Clinical azole cross-resistance by Candida parapsilosis: the upregulation of MDR1 and CDR1-like member gene resulting from MRR1 gain-of-function mutation

Objectives: We describe here the molecular mechanisms underlying the acquisition of azole resistance by C. parapsilosis following fluconazole treatment due to candiduria.Methods: A set of three consecutive C. parapsilosis isolates (CPS-A, CPS-B, CPS-C) were recovered from urine samples of the same patient. Whole-genome sequencing (WGS) and antifungal susceptibility assays were performed and gene expression was quantified by RT-qPCR.Results: The initial isolate CPS-A, was susceptible to all three azoles tested (fluconazole, voriconazole and posaconazole); isolate CPS-B displayed a susceptible-dose dependent phenotype to fluconazole, while isolate CPS-C exhibited a cross-resistance profile to fluconazole and voriconazole. WGS revealed a putative resistance mechanism in isolate CPS-C, associated with a G1810A nucleotide substitution, leading to a G604R change in the Mrr1p transcription factor. Introducing this gain-of-function (GOF) mutation into the susceptible CPS-A isolate results in resistance to fluconazole and voriconazole, as well as upregulation of MRR1 and MDR1. Interestingly, the susceptible dose-dependent phenotype exhibited by isolate CPS-B is associated with increased copy number of a member of the CDR family, CPAR2_304370. Expression of CPAR2_304370 is increased both azole-resistant isolates CPS-B and CPS-C, and in the CPS-A strain expressing the MRR1 gene harboring the GOF mutation.Conclusions: This is the first study showing acquisition of azole cross-resistance in vivo in C. parapsilosis due to a G1810A (G604R) GOF mutation resulting in MRR1 hyperactivation and consequently, MDR1 efflux pump overexpression. We also identified the CPAR2_304370 gene as responsible for the decreased fluconazole susceptibility and as a putative target of the MRR1 GOF mutation.