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¯uconazole 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¯uconazole. A mutation in the calcineurin B regulatory subunit that confers dominant FK506 resistance (CNB1-1/CNB1) abolished FK506±¯uconazole synergism. Candida albicans mutants lacking calcineurin B (cnb1/cnb1) were found to be viable and markedly hypersensitive to¯u-conazole or membrane perturbation with SDS. FK506 was synergistic with¯uconazole 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. Keywords: calcineurin/Candida albicans/cyclosporin A/ uconazole/antifungal drugs IntroductionThe immunosuppressants cyclosporin A (CsA) and FK506 block rejection of transplanted organs by inhibiting signaling pathways required for T-cell activation (Schreiber and Crabtree, 1992). Both drugs are in widespread clinical use and have had a dramatic impact on transplant therapy. Interestingly, CsA and FK506 are natural products of soil-dwelling bacteria or fungi (reviewed in Cardenas et al., 1994). Both drugs are toxic to a variety of fungi, suggesting one natural role might be to inhibit competing microorganisms in the soil (Tropschug et al., 1989;Breuder et al., 1994;Odom et al., 1997a;Arndt et al., 1999). CsA and FK506 exert immunosuppressive and antifungal effects by inhibiting calcineurin (Liu et al., 1991;Foor et al., 1992;Nakamura et al., 1993;Breuder et al., 1994;Odom et al., 1997a;Fox et al., 2001), a conserved Ca 2+ -calmodulin activated protein phosphatase (reviewed in Klee et al., 1998;Hemenway and Heitman, 1999;Aramburu et al., 2000). Calcineurin is a heterodimer comprised of a catalytic A and a regulatory B subunit (Hubbard and Klee, 1989;Anglister et al., 1994;Watanabe et al., 1995). CsA and FK506 do not inhibit calcineurin on their own, but ®rst bind to small, abundant, conserved binding proteins (immunophilins). CsA associates with cyclophilin A, and FK506 with FKBP12, to form protein±drug complexes that inhibit calcineurin by binding to the interface between the A and B subunits (Haddy et al., 1992;Li and Handschumacher, 1993;Milan et al., 1994;Cardenas et al., 1995b;Grif®th et al., 1995;Kawamura and Su, 1995;Kissinger et al., 1995). CsA and FK506 target this unique region of calcineurin and do not inhibit other phosphatases.The mechanisms of action of CsA and FK506 are conserved from fungi to h...

Direct effects of non-antifungal agents used in cancer chemotherapy and organ transplantation on the development and virulence ofCandidaandAspergillusspecies

“…62,71,72 These in vitro results are supported by gene deletion studies showing that calcineurin-defective C. albicans mutants were more sensitive to stress conditions than isogenic wild-type controls, were markedly hypersensitive to azoles, and had attenuated virulence in mice with systemic candidiasis. [73][74][75] Although that hypovirulence is attributed to sensitivity of C. albicans to killing by serum mediated by Ca 2+ , no reported data have indicated that pharmacological inhibition of calcineurin also protects against calcium stress. Interestingly, loss of function crz1 C. albicans mutants, defective in crz1 which encoded a downstream transcription effector of the calcineurin, Crz1, were not sensitive to serum and exhibited no decrease in virulence.…”

“…This interaction extends to the other azoles as well as terbinafine and was observed in both susceptible C. albicans and azole-resistant species, including C. glabrata. [71][72][73] Checkerboard microdilution and time-kill experiments found that at a concentration of 0.625 μg/ ml (i.e., at a concentration achievable in vivo), CsA was synergistic with fluconazole, reducing viable Candida counts by 2 logs. 77 Importantly, testing of Candida isolates obtained from liver transplant recipients receiving tacrolimus showed that treatment with this agent did not select for resistance toward calcineurin inhibitors.…”

Enhancement effect of N-methyl-N″-dodecylguanidine on the vacuole-targeting fungicidal activity of amphotericin B against the pathogenic fungus Candida albicans

“…Cells were grown in YPD medium containing 1% yeast extract (Difco Laboratories, Detroit, MI, USA), 2% Bacto-peptone (Difco Laboratories) and 2% glucose at 30 1C with vigorous shaking. Unless stated otherwise, cells from the overnight-grown culture were collected by centrifugation, washed twice with RPMI 1640 medium with L-glutamine (RPMI 1640 medium), 18 and suspended in the same medium to obtain a final cell density of 1Â10 7 cells ml À1 . Cells were then incubated in the absence or presence of each compound with vigorous shaking at 37 1C.…”

CaIPF14030 negatively modulates intracellular ATP levels during the development of azole resistance in Candida albicans

“…CaIPF14030 is overexpressed during the stepwise acquisition of azole resistance in DSY1024 It has been well documented that calcium can activate the calcineurin pathway and modulate azole activity in C albicans [22,23] . Because the CaIPF14030 gene of C albicans is up-regulated by Ca 2+ in a calcineurin-dependent manner, we investigated the dynamic expression of CaIPF14030 in the DSF7, DSF14, DSF21, and DSF28 strains obtained in our previous study [18] .…”

“…CaIPF14030 was up-regulated by Ca 2+ in a calcineurin-dependent manner. It is well documented that calcium can activate the calcineurin pathway and modulate azole activity in C albicans [22,23] . In addition, the dynamic expression of CaIPF14030 was investigated in the DSF7, DSF14, DSF21, and DSF28 strains obtained in our previous study [18] .…”