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

Abstract

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.

Interactions of FK506 and Rapamycin With FK506 Binding Protein 12 in Opportunistic Human Fungal Pathogens

“…When M. circinelloides is grown in the presence of FK506 it exhibits yeast growth instead of hyphae which shows that calcineurin regulates dimorphism in this fungus (hyphal to yeast transition) (Lee et al, 2013). Mutants lacking functional calcineurin are less virulent than the wild type which suggests that calcineurin is a key target for the treatment of mucormycosis (Lee et al, 2013(Lee et al, , 2015Vellanki et al, 2020). M. circinelloides encodes three calcineurin catalytic subunits (CnaA, CnaB, and CnaC) and one regulatory subunit (CnbR) (Lee et al, 2013).…”

“…It was first reported in C. neoformans that the mutants lacking calcineurin do not survive in conditions that mimic the host environment (Steinbach et al, 2007). Further studies have shown that calcineurin is also required for virulence and pathogenicity in C. albicans (Blankenship et al, 2003;Blankenship and Heitman, 2005), A. fumigatus (Steinbach et al, 2006), and Mucor circinelloides (Lee et al, 2013;Lee et al, 2015;Vellanki et al, 2020). Although, calcineurin is an attractive target for antifungal drug development, there are two key problems.…”

The heterotrimeric G‐protein beta subunit Gpb1 controls hyphal growth under low oxygen conditions through the protein kinase A pathway and is essential for virulence in the fungusMucor circinelloides

“…Mucor stimulates yeast growth, even in aerobic conditions (Kubo & Mihara, 2007;Orlowski, 1991). Accordingly, it has been reported that in M. circinelloides, intracellular cAMP accumulation is higher in yeast compared to hyphal growth (Vellanki et al, 2020). In eukaryotes, it has been reported that cAMP activates the protein kinase A (PKA) pathway by binding to the regulatory subunit (PkaR) that leads to its dissociation from the catalytic subunit (PkaC), which activates several transcription factors that control the final morphological fate of the cell (Choi, Jung, & Kronstad, 2015).…”

A Mucoralean White Collar-1 Photoreceptor Controls Virulence by Regulating an Intricate Gene Network during Host Interactions

“…In this context, most of the current research is based on searching for and characterizing new targets in the physiology of Mucorales that could serve for the development of new antifungal compounds. Different research lines study the role of virulence factors described in Mucorales pathogenesis, such as the CotH proteins, the three components of the high-affinity iron uptake system, and the regulatory proteins involved in dimorphism [ 6 , 7 , 8 , 9 , 10 ]. In addition, several genome-wide strategies have been designed to find new and specific targets in Mucorales, based on the RNAi mechanism of the fungus [ 11 ] and in the host–pathogen transcriptome [ 12 , 13 ].…”