The stepwise acquisition of fluconazole resistance mutations causes a gradual loss of fitness in Candida albicans

The pathogenic yeast Candida albicans can develop resistance to the widely used antifungal agent fluconazole, which inhibits ergosterol biosynthesis. Resistance is often caused by gain-of-function mutations in the transcription factors Mrr1, Tac1 and Upc2, which result in constitutive overexpression of multidrug efflux pumps and ergosterol biosynthesis genes respectively. It is not known how the permanently changed gene expression program in resistant strains affects their fitness in the absence of drug selection pressure. We have systematically investigated the effects of activating mutations in Mrr1, Tac1 and Upc2, individually and in all possible combinations, on the degree of fluconazole resistance and on the fitness of C.?albicans in an isogenic strain background. All combinations of different resistance mechanisms resulted in a stepwise increase in drug resistance, culminating in 500-fold increased fluconazole resistance in strains possessing mutations in the three transcription factors and an additional resistance mutation in the drug target enzyme Erg11. The acquisition of resistance mutations was associated with reduced fitness under non-selective conditions in vitro as well as in vivo during colonization of a mammalian host. Therefore, without compensatory mutations, the inability to appropriately regulate gene expression results in a loss of competitive fitness of drug-resistant C.?albicans strains.