Mucormycosis is an emerging lethal fungal infection in immunocompromised patients. Mucor circinelloides 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 Mucor 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 cnbRΔ 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 bycA (bypass of calcineurin). bycA encodes an amino acid permease. We verified that both the bycAΔ single mutant and the bycAΔ cnbRΔ 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 bycA was significantly higher in the wild-type strain treated with FK506 and in the cnbRΔ mutants but was significantly lower in the wild-type strain without FK506 treatment. These findings suggest that bycA is a negative regulator of hyphal growth and/or a positive regulator of yeast growth in Mucor and that calcineurin suppresses expression of the bycA gene at the mRNA level to promote hyphal growth. BycA is involved in the Mucor hypha-yeast transition as our data demonstrate positive correlations among bycA expression, protein kinase A activity, and Mucor 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. IMPORTANCE Mucor 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 Mucor, 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 Mucor 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 bycA 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 Mucor.

24Mucormycosis is an emerging lethal fungal infection in immunocompromised 25 patients. Mucor circinelloides (Mucor) is a causal agent of mucormycosis and serves as 26 a model system to understand genetics in Mucorales. Calcineurin is a conserved 27 virulence factor in many pathogenic fungi and calcineurin inhibition or deletion of the 28 calcineurin regulatory subunit (CnbR) in Mucor results in a shift from hyphal to yeast 29 growth. We analyzed thirty-six calcineurin inhibitor resistant or bypass mutants that 30 exhibited hyphal growth in the presence of calcineurin inhibitors or in the yeast-locked 31 cnbR mutant background without carrying any mutations in known calcineurin 32 components. We found that a majority of the mutants had altered sequence in a gene, 33 named here bycA (bypass of calcineurin A). bycA encodes an amino acid permease. 34 We verified that both bycA, and the bycA cnbR double mutant are resistant to the 35 calcineurin inhibitor FK506, thereby demonstrating a novel resistance mechanism 36 against calcineurin inhibitors. We also found that the expression of bycA was 37 significantly higher in the wild type with FK506 and in the cnbR mutants, but 38 significantly lower in the wild type without FK506. These findings suggest that bycA is a 39 negative regulator of hyphal growth and/or a positive regulator of yeast growth in Mucor 40 and calcineurin suppresses the bycA gene at the mRNA level to promote hyphal growth. 41BycA is involved in the Mucor hyphal-yeast transition as our data demonstrates a 42 positive correlation between bycA expression, protein kinase A activity, and Mucor 43 yeast-growth. Also, calcineurin activity rather than hyphal morphology primarily 44 contributes to virulence traits including phagosome maturation blockade, host cell 45 damages, and pro-angiogenic growth factor induction during interactions with hosts. 46 3 Importance 47 Mucor is intrinsically resistant to most known antifungals, which makes 48 mucormycosis treatment challenging. Calcineurin is a serine/threonine phosphatase 49 widely conserved across eukaryotes. When calcineurin function is inhibited in Mucor, 50 growth shifts to a less-virulent yeast growth form which makes calcineurin an attractive 51 target for development of new antifungal drugs. Previously we identified two distinct 52 mechanisms through which Mucor can become resistant to calcineurin inhibitors 53 involving Mendelian mutations in the gene for FKBP12, calcineurin A or B subunits and 54 epimutations silencing the FKBP12 gene. Here, we identified a third novel mechanism 55 where loss of function mutations in the amino acid permease encoding the bycA gene 56 contribute to resistance against calcineurin inhibitors. When calcineurin activity is 57 absent, BycA can activate PKA to promote yeast growth via a cAMP-independent 58 pathway. Our data also shows that calcineurin activity, not morphology, primarily 59 contributes to host -pathogen interactions in the pathogenesis of Mucor. 60 61 62