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.IMPORTANCEMucor 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.

Keywords: Mucor; amino acid permease; calcineurin; dimorphism; drug resistance mechanisms; mucormycosis; protein kinase A.

FIG 1

Calcineurin suppressor (CnSp) mutations in the cnbRΔ mutant background restore hyphal growth. (A) Growth of calcineurin suppressor mutants (CnSp1 to CnSp4) and cnbRΔ mutant on a YPD agar plate at 30°C for 4 days postinoculation. While the cnbRΔ mutant shows smaller yeast colonies, the CnSp mutants show larger hyphal colonies. (B) The Mucor WT (R7B), cnbRΔ mutant, or CnSp mutants were grown overnight in liquid YPD medium with shaking at 30°C. Micrographs show that the CnSp mutants exhibit hyphal growth like that seen with the WT (scale bar = 10 μm).

FIG 2

Calcineurin suppressor mutations in the cnaBΔ mutant background confer resistance to calcineurin inhibitors. The images show growth of cyclosporine-resistant mutants (CSR) exhibiting hyphal growth and of cnaBΔ mutants exhibiting yeast growth under conditions of incubation on YPD agar with CsA (100 μg/ml) (top panel) or FK506 (1 μg/ml) (bottom panel) for 4 days at 30°C. CSR1, CSR2, CSR3, and CSR4 are shown. The other CSR mutants exhibited a similar CsA resistance phenotype (data not shown).

FIG 3

The bycAΔ single mutant and bycAΔ cnbRΔ double mutant are resistant to calcineurin inhibitors. (A) After 3 days of growth on solid YPD agar, the cnbRΔ mutant grew as yeast; however, despite no calcineurin function, the bycAΔ cnbRΔ double mutant exhibited hyphal growth like that shown by the WT and the bycAΔ mutant. (B) In the presence of CsA (upper panel; 100 μg/ml), the bycAΔ cnbRΔ double mutant and CnSp4 formed hyphal colonies that were larger than those formed by the WT, indicating resistance to CsA. No major difference in colony size was noted between the bycAΔ mutant and the WT. In the presence of FK506 (lower panel; 1 μg/ml), the WT formed a smaller yeast colony whereas the bycAΔ mutant, bycAΔ cnbRΔ double mutant, and CnSp4 each formed a larger hyphal colony. (C) When Mucor was grown overnight in YPD medium containing FK506 (1 μg/ml) at 30°C with shaking, the bycAΔ mutant, bycAΔ cnbRΔ double mutant, and CnSp4 mutant exhibited resistance to FK506 as evidenced by larger biomass and hyphal morphology, whereas the WT cells were sensitive to FK506 as they not only formed less biomass but also grew as yeast. As expected, the cnbRΔ mutant remained in its yeast-locked form (scale bar = 20 μm).

FIG 4

BycA is involved in the Mucor hypha-yeast transition under aerobic conditions. (A) Reverse transcriptase quantitative PCR showed that in the absence of calcineurin function (cnbRΔ and WT + FK506; yeast morphology), bycA expression was 6-fold higher than the level seen with the WT (hyphal morphology) with calcineurin function, suggesting that calcineurin regulates bycA expression at the mRNA level. When the Mucor WT was grown anaerobically under high-CO₂ conditions (see Materials and Methods), it grew as yeast; however, there was no significant difference in bycA expression compared to the level seen with the WT-hypha group. One-way analysis of variance (ANOVA) data were significant (P = 0.0001). Dunnett’s post hoc test was used to compare cnbRΔ, WT + FK506, WT-yeast results with those seen with the WT-hypha group (*, P < 0.05; ns, not statistically significant; undet., undetected). As expected, no expression of the bycA gene was detected in either of the bycAΔ mutants. (B) When WT, cnbRΔ, bycAΔ, and bycAΔ cnbRΔ mutants were grown anaerobically overnight under high-CO₂ conditions, they all exhibited a yeast morphology, thereby suggesting that neither calcineurin nor BycA has a role in anaerobic morphological pathways (scale bar = 20 μm). (C) Crude protein extracts (0.5 μg) were used to measure overall PKA activity. The WT-hypha and the bycAΔ mutant showed significantly lower PKA activity than WT-yeast (grown anaerobically). In WT + FK506 and the cnbRΔ mutant, PKA activity remained higher; however, the bycAΔ cnbRΔ mutant showed lower PKA activity despite possessing no calcineurin function. One-way ANOVA data were significant (P < 0.0001). Dunnett’s multiple-comparison test was used to compare each group with WT-yeast (*, P < 0.05). (D) Crude extracts from 60 mg biomass were used to measure overall cAMP activity. Yhe WT + FK506 and cnbRΔ strains showed significantly lower cAMP activity than WT-yeast, thereby suggesting that under aerobic conditions, PKA activity is elevated in Mucor yeast in a cAMP-independent manner. One-way ANOVA data were significant (P < 0.0001). Dunnett’s multiple-comparison test was used to compare each group with WT-yeast (*, P < 0.05).

FIG 5

Phagosome maturation in macrophages containing Mucor is dependent on pathogen calcineurin function and not morphology. (A) J774.A1 macrophages (5 × 10⁵) were challenged with Mucor spores or yeast at an MOI of 1 along with LysoTracker Green DND-26 and Hoechst 33342 stain (blue). White arrows indicate where phagosome maturation should be observed in the field (scale bar = 5 μm). DIC, differential interference contrast; DAPI, 4′,6-diamidino-2-phenylindole; LT, LysoTracker. (B) The macrophages containing the cnbRΔ single mutant and the bycAΔ cnbRΔ double mutant underwent significantly higher phagosome maturation than those containing the WT. Data are shown as percent maturation. The numbers (n) of macrophages containing Mucor counted for each group were as follows: WT = 480; bycAΔ mutant = 290; cnbRΔ mutant = 279; bycAΔ cnbRΔ mutant = 413. One-way ANOVA data were significant (P < 0.0001). Dunnett’s multiple-comparison test was used to compare each group to the WT (*, P < 0.05).

FIG 6

Calcineurin mutants cause less endothelial cell damage and FGF-2 protein secretion. A total of 5 × 10³ HUVECs were challenged with Mucor spores or yeast at an MOI of 10. After 24 h, the supernatant was collected. (A) Levels of LDH (indicating cytotoxicity) were quantified. Calcineurin mutants caused less damage than the WT. One-way ANOVA data were significant (P < 0.0001). Dunnett’s multiple-comparison test was used to compare each group to the WT (*, P < 0.05). (B) FGF-2 levels were quantified using ELISA. The WT strain induced significantly higher FGF-2 protein secretion than the calcineurin mutants. One-way ANOVA data were significant (P < 0.0001). Dunnett’s multiple-comparison test was used to compare each group to the WT (*, P < 0.05).

FIG 7

Calcineurin mutants were less virulent in a Galleria mellonella (wax moth) model of mucormycosis. Wax moth larvae (n = 15/group) were inoculated with 1 × 10⁴ Mucor spores or yeast in a mixture containing 2 μl PBS via injection into the last left proleg and were monitored for survival. All wax moth larvae challenged with the WT or the bycAΔ mutant succumbed to mortality within a week. The cnbRΔ mutant was avirulent, whereas about 65% of wax moth larvae inoculated with the bycAΔ cnbRΔ mutant or CnSp4 mutant survived. Results of a log rank (Mantel-Cox) test were statistically significant (P < 0.0001). A pairwise comparison was also performed with the following groups: WT versus bycAΔ mutant (P = 0.82) and WT versus cnbRΔ mutant or bycAΔ cnbRΔ mutant or CnSp4 mutant (*, P < 0.0001).

FIG 8

Calcineurin, BycA, and PKA in the morphogenesis of Mucor under aerobic conditions. Calcineurin is the master regulator of Mucor morphology. Active calcineurin positively regulates hyphal growth and negatively regulates yeast growth. This is achieved by suppressing the expression of the bycA gene, thereby preventing an increase in PKA activity. Calcium-calmodulin activates calcineurin to promote hyphal growth under aerobic conditions. However, when calcineurin is not functional, bycA gene expression is significantly elevated. BycA then activates PKA in a cAMP-independent manner to promote yeast growth. Under anaerobic conditions with high CO₂ levels, PKA is activated through the CO₂-cAMP pathway. Neither BycA nor calcineurin has a defined role in the regulation of Mucor morphology under anaerobic conditions. Can2, carbonyl anhydrase; AC, adenylyl cyclase; CaM, calmodulin.