• azole-resistance
• C. parapsilosis
• 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.