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...

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SummaryCalcineurin is a Ca 21 ±calmodulin-regulated protein phosphatase that is the target of the immunosuppressive drugs cyclosporin A and FK506. Calcineurin is a heterodimer composed of a catalytic A and a regulatory B subunit. In previous studies, the calcineurin A homologue was identified and shown to be required for growth at 378C and hence for virulence of the pathogenic fungus Cryptococcus neoformans.Here, we identify the gene encoding the calcineurin B regulatory subunit and demonstrate that calcineurin B is also required for growth at elevated temperature and virulence. We show that the FKR1-1 mutation, which confers dominant FK506 resistance, results from a 6 bp duplication generating a two-amino-acid insertion in the latch region of calcineurin B. This mutation was found to reduce FKBP12±FK506 binding to calcineurin both in vivo and in vitro. Molecular modelling based on the FKBP12±FK506±calcineurin crystal structure illustrates how this mutation perturbs drug interactions with the phosphatase target. In summary, our studies reveal a central role for calcineurin B in virulence and antifungal drug action in the human fungal pathogen C. neoformans.

Cryptococcus neoformans is a fungal pathogen that causes meningitis in patients immunocompromised by AIDS, chemotherapy, organ transplantation, or high-dose steroids. Current antifungal drug therapies are limited and suffer from toxic side effects and drug resistance. Here, we defined the targets and mechanisms of antifungal action of the immunosuppressant rapamycin in C. neoformans. In the yeast Saccharomyces cerevisiae and in T cells, rapamycin forms complexes with the FKBP12 prolyl isomerase that block cell cycle progression by inhibiting the TOR kinases. We identified the gene encoding a C. neoformans TOR1 homolog. Using a novel two-hybrid screen for rapamycin-dependent TOR-binding proteins, we identified the C. neoformans FKBP12 homolog, encoded by the FRR1 gene. Disruption of the FKBP12 gene conferred rapamycin and FK506 resistance but had no effect on growth, differentiation, or virulence of C. neoformans. Two spontaneous mutations that confer rapamycin resistance alter conserved residues on TOR1 or FKBP12 that are required for FKBP12-rapamycin-TOR1 interactions or FKBP12 stability. Two other spontaneous mutations result from insertion of novel DNA sequences into the FKBP12 gene. Our observations reveal that the antifungal activities of rapamycin and FK506 are mediated via FKBP12 and TOR homologs and that a high proportion of spontaneous mutants in C. neoformans result from insertion of novel DNA sequences, and they suggest that nonimmunosuppressive rapamycin analogs have potential as antifungal agents.Cryptococcus neoformans is an opportunistic fungal pathogen that causes systemic mycosis in immunocompromised patients (20,45). Cryptococcosis in patients with AIDS is characterized by a poor response to therapy and a risk of recurrent disease requiring lifelong suppressive antifungal regimens. Current treatments include amphotericin B, flucytosine, and fluconazole. However, treatment failures and toxicity are common, and new antifungal agents are needed.C. neoformans is sensitive to the immunosuppressants rapamycin, FK506, and cyclosporine (CsA) (48, 49). These compounds are natural products with antifungal activity and are also potent immunosuppressants used to treat graft rejection in transplant recipients (7,38). Rapamycin is a macrolide originally discovered in a screen for antimicrobial activity against Candida albicans and later found to have potent immunosuppressive activity (62).Rapamycin, FK506, and CsA suppress the immune system by inhibiting signal transduction steps required for T-cell activation (for reviews, see references 19 and 59). The mechanisms of action of these compounds have been studied in lymphocytes and Sacharomyces cerevisiae (for reviews, see references 11, 18, 32, and 59). These hydrophobic compounds diffuse into the cell and bind intracellular receptors known as immunophilins. The immunophilins are ubiquitous and conserved from yeast to humans. FK506 and rapamycin bind the immunophilin FKBP12, whereas CsA binds cyclophilin A. Although the cyclophilin and FKBP proteins have no s...

Cryptococcus neoformans is a fungal pathogen that causes meningitis in immunocompromised patients. Its growth is sensitive to the immunosuppressants FK506 and cyclosporin, which inhibit the Ca 2+ ± calmodulin-activated protein phosphatase calcineurin. Calcineurin is required for growth at 37°C and virulence of C.neoformans. We found that calcineurin is also required for mating. FK506 blocks mating of C.neoformans via FKBP12-dependent inhibition of calcineurin, and mutants lacking calcineurin are bilaterally sterile. Calcineurin is not essential for the initial fusion event, but is required for hyphal elongation and survival of the heterokaryon produced by cell fusion. It is also required for hyphal elongation in diploid strains and during asexual haploid fruiting of MATa cells in response to nitrogen limitation. Because mating and haploid fruiting produce infectious basidiospores, our studies suggest a second link between calcineurin and virulence of C.neoformans. Calcineurin regulates ®lamentation and 37°C growth via distinct pathways. Together with studies revealing that calcineurin mediates neurite extension and neutrophil migration in mammals, our ®ndings indicate that calcineurin plays a conserved role in the control of cell morphology.

Candida albicans and Cryptococcus neoformans cause both superficial and disseminated infections in humans.Current antifungal therapies for deep-seated infections are limited to amphotericin B, flucytosine, and azoles. A limitation is that commonly used azoles are fungistatic in vitro and in vivo. Our studies address the mechanisms of antifungal activity of the immunosuppressive drug rapamycin (sirolimus) and its analogs with decreased immunosuppressive activity. C. albicans rbp1/rbp1 mutant strains lacking a homolog of the FK506-rapamycin target protein FKBP12 were found to be viable and resistant to rapamycin and its analogs. Rapamycin and analogs promoted FKBP12 binding to the wild-type Tor1 kinase but not to a rapamycin-resistant Tor1 mutant kinase (S1972R). FKBP12 and TOR mutations conferred resistance to rapamycin and its analogs in C. albicans, C. neoformans, and Saccharomyces cerevisiae. Our findings demonstrate the antifungal activity of rapamycin and rapamycin analogs is mediated via conserved complexes with FKBP12 and Tor kinase homologs in divergent yeasts. Taken together with our observations that rapamycin and its analogs are fungicidal and that spontaneous drug resistance occurs at a low rate, these mechanistic findings support continued investigation of rapamycin analogs as novel antifungal agents.Cryptococcus neoformans and Candida albicans are two common opportunistic fungal pathogens. Current antifungal agents in clinical use include amphotericin B, fluconazole, and flucytosine (29), which have side effects, lack fungicidal activity, or lack activity against emerging resistant mutants (66). Thus, additional antifungal agents are needed.Several new antifungal agents are in development. The candins are 1,3-␤-glucan synthase inhibitors and are potently active against Candida species and Aspergillus fumigatus (23,40,51,52). The pneumocandin caspofungin acetate-MK-0991 is in phase III clinical trials for candida infections and has been approved for refractory aspergillosis. Broad-spectrum triazoles, including voriconazole, posaconazole, and ravuconazole, are being studied in human trials (50). Combination therapy with different antifungal agents may also improve therapy (20,28,62).Rapamycin (sirolimus) is a natural product of the bacterium Streptomyces hygroscopicus originally discovered in a screen for antimicrobial activity against C. albicans and later found to have potent immunosuppressive activity (4, 63). Rapamycin diffuses into the cell and associates with the peptidylprolyl isomerase FKBP12. Rapamycin inhibits FKBP12 enzymatic activity; however, this inhibition is not related to immunosuppressive or antifungal activity. The targets of the FKBP12-rapamycin complex are the TOR kinases. Two TOR proteins, Tor1 and Tor2, have been characterized in the yeasts S. cerevisiae and Schizosaccharomyces pombe, and a single TOR homolog has been identified in C. albicans, C. neoformans, Drosophila melanogaster, and humans (10,18,33,39,49,56,64). The Tor kinase has been functionally conserved from yeast to ...

Cryptococcus neoformans is an opportunistic fungal pathogen that causes life-threatening infections of the central nervous system. Existing therapies include amphotericin B, fluconazole, and flucytosine, which are limited by toxic side effects and the emergence of drug resistance. We recently demonstrated that the protein phosphatase calcineurin is required for growth at 37°C and virulence of C. neoformans. Because calcineurin is the target of potent inhibitors in widespread clinical use, cyclosporine and FK506 (tacrolimus), it is an attractive drug target for novel antifungal agents. Here we have explored the synergistic potential of combining the calcineurin inhibitor FK506 or its nonimmunosuppressive analog, L-685,818, with other antifungal agents and examined the molecular basis of FK506 action by using genetically engineered fungal strains that lack the FK506 target proteins FKBP12 and calcineurin. We demonstrate that FK506 exhibits marked synergistic activity with the H ؉ ATPase inhibitor bafilomycin A 1 via a novel action distinct from calcineurin loss of function. FK506 also exhibits synergistic activity with the pneumocandin MK-0991/caspofungin acetate (formerly L-743,873), which targets the essential ␤-1,3 glucan synthase, and in this case, FK506 action is mediated via FKBP12-dependent inhibition of calcineurin. Finally, we demonstrate that FK506 and fluconazole have synergistic activity that is independent of both FKBP12 and calcineurin and may involve the known ability of FK506 to inhibit multidrug resistance pumps, which are known to export azoles from fungal cells. In summary, our studies illustrate the potential for synergistic activity of a variety of different drug combinations and the power of molecular genetics to define the mechanisms of drug action, as well as identify a novel action of FK506 that could have profound implications for therapeutic or toxic effects in other organisms, including humans.Cryptococcus neoformans is a common cause of life-threatening opportunistic infections in immunocompromised hosts, especially in patients infected with the human immunodeficiency virus and recipients of organ transplants (5, 29). Although azoles and amphotericin B are currently acceptable therapies for patients with cryptococcal meningitis, the success of these treatments remains suboptimal. New antifungal agents are needed for the effective management of this pathogenic fungus. Evaluation of the synergistic activity of new antifungal compounds through in vitro susceptibility testing can help to establish guidelines for the potential clinical application of new therapies.Virulence factors that have been defined for C. neoformans and that could be targeted for therapeutic intervention include production of a polysaccharide capsule, synthesis of melanin, prototrophy, and ability to grow at 37°C (13,24,42,47). We recently demonstrated that the protein phosphatase calcineurin is required for C. neoformans growth at 37°C, and as a consequence, mutant strains lacking calcineurin are avirulent in animal ...

The calcineurin gene was cloned and disrupted in serotype D strains of Cryptococcus neoformans. Serotype A and serotype D calcineurin mutants were inviable at 37°C and avirulent in mice, whereas only serotype A mutants were cation stress sensitive. Thus, calcineurin plays conserved and divergent roles in serotype A and serotype D strains.Cryptococcus neoformans is an encapsulated basidiomycete that is the most common cause of systemic mycosis in AIDS patients. C. neoformans strains are classified into five serotypes (A, B, C, D, and AD) and two varieties: C. neoformans var. neoformans (serotypes A, D, and AD) and C. neoformans var. gattii (serotypes B and C). Serotype A and serotype D strains exhibit significant variation and may represent distinct varieties that have diverged in ϳ18 million years of evolution (8,9,16,19,25). C. neoformans virulence factors include the capsule (3-5), melanin (22), prototrophy (17), and growth at 37°C (14). The protein phosphatase calcineurin is required for C. neoformans growth at 37°C and virulence (14).Calcineurin is a Ca 2ϩ -calmodulin-activated phosphatase with catalytic and regulatory subunits (10). Calcineurin is the target of the T-cell-specific immunosuppressants cyclosporine (CsA) and tacrolimus (FK506). CsA and FK506 suppress the immune system by inhibiting calcineurin and preventing gene expression during T-cell activation. The antifungal activities of CsA and FK506 are mediated by a similar mechanism involving fungal homologs of calcineurin and the drug-binding proteins cyclophilin A and FKBP12 (1,2,6,7,14,15).Calcineurin has been identified from several fungi and regulates cell cycle progression in Aspergillus nidulans (21), hyphal elongation and growth in Neurospora crassa (11,20), and mating and cytokinesis in Schizosaccharomyces pombe (18,28). In Saccharomyces cerevisiae, calcineurin is required for recovery from pheromone arrest (12, 27) and regulates cation homeostasis and cell wall biosynthesis via the transcription factor Crz1 (1,13,23,24).The calcineurin gene has been identified, sequenced, and disrupted by homologous recombination in C. neoformans serotype A strain H99 (14). Calcineurin is essential for growth at 37°C, virulence in a rabbit model of cryptococcal meningitis, and cation homeostasis (14). Here, we isolated and disrupted the gene encoding the calcineurin A catalytic subunit (CNA1) from the congenic serotype D strains of C. neoformans and compared the functions of calcineurin in serotype A and serotype D strains.Identification, sequence, and disruption of the serotype D calcineurin A CNA1 gene. The CNA1 gene encoding calcineurin A was cloned from the serotype D strain JEC21 by PCR amplification with primers to conserved sequences in the serotype A CNA1 gene. A 1.8-kb CNA1 gene fragment was sequenced, revealing identity to calcineurin genes, and used in Southern blot analysis to show that the CNA1 gene is contained on an 8-kb EagI fragment. The gene was recovered from a size-selected EagI genomic library and sequenced. There were seven amino acid...