Contribution of Clinically Derived Mutations in the Gene Encoding the Zinc Cluster Transcription Factor Mrr2 to Fluconazole Antifungal Resistance and CDR1 Expression in Candida albicans

Abstract

Mutations in genes encoding zinc cluster transcription factors (ZCFs) such as TAC1, MRR1, and UPC2 play a key role in Candida albicans azole antifungal resistance. Artificial activation of the ZCF Mrr2 has shown increased expression of the gene encoding the Cdr1 efflux pump and resistance to fluconazole. Amino acid substitutions in Mrr2 have recently been reported to contribute to fluconazole resistance in clinical isolates. In the present study, 57 C. albicans clinical isolates with elevated fluconazole MICs were examined for mutations in MRR2 and expression of CDR1. Mutations in MRR2 resulting in 15 amino acid substitutions were uniquely identified among resistant isolates, including 4 substitutions (S466L, A468G, S469T, T470N) previously reported to reduce fluconazole susceptibility. Three additional, novel amino acid substitutions (R45Q, A459T, V486M) were also discovered in fluconazole-resistant isolates. When introduced into a fluconazole-susceptible background, no change in fluconazole MIC or CDR1 expression was observed for any of the mutations found in this collection. However, introduction of an allele leading to artificial activation of Mrr2 increased resistance to fluconazole as well as CDR1 expression. Moreover, Mrr2 amino acid changes reported previously to have the strongest effect on fluconazole susceptibility and CDR1 expression also exhibited no differences in fluconazole susceptibility or CDR1 expression relative to the parent strain. While all known fluconazole resistance mechanisms are represented within this collection of clinical isolates and contribute to fluconazole resistance to different extents, mutations in MRR2 do not appear to alter CDR1 expression or contribute to resistance in any of these isolates.

Fructose Induces Fluconazole Resistance in Candida albicans through Activation of Mdr1 and Cdr1 Transporters

“…[34]. In C. albicans, the tr scriptional regulation of the CDR1 and CDR2 genes overlaps: Mrr2, Upc2, Ndt80, Znc1 act as positive regulators [35,36], and Flo8 as a negative regulator [36] of both gen However, our results suggest that the disruption of either C. albicans ABC transpor does not induce the production of the remaining protein (Figure 3). Cells cultured in a medium with glucose, in contrast to those cultured with fruct did not synthesize Mdr1p, which was the reason for the absence of the Mdr1p-GFP sig during the microscopic observations and the lack of Mdr1p detected by Western (Figure 4).…”

“…[ 34 ]. In C. albicans , the transcriptional regulation of the CDR1 and CDR2 genes overlaps: Mrr2, Upc2, Ndt80, and Znc1 act as positive regulators [ 35 , 36 ], and Flo8 as a negative regulator [ 36 ] of both genes. However, our results suggest that the disruption of either C. albicans ABC transporters does not induce the production of the remaining protein ( Figure 3 ).…”

Rapid Hypothesis Testing in Candida albicans Clinical Isolates Using a Cloning-Free, Modular, and Recyclable System for CRISPR-Cas9 Mediated Mutant and Revertant Construction

“…BglI digested, phosphatase-treated, and heat-inactivated pBSS2 plasmid containing the SAT1 -flipper disruption cassette from pSF2 was used as the template for a PCR with primers FLPPrACT1-F and PrACT1-R-PstI ( Fig. 1A ) ( 7 , 35 ). This reaction generated a fragment containing the flip recombinase coding sequence ( FLP ), ACT1 terminator ( ACT1 t), and ACT1 promoter (Pr ACT1 ) which was then purified over a column (ThermoScientific) and digested with SalI and PstI.…”

CRISPR-Based Genetic Manipulation of Candida Species: Historical Perspectives and Current Approaches

“…Studies have investigated polyene susceptibility using CRISPR in C. albicans (Min et al, 2018 ; Huang C.-Y. et al, 2019 ) as well as probed the underlying mechanisms of azole susceptibility in C. albicans (Vyas et al, 2015 ; Liu and Myers, 2017a , b ; Chen et al, 2018 ; Shapiro et al, 2018b ; Nishimoto et al, 2019 ), and other Candida species such as C. orthopsilosis (de San Vicente et al, 2019 ; Morio et al, 2019 ), and the highly azole-resistant C. auris (Kim et al, 2019 ; Rybak et al, 2019 , 2020 ). Similar studies, using mutants created with CRISPR-Cas9, have screened for genes involved in echinocandin susceptibility in C. albicans (Lee et al, 2018 ; Lagree et al, 2020 ) and C. glabrata (Hou et al, 2019 ).…”