Calcineurin is essential for survival during membrane stress in Candida albicans

Abstract

The immunosuppressants cyclosporin A (CsA) and FK506 inhibit the protein phosphatase calcineurin and block T-cell activation and transplant rejection. Calcineurin is conserved in microorganisms and plays a general role in stress survival. CsA and FK506 are toxic to several fungi, but the common human fungal pathogen Candida albicans is resistant. However, combination of either CsA or FK506 with the antifungal drug fluconazole that perturbs synthesis of the membrane lipid ergosterol results in potent, synergistic fungicidal activity. Here we show that the C.albicans FK506 binding protein FKBP12 homolog is required for FK506 synergistic action with fluconazole. A mutation in the calcineurin B regulatory subunit that confers dominant FK506 resistance (CNB1-1/CNB1) abolished FK506-fluconazole synergism. Candida albicans mutants lacking calcineurin B (cnb1/cnb1) were found to be viable and markedly hypersensitive to fluconazole or membrane perturbation with SDS. FK506 was synergistic with fluconazole against azole-resistant C.albicans mutants, against other Candida species, or when combined with different azoles. We propose that calcineurin is part of a membrane stress survival pathway that could be targeted for therapy.

Fig. 1. FKBP12 and calcineurin are the targets of FK506–fluconazole synergism. The RBP1/RBP1 wild-type (SC5314), rbp1/RBP1 heterozygous mutant (YAG116), rbp1/rbp1 homozygous mutant (YAG171) and CNB1-1/CNB1 heterozygous mutant strains (YAG237) were grown on YPD medium alone or with 1 µg/ml FK506, 50 µg/ml fluconazole, or 1 µg/ml FK506 and 50 µg/ml fluconazole for 72 h at 37°C.

Fig. 2. Membrane perturbations enhance uptake and toxicity of CsA and FK506 in C.albicans. (A) Fluconazole treatment increases cell uptake of [³H]CsA and [³H]FK506. Candida albicans strain SC5314 was grown overnight in YPD medium with or without fluconazole (50 µg/ml) at 30°C. [³H]CsA or [³H]FK506 uptake was measured as described in Materials and methods. The means of two independent experiments (three assays in total) were plotted as the fold of the base line concentration of [³H]CsA or [³H]FK506 that was taken up by C.albicans cells growing without fluconazole. (B) Five-fold serial dilutions of C.albicans wild-type (CAI4), erg6/erg6 homozygous mutant (KPC8), erg6/ERG6 heterozygous mutant (KPC1) and erg11/ERG11 heterozygous mutant (4A) strains were spotted onto YPD solid medium with or without CsA (100 µg/ml), FK506 (1 µg/ml) or fluconazole (50 µg/ml) and incubated at 37°C for 48 h.

Fig. 3. Disruption of the C.albicans calcineurin B genes. (A) Disruption alleles of the CNB1 gene. One CNB1 wild-type allele was replaced by transformation and homologous integration with the UAU1 cassette in strains DAY343 (CNB1/cnb1 #1) and DAY345 (CNB1/cnb1 #2). ‘U’ indicates the ura3 portions and ‘A’ the ARG4 portions of the UAU1 cassette. Arg⁺ Ura⁺ isolates containing the homozygous cnb1/cnb1 mutation [strains DAY364 (cnb1/cnb1 #1) and DAY365 (cnb1/cnb1 #2)] were identified from each heterozygous strain. (B) PCR verification of the CNB1 gene deletions. Genomic DNA from the wild-type CNB1/CNB1 strain BWP17, the cnb1/CNB1 heterozygous strains DAY343 and DAY345, and the cnb1/cnb1 strains DAY364 and DAY365 was PCR amplified with primers PR146/147 to the CNB1 gene (515 bp product) or primers to the RBP1 gene (375 bp product) as a control. (C) Southern blot analysis of CNB1 alleles. Genomic DNA from the wild-type CNB1/CNB1 strain BWP17, the cnb1/CNB1 heterozygous strains DAY343 and DAY345 and the cnb1/cnb1 homozygous strains DAY364 and DAY365 was cleaved with EcoRV (E) and analyzed by Southern blotting with probes to the URA3 or CNB1 gene. The URA3 probe hybridized to the CNB1 disruption allele in strains DAY343 and DAY345 (∼6.2 kb) and to cnb1 disruption alleles in strain DAY364 and DAY365 (∼9.0, 6.2 and 3.6 kb). An interchromosomal recombination event between the proximal portion of the URA3 gene in one allele and the distal portion of the URA3 gene in the other yielded one cnb1Δ::URA3 allele and one larger ∼9 kb tandem cnb1Δ::ura3-ARG4-ura3-ARG4-ura3 duplication in strain DAY364. Positions of DNA markers are shown on the left in kb. P1 and P2 indicate probes used to detect the CNB1 and URA3 genes.

Fig. 4. Candida albicans calcineurin mutant strains are viable and fluconazole hypersensitive. The wild-type (BWP17), cnb1/CNB1 heterozygous (DAY343, DAY345) and cnb1/cnb1 homozygous (DAY364, DAY365) mutants, and the cnb1/cnb1 + CNB1 complemented strain MCC85 were grown on YPD solid medium with or without CsA (100 µg/ml), FK506 (10 µg/ml) and fluconazole (50 µg/ml), and incubated at 37°C for 72 h.

Fig. 5. Calcineurin protects C.albicans from membrane stress exerted by other azoles or SDS. (A) Disks were spotted with H₂O (0), 5 µg fluconazole (F), 160 µg ketoconazole (K), 16 µg itraconazole (I), 80 ng posaconazole (P), or 60 ng voriconazole (V) and placed on the surface of YPD solid medium containing ∼1 × 10⁶ cells of C.albicans in top agar with no drug, 100 µg/ml cyclosporin A, or 1 µg/ml FK506. Strains were wild-type (SC5314), the cnb1/cnb1 mutant strain (DAY364), or the cnb1/cnb1 + CNB1 complemented strain (MCC85). Cells were incubated at 37°C for 3 days and photographed. (B) Disks containing water (0), 2 or 5 μg of FK506 or 100 or 250 μg of CsA were placed on YPD medium without (–) or with 0.01% SDS and containing ∼1 × 10⁶ cells of C.albicans in top agar. Strains were wild-type (SC5314), the cnb1/cnb1 mutant strain (DAY364), or the cnb1/cnb1 + CNB1 complemented strain (MCC85). Cells were incubated at 37°C for 3 days and photographed.

Fig. 6. ERG6 gene induction in response to azoles is calcineurin independent. Transcription of the ERG6 gene was assayed in wild-type (SC5314) and cnb1/cnb1 mutant cells (DAY364) by northern blotting. Liquid cultures of cells were harvested after 8 h of either (A) no treatment, (B) 10 µg/ml fluconazole, or (C) 10 µg/ml fluconazole + 1 µg/ml FK506.

Fig. 7. Some azole resistant strains are sensitive to FK506 + fluconazole. Wild-type strain SC5314, rbp1/rbp1 mutant strain YAG171 and six representative C.albicans isolates, 12–17, from a series of increasingly fluconazole-resistant isolates from an HIV patient were plated on YPD medium alone or containing 10 µg/ml FK506, 50 µg/ml fluconazole, or both 10 µg/ml FK506 and 50 µg/ml fluconazole. Previously described changes in the expression pattern of ERG11 mRNA and CDR1/2 mRNAs are labeled.

Fig. 8. FK506–fluconazole synergism in other fungi. (A) Wild-type and an rbp1/rbp1 mutant C.albicans and isolates of C.glabrata, C.krusei and C.tropicalis strains were grown on YPD medium alone (–), or with 100 µg/ml CsA, 10 µg/ml FK506, 10 µg/ml fluconazole (Flu), 100 µg/ml CsA + 10 µg/ml fluconazole, or 10 µg/ml FK506 + 10 µg/ml fluconazole for 48 h at 37°C. (B) Cells (∼1 × 10⁶) of the C.glabrata and C.krusei strains used in (A) were plated in top agar on YPD rich medium alone (–), or containing 100 µg/ml CsA or 10 µg/ml FK506. Discs containing 5 µg fluconazole, 160 ng ketoconazole, 16 µg itraconazole, 80 ng posaconazole, 60 ng voriconazole or water were placed on the top agar and the cells were incubated for 48 h at 37°C and photographed.