PKC Signaling Regulates Drug Resistance of the Fungal Pathogen Candida albicans via Circuitry Comprised of Mkc1, Calcineurin,and Hsp90

Fungal pathogens exploit diverse mechanisms to survive exposure to antifungal drugs. This poses concern given the limited number of clinically useful antifungals and the growing population of immunocompromised individuals vulnerable to life-threatening fungal infection. To identify molecules that abrogate resistance to the most widely deployed class of antifungals, the azoles, we conducted a screen of 1,280 pharmacologically active compounds. Three out of seven hits that abolished azole resistance of a resistant mutant of the model yeast Saccharomyces cerevisiae and a clinical isolate of the leading human fungal pathogen Candida albicans were inhibitors of protein kinase C (PKC), which regulates cell wall integrity during growth, morphogenesis, and response to cell wall stress. Pharmacological or genetic impairment of Pkc1 conferred hypersensitivity to multiple drugs that target synthesis of the key cell membrane sterol ergosterol, including azoles, allylamines, and morpholines. Pkc1 enabled survival of cell membrane stress at least in part via the mitogen activated protein kinase (MAPK) cascade in both species, though through distinct downstream effectors. Strikingly, inhibition of Pkc1 phenocopied inhibition of the molecular chaperone Hsp90 or its client protein calcineurin. PKC signaling was required for calcineurin activation in response to drug exposure in S. cerevisiae. In contrast, Pkc1 and calcineurin independently regulate drug resistance via a common target in C. albicans. We identified an additional level of regulatory control in the C. albicans circuitry linking PKC signaling, Hsp90, and calcineurin as genetic reduction of Hsp90 led to depletion of the terminal MAPK, Mkc1. Deletion of C. albicans PKC1 rendered fungistatic ergosterol biosynthesis inhibitors fungicidal and attenuated virulence in a murine model of systemic candidiasis. This work establishes a new role for PKC signaling in drug resistance, novel circuitry through which Hsp90 regulates drug resistance, and that targeting stress response signaling provides a promising strategy for treating life-threatening fungal infections.     

The yeast mRNA-binding protein Cth2 post-transcriptionally modulates ergosterol biosynthesis in response to iron deficiency

Sterol synthesis is an iron-dependent metabolic pathway in eukaryotes. Consequently, fungal ergosterol biosynthesis (ERG) is down-regulated in response to iron deficiency. In this report, we show that, upon iron limitation or overexpression of the iron-regulated mRNA-binding protein Cth2, the yeast Saccharomyces cerevisiae down-regulates the three initial enzymatic steps of ergosterol synthesis (ERG1, ERG7 and ERG11). Mechanistically, we show that Cth2 protein limits the translation and promotes the decrease in the mRNA levels of these specific ERG genes, which contain consensus Cth2-binding sites defined as AU-rich elements (AREs). Thus, expression of CTH2 leads to the accumulation of initial sterol intermediates, such as squalene, and to the drop of ergosterol levels. Changes in CTH2 expression levels disturb the response of yeast cells to stresses related to membrane integrity such as high ethanol and sorbitol concentrations. Therefore, CTH2 should be considered as a critical regulatory factor of ergosterol biosynthesis during iron deficiency.     

Molecular basis of resistance to azole antifungals

The increased incidence of invasive mycoses and the emerging problem of antifungal drug resistance has prompted investigations of the underlying molecular mechanisms, particularly for the azole compounds central to current therapy. The target site for the azoles is the ERG11 gene product, the cytochrome P450 lanosterol 14alpha-demethylase, which is part of the ergosterol biosynthetic pathway. The resulting ergosterol depletion renders fungal cells vulnerable to further membrane damage. Development of azole resistance in fungi may occur through increased levels of the cellular target, upregulation of genes controlling drug efflux, alterations in sterol synthesis and decreased affinity of azoles for the cellular target. Here, we review the adaptative changes in fungi, in particular Candida albicans, in response to inhibitors of ergosterol biosynthesis. The molecular mechanisms of azole resistance might help in devising more effective antifungal therapies.     

牛源近平滑念珠菌对氟康唑耐药性研究

以牛源近平滑念珠菌（Candida parapsilosis）为试验菌株,采用微量稀释法进行药物敏感性试验,PCR扩增测序检测ERG11基因突变,Realtime PCR检测ERG11、CDR1、MDR1、MRR1基因的mRNA表达量,探讨耐药相关基因在牛源近平滑念珠菌耐唑类药物中的作用,为牛源近平滑念珠菌的耐药研究提供参考。结果表明,近平滑念珠菌对5-氟胞嘧啶、两性霉素Ｂ的敏感率均高于75%,对唑类药物的耐药率均高于50%,其中对氟康唑的耐药率最高,达58.3%;所有菌株的ERG11基因中均检测出错义突变A395T,耐氟康唑和剂量依赖菌株的ERG11基因中检测出同义突变T591C;氟康唑耐药组ERG11、CDR1、 MDR1、MRR1基因表达水平均显著高于敏感组（P<0.05）。牛源近平滑念珠菌对唑类抗真菌药物的耐药率较高且具有多重耐药性。牛源近平滑念珠菌ERG11基因中的T591C突变以及ERG11、CDR1、MDR1、MRR1基因的高表达都可能在其对氟康唑耐药性的产生中起到一定的作用。     

Ergosterol promotes pheromone signaling and plasma membrane fusion in mating yeast

Ergosterol depletion independently inhibits two aspects of yeast mating: pheromone signaling and plasma membrane fusion. In signaling, ergosterol participates in the recruitment of Ste5 to a polarized site on the plasma membrane. Ergosterol is thought to form microdomains within the membrane by interacting with the long acyl chains of sphingolipids. We find that although sphingolipid-free ergosterol is concentrated at sites of cell-cell contact, transmission of the pheromone signal at contact sites depends on a balanced ratio of ergosterol to sphingolipids. If a mating pair forms between ergosterol-depleted cells despite the attenuated pheromone response, the subsequent process of membrane fusion is retarded. Prm1 also participates in membrane fusion. However, ergosterol and Prm1 have independent functions and only prm1 mutant mating pairs are susceptible to contact-dependent lysis. In contrast to signaling, plasma membrane fusion is relatively insensitive to sphingolipid depletion. Thus, the sphingolipid-free pool of ergosterol promotes plasma membrane fusion.     

Hsp90 Governs Echinocandin Resistance in the Pathogenic Yeast Candida albicans via Calcineurin

Candida albicans is the leading fungal pathogen of humans, causing life-threatening disease in immunocompromised individuals. Treatment of candidiasis is hampered by the limited number of antifungal drugs whose efficacy is compromised by host toxicity, fungistatic activity, and the emergence of drug resistance. We previously established that the molecular chaperone Hsp90, which regulates the form and function of diverse client proteins, potentiates resistance to the azoles in C. albicans and in the model yeast Saccharomyces cerevisiae. Genetic studies in S. cerevisiae revealed that Hsp90''s role in azole resistance is to enable crucial cellular responses to the membrane stress exerted by azoles via the client protein calcineurin. Here, we demonstrate that Hsp90 governs cellular circuitry required for resistance to the only new class of antifungals to reach the clinic in decades, the echinocandins, which inhibit biosynthesis of a critical component of the fungal cell wall. Pharmacological or genetic impairment of Hsp90 function reduced tolerance of C. albicans laboratory strains and resistance of clinical isolates to the echinocandins and created a fungicidal combination. Compromising calcineurin function phenocopied compromising Hsp90 function. We established that calcineurin is an Hsp90 client protein in C. albicans: reciprocal co-immunoprecipitation validated physical interaction; Hsp90 inhibition blocked calcineurin activation; and calcineurin levels were depleted upon genetic reduction of Hsp90. The downstream effector of calcineurin, Crz1, played a partial role in mediating calcineurin-dependent stress responses activated by echinocandins. Hsp90''s role in echinocandin resistance has therapeutic potential given that genetic compromise of C. albicans HSP90 expression enhanced the efficacy of an echinocandin in a murine model of disseminated candidiasis. Our results identify the first Hsp90 client protein in C. albicans, establish an entirely new role for Hsp90 in mediating resistance to echinocandins, and demonstrate that targeting Hsp90 provides a promising therapeutic strategy for the treatment of life-threatening fungal disease.     

The genetic basis of fluconazole resistance development in Candida albicans

Infections by the opportunistic fungal pathogen Candida albicans are widely treated with the antifungal agent fluconazole that inhibits the biosynthesis of ergosterol, the major sterol in the fungal plasma membrane. The emergence of fluconazole-resistant C. albicans strains is a significant problem after long-term treatment of recurrent oropharyngeal candidiasis (OPC) in acquired immunodeficiency syndrome (AIDS) patients. Resistance can be caused by alterations in sterol biosynthesis, by mutations in the drug target enzyme, sterol 14alpha-demethylase (14DM), which lower its affinity for fluconazole, by increased expression of the ERG11 gene encoding 14DM, or by overexpression of genes coding for membrane transport proteins of the ABC transporter (CDR1/CDR2) or the major facilitator (MDR1) superfamilies. Different mechanisms are frequently combined to result in a stepwise development of fluconazole resistance over time. The MDR1 gene is not or barely transcribed during growth in vitro in fluconazole-susceptible C. albicans strains, but overexpressed in many fluconazole-resistant clinical isolates, resulting in reduced intracellular fluconazole accumulation. The activation of the gene in resistant isolates is caused by mutations in as yet unknown trans-regulatory factors, and the resulting constitutive high level of MDR1 expression causes resistance to other toxic compounds in addition to fluconazole. Disruption of both alleles of the MDR1 gene in resistant C. albicans isolates abolishes their resistance to these drugs, providing genetic evidence that MDR1 mediates multidrug resistance in C. albicans.     

Calcineurin is essential for survival during membrane stress in Candida albicans

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.     

Mutual co-regulation between GPI-N-acetylglucosaminyltransferase and ergosterol biosynthesis in Candida albicans

A novel co-regulation exists between the first step of GPI (glycosylphosphatidylinositol) anchor biosynthesis and the rate-determining step of ergosterol biosynthesis in Candida albicans. Depleting CaGpi19p, an accessory subunit of the enzyme complex that initiates GPI biosynthesis, down-regulates ERG11, altering ergosterol levels and drug response. This effect is specific to CaGpi19p depletion and is not due to cell wall defects or GPI deficiency. Additionally, down-regulation of ERG11 down-regulates CaGPI19 and GPI biosynthesis.     

Arv1 lipid transporter function is conserved between pathogenic and nonpathogenic fungi

The lipid transporter Arv1 regulates sterol trafficking, and glycosylphosphatidylinositol and sphingolipid biosyntheses in Saccharomyces cerevisiae. ScArv1 contains an Arv1 homology domain (AHD) that is conserved at the amino acid level in the pathogenic fungal species, Candida albicans and Candida glabrata. Here we show S. cerevisiae cells lacking Arv1 are highly susceptible to antifungal drugs. In the presence of drug, Scarv1 cells are unable to induce ERG gene expression, have an altered pleiotrophic drug response, and are defective in multi-drug resistance efflux pump expression. All phenotypes are remediated by ectopic expression of CaARV1 or CgARV1. The AHDs of these pathogenic fungi are required for specific drug tolerance, demonstrating conservation of function. In order to understand how Arv1 regulates antifungal susceptibility, we examined sterol trafficking. CaARV1/CgARV1 expression suppressed the sterol trafficking defect of Scarv1 cells. Finally, we show that C. albicansarv1/arv1 cells are avirulent using a BALB/c disseminated mouse model. We suggest that overall cell survival in response to antifungal treatment requires the lipid transporter function of Arv1.