A Novel Resistance Pathway for Calcineurin Inhibitors in the Human-Pathogenic Mucorales Mucor circinelloides.

Mucormycosis is an emerging lethal fungal infection in immunocompromised patients. is a causal agent of mucormycosis and serves as a model system to understand genetics in Mucorales. Calcineurin is a conserved virulence factor in many pathogenic fungi, and calcineurin inhibition or deletion of the calcineurin regulatory subunit (CnbR) in results in a shift from hyphal to yeast growth. We analyzed 36 calcineurin inhibitor-resistant or bypass mutants that exhibited hyphal growth in the presence of calcineurin inhibitors or in the yeast-locked Δ mutant background without carrying any mutations in known calcineurin components. We found that a majority of the mutants had altered sequence in a gene, named here (pass of lcineurin). encodes an amino acid permease. We verified that both the Δ single mutant and the Δ Δ double mutant are resistant to calcineurin inhibitor FK506, thereby demonstrating a novel mechanism of resistance against calcineurin inhibitors. We also found that the level of expression of was significantly higher in the wild-type strain treated with FK506 and in the Δ mutants but was significantly lower in the wild-type strain without FK506 treatment. These findings suggest that is a negative regulator of hyphal growth and/or a positive regulator of yeast growth in and that calcineurin suppresses expression of the gene at the mRNA level to promote hyphal growth. BycA is involved in the hypha-yeast transition as our data demonstrate positive correlations among expression, protein kinase A activity, and yeast growth. Also, calcineurin, independently of its role in morphogenesis, contributes to virulence traits, including phagosome maturation blockade, host cell damages, and proangiogenic growth factor induction during interactions with hosts. is intrinsically resistant to most known antifungals, which makes mucormycosis treatment challenging. Calcineurin is a serine/threonine phosphatase that is widely conserved across eukaryotes. When calcineurin function is inhibited in , growth shifts to a less virulent yeast growth form, which makes calcineurin an attractive target for development of new antifungal drugs. Previously, we identified two distinct mechanisms through which can become resistant to calcineurin inhibitors involving Mendelian mutations in the gene for FKBP12, including mechanisms corresponding to calcineurin A or B subunits and epimutations silencing the FKBP12 gene. Here, we identified a third novel mechanism where loss-of-function mutations in the amino acid permease corresponding to the gene contribute to resistance against calcineurin inhibitors. When calcineurin activity is absent, BycA can activate protein kinase A (PKA) to promote yeast growth via a cAMP-independent pathway. Our data also show that calcineurin activity contributes to host-pathogen interactions primarily in the pathogenesis of