Triazole Evolution of Candida parapsilosis Results in CrossResistance to Other Antifungal Drugs, Influences Stress Responses, and Alters Virulence in an Antifungal DrugDependent Manner

The number of invasive infections caused by Candida species is increasing worldwide. The incidence of candidiasis cases caused by non-albicans Candida species, such as Candida parapsilosis, is also increasing, and non-albicans Candida species are currently responsible for more invasive infections than C. albicans. Additionally, while the development of azole resistance during invasive disease with C. albicans remains uncommon, azole-resistant C. parapsilosis strains are frequently isolated in the hospital setting. In this study, we applied direct selection to generate azole-adapted and azole-evolved C. parapsilosis strains in order to examine the effect of azole resistance development on fungal viability and pathogenesis progression. Depending on the drug applied, the different evolved strains developed distinct cross-resistance patterns: the fluconazole-evolved (FLU^EVO) and voriconazole-evolved (VOR^EVO) strains gained resistance to fluconazole and voriconazole only, while po-saconazole evolution resulted in cross-resistance to all azoles and the posaconazole-evolved (POS^EVO) strains showed higher echinocandin MIC values than the FLU^EVO and VOR^EVO strains. Whole-genome sequencing results identified the development of different resistance mechanisms in the evolved strains: the FLU^EVO and VOR^EVO strains harbored amino acid substitutions in Mrr1p (A808T and N394Y, respectively), and the POS^EVO strain harbored an amino acid change in Erg3p (D14Y). By revealing increased efflux pump activity in both the FLU^EVO and the VOR^EVO strains, along with the altered sterol composition of the POS^EVO strain, we now highlight the impact of the above-mentioned amino acid changes in C. parapsilosis azole resistance development. We further revealed that the virulence of this species was only slightly or partially affected by fluconazole and voriconazole adaptation, while it significantly decreased after posaconazole adaptation. Our results suggest that triazole adaptation can result in azole cross-resistance and that this process may also result in virulence alterations in C. parapsilosis, depending on the applied drug. IMPORTANCE Candida parapsilosis causes life-threatening fungal infections. In the last 2 decades, the increasing number of azole-resistant C. parapsilosis clinical isolates has been attributable to the overuse and misuse of fluconazole, the first-line antifungal agent most commonly used in several countries. To date, the range of applicable antifungal drugs is limited. As a consequence, it is essential to understand the possible mechanisms of antifungal resistance development and their effect on virulence in order to optimize antifungal treatment strategies in the clinical setting. Our results revealed that the prolonged exposure to azoles resulted not only in azole resistance but also in cross-resistance development. Our data further indicate that resistance development may occur through different mechanisms that can also alter the virulence of C. parapsilosis. These results highlight the consequences of prolonged drug usage and suggest the need for developing alternative antifungal treatment strategies in clinical practice.