antifungal Agents; C094693:CDR1 protein, Candida albicans; D017931:DNA Primers; D004271:DNA, Fungal; D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D015725:Fluconazole