Calcineurin orchestrates dimorphic transitions, antifungal drug responses and host-pathogen interactions of the pathogenic mucoralean fungus Mucor circinelloides

Abstract

Calcineurin plays essential roles in virulence and growth of pathogenic fungi and is a target of the natural products FK506 and Cyclosporine A. In the pathogenic mucoralean fungus Mucor circinelloides, calcineurin mutation or inhibition confers a yeast-locked phenotype indicating that calcineurin governs the dimorphic transition. Genetic analysis in this study reveals that two calcineurin A catalytic subunits (out of three) are functionally diverged. Homology modeling illustrates modes of resistance resulting from amino substitutions in the interface between each calcineurin subunit and the inhibitory drugs. In addition, we show how the dimorphic transition orchestrated by calcineurin programs different outcomes during host-pathogen interactions. For example, when macrophages phagocytose Mucor yeast, subsequent phagosomal maturation occurs, indicating host cells respond appropriately to control the pathogen. On the other hand, upon phagocytosis of spores, macrophages fail to form mature phagosomes. Cytokine production from immune cells differs following exposure to yeast versus spores (which germinate into hyphae). Thus, the morphogenic transition can be targeted as an efficient treatment option against Mucor infection. In addition, genetic analysis (including gene disruption and mutational studies) further strengthens the understanding of calcineurin and provides a foundation to develop antifungal agents targeting calcineurin to deploy against Mucor and other pathogenic fungi.

Figure 1

Phenotypes of cnaBΔ mutants. (A) The cnaBΔ mutants are even more hypersensitive to CsA compared to cnaAΔ mutants, which are themselves CsA hypersensitive compared to wild-type (Lee et al., 2013). In contrast, unlike cnaAΔ mutants, the cnaBΔ mutants are not hypersensitive to FK506 or SDS. Four different cnaBΔ mutants (from left to right: MSL22, MSL23, MSL24, and MSL25) were tested and the isolate MSL22 (cnaBΔ1) was independently isolated compared to the others (cnaBΔ2). Each strain was incubated for two days at room temperature on YPD media or YPD media supplemented with CsA (100 μg/ml), and SDS (0.02%). For YPD media supplemented with FK506 (0.1 μg/ml), strains were grown for three days. The R7B strain served as the wild-type control. Note that the cnaAΔ mutants grow slower than wild-type. (B) cnaBΔ mutants grow exclusively as yeast in liquid culture, whereas wild-type and cnaAΔ mutants are able to produce hyphae in the presence of CsA. (C) In virulence tests in wax moth larva, two independently derived cnaBΔ mutants (MSL22 and MSL23) were fully virulent and were not significantly different from wild-type strain R7B (WT vs. MSL22, p=0.47761; WT vs. MSL23, p=0.3458). Each larva (N=10 per strain) was infected with 1 × 10⁴ spores.

Figure 2

Homology modeling of calcineurin complex (CnaA-CnbR-FK506-FKBP12) of M. circinelloides and effect of amino acid substitutions on FK506 susceptibility. Violet is for CnbR; cyan for CnaA; orange for FKBP12; pink for FK506. (A) The calcineurin complex with the catalytic A subunit (CnaA), regulatory B subunit (CnbR), and FKBP12-FK506 was built based on the human calcineurin ternary complex X-ray crystal structure (Protein Data Bank ID: 1TCO). The FKBP12-FK506 complex binds to the interface of the CnaA and CnbR subunits. Interestingly amino acid substitutions in the spontaneous FK506-resistant mutants occurred in residues of the CnaA or CnbR subunits that are predicted to lie in the binding pocket between FKBP12-FK506 and the two calcineurin subunits. (B) The presence of a larger aromatic group in Tyr at the 125^th position in the CnbR-1 mutant (N125Y) is predicted to cause steric clashes with residues F47 and Q48 of FKBP12. An additional amino acid after N129 of CnbR in the CnbR-2 mutant (N129_QinsH) likely extends the latch loop (Fox et al., 2001), resulting in steric clashes with the 40’s loop of FKBP12. The V122F substitution in the CnbR-3 mutant results in the addition of an aromatic group at the 122^nd residue of CnbR, predicted to cause a strong steric clash disrupting the interaction due to the closer distance between residue F122 and FK506 (~0.5 A). (C) The serine substitution of threonine in the CnaA-1 mutant (S378T) introduces a methyl group at the interface that is predicted to result in a steric clash between L118 of CnbR and the FKBP12-FK506 complex. The substitution of asparagine to aspartate in the CnaA-2 (N370D) mutant replaces the nitrogen group with an oxygen and the reversed charge at this position may introduce a repulsive effect between CnaA and FKBP12. The tryptophan at the 377^th residue may participate in a hydrophobic interaction between FK506 and CnaA and the W377L substitution in the CnaA-3 mutant could result in a weaker interaction caused by disruption of the hydrophobic pocket at the interface.

Figure 3

Generation of FK506 resistant mutants by site-directed mutagenesis of the catalytic A subunit genes. (A) An asparagine as the 370^th residue of CnaA is likely one of the crucial amino acids in the interaction between the catalytic A subunit and FKBP12 during formation of the inhibited ternary calcineurin complex. N370 is conserved in the two other catalytic A subunits CnaB (N369) and CnaC (N342). Mutagenic oligomers to generate the N to D substitution at the corresponding residues of the CnaA, CnaB, and CnaC subunits were designed. (B) The CNAA^N370D and CNAB^N369D mutants obtained exhibited resistance to FK506 (hyphal growth), whereas the wild-type was sensitive to FK506 (yeast growth). CNAC^N342D mutants were not isolated from this experiment.

Figure 4

In vitro synergistic drug sensitivity of calcineurin mutants. (A) The cnbRΔ mutants lacking the calcineurin B regulatory subunit were found to be more sensitive to Ambisome, mycamine, and posaconazole compared to wild-type. The MICs of Ambisome for wild-type and the cnbRΔ mutants do not differ; however, the MIC of posaconazole for wild-type was higher than that of the cnbRΔ mutants. Interestingly, wild-type was able to grow regardless of the presence of mycamine (up to 512 μg/ml), however, the MIC of mycamine for the cnbRΔ mutants is significantly lower. (B) In cnaAΔ mutants lacking the CnaA catalytic subunit, addition of FK506 decreased the MIC’s for wild-type and cnaAΔ, in which the cnaAΔ mutants are not more sensitive to Ambisome compared to wild-type. However, the cnaAΔ mutants displayed a much lower MIC for mycamine in combination with FK506, compared to wild-type. The highest concentration of mycamine tested did not inhibit the growth of wild-type or the cnaAΔ mutants without the addition of FK506. (C) cnaBΔ mutants also exhibited higher sensitivity to Ambisome and mycamine in the presence of a low amount of FK506 as observed in the cnaAΔ mutants. The MIC of Ambisome was 0.5 or 0.125 (μg/ml) in the presence of FK506 (0.01 or 0.075 μg/ml, respectively) and the MIC of mycamine was 64 (μg/ml) in the presence of FK506 (0.075 μg/ml). However, unlike the cnaAΔ mutants, the sensitivity of the cnaBΔ mutants to mycamine was not higher than wild-type.

Figure 5

Phagosomal maturation occurs in murine macrophage cell line J774.1 and bone marrow-derived primary murine macrophage cells containing Mucor yeast but not spores. (A) The murine macrophages were co-cultured with spores or yeast (cnbRΔ yeast-locked mutant or wild-type yeast) and in tests of cathepsin B activity in the phagosomes, red fluorescent signals surrounding the yeast cells (cnbRΔ yeast or wild-type yeast) were observed after 45 minute incubation. However, phagosomes in macrophages containing live Mucor spores did not display red fluorescent signals. The red fluorescent signals are only emitted when cathepsin B is activated, by which its substrate MR-(RR)2 is cleaved. The activation of cathepsin B results from phagosomal acidification. MR: magic red signal for active cathepsin B; H: Hoechst 33342; DIC: differential interference contract. (B) Bone marrow-derived murine macrophages containing cnbRΔ yeast-locked mutant cells underwent phagosome maturation; however, macrophages containing spores did not undergo phagosome maturation, which is similar to the observation in J774.1 macrophage cell lines. (C) Phagosomes containing UV-inactivated spores exhibit red fluorescent signals surrounding spores, indicating phagosome maturation occurred. yeast cells. Scale = 10 μm. (D) Comparison of matured or nonmatured phagosomes in J774.1 macrophage cells containing yeast or spores. Based on monitoring cathesin B activity, ~90% of yeast-containing phagosomes (151 out of 168) underwent maturation; whereas, upon challenge with spores of two different M. circinelloides isolates (R7B and NRRL3631), a majority of the phagosomes [~94% (98 out of 104) or ~95% (54 out of 57), respectively] did not undergo maturation. When stained with LysoTracker Red DND-99, ~86% (119 out of 139) of phagosomes containing yeast were acidified, representing phagosome maturation; in contrast, ~80% (61 out of 76) of phagosomes containing spores of the R7B strain did not undergo maturation.

Figure 6

Cell wall structures of different growth stages of Mucor imaged via transmission electron microscopies (TEM). The spores, hyphae, and yeast (wild-type and cnbRΔ) have cell walls with an electron dense outer layer. However, interestingly, the wild-type yeast generated in anaerobic conditions have thicker outer layers compared to the cnbRΔ mutant yeast. Scale = 0.5 μm for the rectangular insert or 1 μm for the larger image.

Figure 7

Differential cytokine induction by spores/hyphae and yeast. (A) The amounts of the angiogenesis growth factor FGF-2 produced by seven LCLs co-cultured with wild-type, yeast, and the other pathogenic fungi for 24 hrs are plotted. The mean and SEM for the seven LCLs is in red, while the mean from two experiments for each individual LCL is represented by each black dot. An increased amount of FGF-2 was observed when co-cultured with Mucor spores/hyphae; however, the yeast locked cnbRΔ mutant did not induce production of FGF-2 (p=0.04 by paired t-test). Other pathogenic fungi, including A. fumigatus (Af), R. delemar (Rd), and C. albicans (Ca), also induced FGF-2 from the LCLs, suggesting that production of FGF-2 is a host-response to hyphal growth by pathogenic fungi. (B) FGF-2 was also induced in BMMs co-cultured with Mucor spores/hyphae (p=0.02 by t-test) but not with the yeast locked cnbRΔ mutant (p=0.22). Data for each condition are from at least 7 biological replicates recorded over two independent experiments. (C and D) the proinflammatory cytokines MIP1α and IL-8 were induced from the THP-1 human monocyte cells by Mucor spores/hyphae but not by yeast. Data are from 4 biological replicates recorded over two independent experiments. (D) IL-8 induction in the LCL 18524 cell line is lower with Mucor spores/hyphae but the suppression of IL-8 production is not observed when the 18524 cells were co-cultured with the yeast-locked mutant. Data are from 4 biological replicates recorded over two independent experiments.

Figure 8

Host immune cell death is induced by Mucor. (A) J774.1 macrophage cells containing Mucor spores and those not containing spores (bystanders) both underwent cell death (Upper). Uninfected bystander bone marrow-derived murine macrophage cells also underwent cell death (Bottom). Scale=10 μm. (B) Flow cytometry to detect apoptotic cells with Annexin V was conducted. Total dead cells were also stained with 7-AAD. As a positive control cells were treated with staurosporine (STS) to induce apoptosis. Both macrophage cells after STS or Mucor spores treatment exhibited a similar staining pattern, indicating that cell death occurred via apoptosis.

Figure 9

Roles of calcineurin in the dimorphism, virulence, and host-pathogen interactions. Calcineurin plays a central role in the dimorphic transition, in which the inhibitions of calcineurin by FK506 or mutations enforce Mucor to grow exclusively as yeast. cnaAΔ mutants are sensitive to SDS, indicating that calcineurin is involved in cell wall integrity. Spore size is related to the virulence of Mucor: larger spores are more virulent than smaller spores. The cnaAΔ mutants produce larger spores than wild-type, demonstrating that calcineurin regulates Mucor spore size. Upon phagocytosis, macrophages undergo phagosome maturation with yeast Mucor but not with spores, suggesting that the yeast-hypha dimorphism results in different host-pathogen interaction.