Triazole Evolution of Candida parapsilosis Results in Cross-Resistance to Other Antifungal Drugs, Influences Stress Responses, and Alters Virulence in an Antifungal Drug-Dependent Manner

Abstract

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 (FLUEVO) and voriconazole-evolved (VOREVO) strains gained resistance to fluconazole and voriconazole only, while posaconazole evolution resulted in cross-resistance to all azoles and the posaconazole-evolved (POSEVO) strains showed higher echinocandin MIC values than the FLUEVO and VOREVO strains. Whole-genome sequencing results identified the development of different resistance mechanisms in the evolved strains: the FLUEVO and VOREVO strains harbored amino acid substitutions in Mrr1p (A808T and N394Y, respectively), and the POSEVO strain harbored an amino acid change in Erg3p (D14Y). By revealing increased efflux pump activity in both the FLUEVO and the VOREVO strains, along with the altered sterol composition of the POSEVO 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.

Narrow mutational signatures drive acquisition of multidrug resistance in the fungal pathogen Candida glabrata

“…Importantly, the link between ERG3 and cross-resistance may not be restricted to C. glabrata as ERG3 mutations leading to the depletion of ergosterol and the accumulation of less toxic sterols when ERG11 is inhibited have been implicated in cross-resistance between azoles and polyenes in S. cerevisiae and C. albicans 40 , 41 , 42 , 43 and between echinocandins and azoles in C. parapsilosis . 44 , 45 In addition, acquisition of ERG3 mutations upon echinocandin exposure has also been described in C. auris . 46 Why ERG3 mutations are often acquired under exposure to ani and how they contribute to resistance to flz remain unclear and need further attention.…”

Experimental Evolution of Multidrug Resistance in Neurospora crassa under Antifungal Azole Stress

“…In the clinic setting, multidrug resistance has been generally considered as a consequence of applying different kinds of antifungals for the control of fungal infections [1,16]. However, in pathogenic yeast C. parapsilosis, cross-resistance to posaconazole, FLC, VRC, ITC, and echinocandin was developed under posaconazole stress by experimental evolution [47]. Thus, multidrug resistance in fungi can occur under stress of a single azole drug.…”

New Biological Targets in Fungi and Novel Molecule under Development: A Review

“…This heterocycle, which is also substituted in position 1 by the same side chain, is most often found in antifungal azoles with a systemic effect [21]. Antifungal azoles act at the level of membrane sterols by blocking the biosynthesis of ergosterol following the inhibition of 14α-sterol demethylase, a cytochrome P450 enzyme responsible for the transformation of lanosterol into ergosterol, which is essential for the construction of the fungal membrane [22,23]. The oxidative reaction would take place as a result of the interaction between the azoles at their pyridine nitrogen atom in position 3 or 4 and the heme iron of cytochrome P450.…”

“…This complex thus formed would be at the origin of the blocking of the site of occupation of oxygen, hence the oxidative action. The latter would result in ergosterol depletion and accumulation of lanosterol and other sterols methylated [22] at position 14. These changes render the membrane more fragile and alter the activity of several membrane-bound enzymes responsible for the fungistatic activities of azoles.…”