Study of the role of the C2585G mutation of the CPAR2_807270 gene in the development of azole resistance in Candida parapsilosis by gene editing

Candida parapsilosis is an opportunistic pathogen that causes invasive infections mainly in immunocompromised patients and premature newborns. Its incidence is increasing worldwide alongside with its resistance rates to antifungal drugs, especially azole compounds. Currently, at the Azienda Ospedaliero-Universitaria Pisana, Pisa-Italy there is an ongoing clinical outbreak associated to azole-resistant C. parapsilosis. Whole Genome Sequencing analyses performed on 16 clinical strains pointed out that azole-resistant clades were associated to the presence of the A395T mutation (Y132F amino acid substitution), which is widely known to be correlated with azole-resistance. In addition a new mutation, the C2585G leading to the S862C amino acid substitution in the CpMRR1 (CPAR2_807270) gene was present only in resistant strains (n=13). CpMrr1 is a transcriptional regulator of the CpMDR1 gene that encodes for a transport membrane protein. The aim of this study was to describe the contribution of the C2585G mutation in the CpMRR1 gene to the development of azole resistance in susceptible strains of C. parapsilosis. CRISPR-Cas9 gene editing strategy based on the use of an episomal plasmid has been used to induce a single nucleotide mutation in the CpMRR1 target gene. Sanger sequencing analyses confirmed the acquisition of the C2585G mutation in the CpMRR1 gene both in homozygosis and heterozygosis. Mutant clones were tested with E-test and broth microdilution methods to assess the antifungal susceptibility profiles. Fluconazole and voriconazole resistance were observed in all homozygous clones, while heterozygous mutants were resistant only to fluconazole. Real-Time PCR analysis demonstrated the over- expression of the CpMRR1 gene. Interestingly, the gene expression in homozygous mutants was higher than heterozygous and parental strains. Growth curve evaluation assessed the ability of mutant strains to replicate in liquid media. The acquisition of the C2585G mutation in the CpMRR1 gene affected the growth rate and fitness of the homozygous mutant clones, while heterozygous mutants had a growth rate similar to the parental strain. To the best of our knowledge this is the first study to confirm the contribution of the C2585G mutation in the CpMRR1 gene to the development of azole resistance in C. parapsilosis.