A novel resistance pathway for calcineurin inhibitors in the human pathogenic MucoralesMucor circinelloides

Abstract

AbstractMucormycosis is an emerging lethal fungal infection in immunocompromised patients.Mucor circinelloidesis 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) inMucorresults in a shift from hyphal to yeast growth. We analyzed thirty-six calcineurin inhibitor resistant or bypass mutants that exhibited hyphal growth in the presence of calcineurin inhibitors or in the yeast-lockedcnbRΔ 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 herebycA(bypass ofcalcineurinA).bycAencodes an amino acid permease. We verified that bothbycAΔ, and thebycAΔcnbRΔ double mutant are resistant to the calcineurin inhibitor FK506, thereby demonstrating a novel resistance mechanism against calcineurin inhibitors. We also found that the expression ofbycAwas significantly higher in the wild type strain treated with FK506 and in thecnbRΔ mutants, but significantly lower in the wild type without FK506. These findings suggest thatbycAis a negative regulator of hyphal growth and/or a positive regulator of yeast growth inMucorand calcineurin suppresses thebycAgene at the mRNA level to promote hyphal growth. BycA is involved in theMucorhyphal-yeast transition as our data demonstrates a positive correlation betweenbycAexpression, protein kinase A activity, andMucoryeast-growth. Also calcineurin, independent of its role in morphogenesis, contributes to virulence traits including phagosome maturation blockade, host cell damages, and pro-angiogenic growth factor induction during interactions with hosts.ImportanceMucoris intrinsically resistant to most known antifungals, which makes mucormycosis treatment challenging. Calcineurin is a serine/threonine phosphatase widely conserved across eukaryotes. When calcineurin function is inhibited inMucor, 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 whichMucorcan become resistant to calcineurin inhibitors involving Mendelian mutations in the gene for FKBP12, 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 encoding thebycAgene contribute to resistance against calcineurin inhibitors. When calcineurin activity is absent, BycA can activate PKA to promote yeast growth via a cAMP-independent pathway. Our data also shows that calcineurin activity, primarily contributes to host - pathogen interactions in the pathogenesis ofMucor.