Distinct Roles of Candida albicans Drug Resistance Transcription Factors TAC1 , MRR1 , and UPC2 in Virulence

Abstract

Azoles are widely used in antifungal therapy in medicine. Resistance to azoles can occur in Candida albicans principally by overexpression of multidrug transporter gene CDR1, CDR2, or MDR1 or by overexpression of ERG11, which encodes the azole target. The expression of these genes is controlled by the transcription factors (TFs) TAC1 (involved in the control of CDR1 and CDR2), MRR1 (involved in the control of MDR1), and UPC2 (involved in the control of ERG11). Several gain-of-function (GOF) mutations are present in hyperactive alleles of these TFs, resulting in the overexpression of target genes. While these mutations are beneficial to C. albicans survival in the presence of the antifungal drugs, their effects could potentially alter the fitness and virulence of C. albicans in the absence of the selective drug pressure. In this work, the effect of GOF mutations on C. albicans virulence was addressed in a systemic model of intravenous infection by mouse survival and kidney fungal burden assays. We engineered a set of strains with identical genetic backgrounds in which hyperactive alleles were reintroduced in one or two copies at their genomic loci. The results obtained showed that neither TAC1 nor MRR1 GOF mutations had a significant effect on C. albicans virulence. In contrast, the presence of two hyperactive UPC2 alleles in C. albicans resulted in a significant decrease in virulence, correlating with diminished kidney colonization compared to that by the wild type. In agreement with the effect on virulence, the decreased fitness of an isolate with UPC2 hyperactive alleles was observed in competition experiments with the wild type in vivo but not in vitro. Interestingly, UPC2 hyperactivity delayed filamentation of C. albicans after phagocytosis by murine macrophages, which may at least partially explain the virulence defects. Combining the UPC2 GOF mutation with another hyperactive TF did not compensate for the negative effect of UPC2 on virulence. In conclusion, among the major TFs involved in azole resistance, only UPC2 had a negative impact on virulence and fitness, which may therefore have consequences for the epidemiology of antifungal resistance.

Competitive Fitness of Fluconazole-Resistant Clinical Candida albicans Strains

“…We found that, when introduced into strain SC5314, all tested Mrr1 GOF mutations (eight different mutations) as well as GOF mutations in Tac1 and Upc2 caused a significant fitness defect during growth in YPD medium. In contrast, Lohberger et al did not observe an effect of GOF mutations in these TFs on in vitro fitness in the same strain background (8). Of note, these authors used a modified yeast extract-peptone-dextrose medium containing only half the standard amount of peptone (1% instead of 2%) and yeast extract (0.5% instead of 1%), so the absence of an observable fitness defect in this medium may reflect the absence of a fitness defect in minimal medium in the study by Sasse et al (26).…”

“…The competitive fitness was further reduced in strains containing two or all three hyperactive TFs, and the latter also exhibited a strong fitness defect in a mouse model of gastrointestinal colonization. A separate study similarly reported on the decreased virulence of genetically engineered strains with hyperactive forms of Upc2 and, to a lesser extent, Tac1 in a mouse model of disseminated candidiasis (8). Some fluconazoleresistant clinical C. albicans isolates with GOF mutations in MRR1 or TAC1 were also found to be less virulent than fluconazole-susceptible isolates from the same patients in mouse models of systemic or oral candidiasis (27,28).…”

“…Of note, these authors used a modified yeast extract-peptone-dextrose medium containing only half the standard amount of peptone (1% instead of 2%) and yeast extract (0.5% instead of 1%), so the absence of an observable fitness defect in this medium may reflect the absence of a fitness defect in minimal medium in the study by Sasse et al (26). In addition, cocultures were started with 3.7 ϫ 10 6 cells/ml in the study by Lohberger et al (8), which amounts to only ca. 7 generations until stationary phase is reached, whereas the cocultures were grown for FIG 5 Competitive fitness of fluconazole-susceptible isolates F2, B3, 5833, and TW1 (light gray bars) and matched fluconazoleresistant isolates F5, B4, 6692, and TW17 (dark gray bars), respectively, in a mouse model of disseminated candidiasis.…”

“…The most commonly observed resistance mechanisms are mutations in the drug target enzyme Erg11, which reduce drug binding, and gain-of-function (GOF) mutations in the zinc cluster transcription factors (ZnTFs) Mrr1, Tac1, and Upc2, which result in changes in gene expression that promote drug resistance (2-4). Specifically, GOF mutations in Mrr1 cause overexpression of the multidrug efflux pump MDR1 and additional genes that contribute to fluconazole resistance (5)(6)(7)(8)(9)(10)(11). Similarly, GOF mutations in Tac1 confer fluconazole resistance by mediating overexpression of the multidrug efflux pumps CDR1 and CDR2 and other Tac1 target genes (12)(13)(14)(15)(16)(17).…”

The Etest Performed Directly on Blood Culture Bottles Is a Reliable Tool for Detection of Fluconazole-Resistant Candida albicans Isolates

“…However, CA-2 and CA-3 had higher expression levels of CDR2 and were also resistant to voriconazole and posaconazole (data not shown). The lack of a clear correlation between phenotypic and molecular resistance was not surprising, as the molecular explanation for fluconazole resistance in Candida is based on single mechanisms or simultaneous multiple mechanisms, and there might be other unknown underlying mechanisms that play a role in the resistance of these isolates (33)(34)(35)(36). Furthermore, there may be an association between the specific resistance mechanisms and the anatomical site at which the isolate has become resistant (37).…”