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article addendum Autophagic Punctum Virulence 1:6, 1-4; November/December 2010; © 2010 Landes Bioscience This manuscript has been published online, prior to printing. Once the issue is complete and page numbers have been assigned, the citation will change accordingly. Beyond ergosterol Linking pH to antifungal mechanisms Yong-Qiang Zhang and Rajini Rao* Department of Physiology; The Johns Hopkins University School of Medicine; Baltimore, MD USA M any antifungal drugs including the highly successful azoles target the fungal-specific sterol, ergosterol, yet the molecular identity of cellular pathways mediating antifungal activity remained obscure. A recent study on the requirement of ergosterol in vacuolar H+-ATPase (V-ATPase) function uncovered a critical role for ion homeostasis downstream of azole inhibition of ergosterol biosynthesis. Here we review the functional link between regulation of V-ATPase by fungal membrane lipid components and discuss contributions of V-ATPase function to pathogenicity. The importance of pH homeostasis in virulence highlights its potential as target in antifungal chemotherapy. Key words: pH homeostasis, V-ATPase, ergosterol, vacuole, Candida albicans, yeast Abbreviations: abbreviations Submitted: 09/07/10 Accepted: 09/29/10 Previously published online: www.landesbioscience.com/journals/ virulence/article/13802 Correspondence to: Rajini Rao; Email: [email protected] Addendum to: Zhang YQ, Gamarra S, GarciaEffron G, Park S, Perlin DS, Rao R. Requirement for ergosterol in V-ATPase function underlies antifungal activity of azole drugs. PLoS Pathog 2010; 6:e1000939. www.landesbioscience.com Sterols are major lipid components in membranes. Besides plasma membrane, where the bulk of sterols reside, sterols have been found in intracellular organelles including peroxisomes, mitochondria, vacuoles/lysosome, ER and trans-Golgi network. The presence of sterols in membranes modulates their thickness, fluidity and permeability. Their preferential association with sphingolipids to form ‘raft’ like domains confers membrane heterogeneity and facilitates functions of raftassociated proteins. Fungal cells contain a unique sterol, ergosterol. Deletion of genes responsible for early steps in the ergosterol biosynthetic pathway is lethal, demonstrating that ergosterol plays an essential role in fungal cells. In the battle against pathogenic fungi, ergosterol provides the most common and effective target for several classes of antimycotics, including azoles, allylamines, morpholines and polyenes.1-3 Yet despite this importance, the specific cellular pathways impacted by ergosterol in mediating cytotoxicity of antifungal agents remain obscure. Recently, we uncovered a plausible cellular mechanism for azole toxicity that links ergosterol to pH homeostasis.4 The pleiotropic phenotypes of erg mutants defective in the late steps of ergosterol biosynthesis offer insight into specific cellular processes modulated by sterols. Thus, erg mutants are defective in plasma membrane fusion and pheromone signaling,5 in lipid raft association of beta1,3-glucanosyltransferase Gas1p,6 and in plasma membrane targeting of tryptophan permease Tat2p and the marker protein Fus1-Mid2-GFP.7,8 Ergosterol is also important for endocytosis as demonstrated by defects seen in erg2Δerg6Δ and erg3Δerg6Δ double mutants.9 These clearly demonstrate that sterols are critical for plasma membrane biogenesis and function. In addition, erg mutants exhibit defects in vacuolar fusion10 and in mitochondrial morphogenesis and maintenance11 pointing to a critical role in organelle biogenesis. Some erg mutants are also associated with ion-related phenotypes. For example, growth of erg24Δ is dependent on external Ca 2+ and is enhanced by Mg2+ 12 and erg6Δ shows enhanced uptake of Li+ and Na+ ions.13 The mechanistic basis for many of these phenotypes, nevertheless, is poorly understood. Clearly our understanding of the biochemical and cellular role of ergosterol is still limited. We initially observed a striking overlap between multiple erg phenotypes and those associated with vma mutants, lacking the vacuolar H+ pumping ATPase. Thus, in a Virulence1 Figure 1. Ergosterol removal alters V-ATPase conformation. Vacuolar vesicles were isolated from log phase cells of S. cerevisiae strain BY4742 as described previously4 and incubated on ice with or without methylbetacyclodextrin for 40 minutes. Vesicles were then spun down and endogenous proteolysis was analyzed by immunoblotting against V-ATPase subunits Vph1 and Vma2. genome-wide screen of hypersensitivity to amiodarone, an antifungal agent known to disrupt cellular Ca 2+ and H+ homeostasis, we recovered multiple vma and erg mutants.14 A closer examination revealed that erg mutants as exemplified by erg24Δ (lacking C-14 sterol reductase), resembled vma mutants in hypersensitivity to Zn2+ toxicity, inhibitors of the Ca 2+ -activated phosphatase calcineurin (FK506) and a cell wall disrupting agent (calcofluor white). More significantly, erg mutants exhibited the classical vma phenotype: an inability to grow in alkaline medium, consistent with an inability to acidify the vacuole. Measurement of vacuole pH in situ confirmed that erg mutants failed to acidify vacuoles, essentially indistinguishable from vma mutants lacking a functional H+ pump. Finally, purified vacuoles showed severe impairment of H+ pumping and ATP hydrolysis rates in erg24Δ. Taken together, these findings indicated a critical requirement for ergosterol in V-ATPase function.4 Three classes of antifungals (azoles, morpholines and allylamines) target enzymes catalyzing early reactions in the 2 ergosterol biosynthesis pathway. The most widely used antifungal fluconazole targets the lanosterol demethylase Erg11p, immediately upstream from Erg24p. We found that fluconazole treatment of S. cerevisiae or Candida albicans phenocopied deletion of downstream ERG genes in eliciting vacuolar alkalinization and inhibition of V-ATPase. Strikingly, feeding S. cerevisiae with exogenous ergosterol restored both fungal growth and vacuolar acidification in fluconazole treated cells. Several lines of evidence suggest direct modulation of V-ATPase by ergosterol. First, ergosterol depletion in erg24Δ or by fluconazole treatment (20 μg/ml, 6 h) did not alter V-ATPase expression or its localization on vacuolar membrane.4 Second, ergosterol depletion did not dissociate V1 and Vo domains, a mechanism by which glucose rapidly regulates V-ATPase activity. Upon acute removal of ergosterol from purified vacuolar vesicles by methyl-betacyclodextrin, both V1 and V0 domains remained membrane-associated yet V-ATPase activity was lost. Interestingly, V-ATPase subunits exhibited distinct proteolytic patterns following ergosterol extraction, including enhanced degradation of specific fragments and appearance of novel peptide bands (Fig. 1). This indicates that V-ATPase adopts a different conformation in the absence of ergosterol that may correlate with loss of activity. Membrane protein function is tightly coupled to membrane lipid composition. Indeed, association of V-ATPase with cholesterol has been documented in mammalian cells15,16 and incorporation of cholesterol into membranes stimulated V-ATPase activity in osteoclasts.17 Phosphatidylinositol phosphates, sphingolipids and cardiolipin have also been reported to regulate V-ATPase function, albeit by distinct mechanisms. In renal epithelial cells, alteration in phosphatidylinositol phosphate composition affected V-ATPase assembly and trafficking.18 Cardiolipin is located in mitochondrial membrane and its effect on V-ATPase suggests functional cross-talk between vacuoles and mitochondria.19 The role of sphingolipids in yeast V-ATPase regulation has been characterized in detail. It was shown that depletion of sphingolipids with C26 acyl group disrupted Virulence structural integrity of V1–Vo assembly and disabled V1 domain function.20 This is in contrast to our findings that ergosterol depletion did not impair V-ATPase assembly or domain integrity, although it may have altered protein conformation. Thus, although intimately associated in membranes, ergosterol and sphingolipids have distinct effects on protein function. Ergosterol has rigid planar rings whereas sphingolipids display flexible long hydrocarbon chains. This structural difference may dictate their preferential interaction with distinct protein motifs, thereby modulating protein function by different mechanisms. The importance of the V-ATPase in fungal physiology and virulence cannot be underestimated. The V-ATPase maintains acidic pH within the vacuole to activate lysosomal enzymes for protein processing and degradation, and generates a proton motive force across endomembranes for the transport, sequestration and detoxification of metabolites, ions and drugs.21,22 Besides the vacuole, V-ATPase is broadly distributed in the endomembrane system, including the Golgi, secretory vesicles, early and late endosomes23 where its function is critical for endocytosis, vesicle trafficking, uncoupling of ligand-receptor complexes, and pH-driven exocytosis. Impairment of V-ATPase function, thus, could affect surface expression and secretion of proteins such as superoxide dismutase (Sod5p) and lipases, which play important roles in facilitating infection.24,25 In addition, Ca 2+ rich, alkaline serum conditions of the mammalian host present a hostile environment for fungal cells. Defective pH and Ca 2+ -homeostasis would render fungal pathogens susceptible to detrimental ion fluxes encountered in the host. Therefore, disruption of V-ATPase activity downstream from antifungal drug inhibition of ergosterol biosynthesis should cripple multiple cellular functions required for pathogenesis. Indeed, both erg24 -/- and vma7-/- null mutants of C. albicans were shown to be avirulent in mouse models of Candidiasis.26,27 Thus, while ergosterol depletion may well impact additional cellular targets that remain to be elucidated at a molecular level, loss of V-ATPase function is sufficient to account for loss of virulence. Volume 1 Issue 6 Another critical consequence of disabling V-ATPase is inhibition of filamentous growth, as was observed for the vma7-/- strain of C. albicans in the presence of serum or liquid Spider medium (with mannitol).26 Fluconazole treatment and ERG3 deletion have both been shown to inhibit filamentation,28,29 which can now be explained, at least in part, due to impaired V-ATPase function. The ability to transition from yeast to hyphae is a critical virulence factor for polymorphic pathogenic fungi, such as C. albicans.30 To infect their hosts, pathogenic fungi first have to penetrate host epithelial barriers. Once in the bloodstream, fungal cells must survive the attack by macrophages and dendritic cells and subvert innate immune system. A recent report shows that C. albicans cells invade oral and gastrointestinal tractepithelia in hyphal form.31 Another study shows that wild-type C. albicans cells are more capable than a filamentation-defective mutant (efg1Δ/cph1Δ) in blocking phagosomal maturation and acidification.32 Consistent with these findings, we observe that vma7-/- mutants of C. albicans fail to colonize Caco-2 epithelial models in culture and are efficiently eliminated by murine J774A.1 macrophage cells (Fig. 2). Given the significance of filamentation in virulence, the molecular basis underlying the requirement for V-ATPase function in filamentation is a critical area in the study of fungal pathogenesis. The requirement of ergosterol for V-ATPase function suggests that combining azole drugs with other agents that can trigger harmful ion fluxes would have synergistic antifungal effect. The antiarrhythmia drug amiodarone elicits a surge of cytosolic H+ and Ca 2+ that triggers ion stress, cell cycle arrest and fungal death,33,34 and exhibits synergism with azoles in vitro.14,35,36 We showed that pretreatment with fluconazole exacerbated amiodarone induced H+ and Ca 2+ surges in S. cerevisiae and impaired vacuole function in C. albicans.4 Consistent with increased ion stress from the drug combination, we observed lysis of C. albicans cells in vitro (Fig. 3) and significant reductions in microbial kidney burden in a murine Candidiasis.35 Maintaining different organelles at distinct pH is critical for multiple cellular events in all eukaryotic cells. The Figure 2. C. albicans vma7-/- mutant cells are eliminated by macrophages and fail to colonize epithelial cells. Murine macrophage cells (J774A.1) and human colorectal epithelial c ells (Caco-2) were grown in DMEM with 10% fetal bovine serum to confluence (37ºC, 5% CO2). Log phase C. albicans WT (CNC44) and vma7Δ/vma7Δ cells (VHU4)26 were inoculated into mammalian cultures at multiplicity of infection of 1/1000 and incubated for 20 hours. Note the inability of vma-/- strains to form hyphae and colonize host cells. Figure 3. Combination of fluconazole and amiodarone induces lysis of C. albicans cells. Early log phase cells of C. albicans SC5314 were grown in YPD or YPD plus 3.3 μg/ml fluconazole for 6 hours at 30ºC. Cells were collected and incubated in YPD plus 5% fetal bovine serum with or without amiodarone (10 μM; AMD) and fluconazole (3.3 μg/ml; Fluc) for 2 hours at 37ºC. Fewer cells and increased lysis can be observed in the presence of both amiodarone and fluconazole. www.landesbioscience.comVirulence 3 importance of pH homeostasis in fungal pathogenicity is illustrated by the avirulence of V-ATPase null mutants. Future studies of other proteins involved in ion homeostasis will provide in-depth understanding of the role of intracellular ion traffic in specific aspects of pathogenicity, such as secretion of virulence factors, filamentation, biofilm formation, subverting host innate immunity, and adaptation of parasitic life styles. Meanwhile, the success of azole drugs and the realization of their effect on V-ATPase function demonstrate the effectiveness of targeting pH homeostasis in fungal pathogens. The therapeutic potential of combining agents that weaken maintenance of pH homeostasis and those disrupt ion fluxes remain to be further explored in the future. References 1. Hitchcock CA, Dickinson K, Brown SB, Evans EG, Adams DJ. Interaction of azole antifungal antibiotics with cytochrome P-450-dependent 14 alphasterol demethylase purified from Candida albicans. Biochem J 1990; 266:475-80. 2. Ryder NS. Effect of allylamine antimycotic agents on fungal sterol biosynthesis measured by sterol sidechain methylation. J Gen Microbiol 1985; 131:1595602. 3. Marcireau C, Guilloton M, Karst F. In vivo effects of fenpropimorph on the yeast Saccharomyces cerevisiae and determination of the molecular basis of the antifungal property. Antimicrob Agents Chemother 1990; 34:989-93. 4. Zhang YQ, Gamarra S, Garcia-Effron G, Park S, Perlin DS, Rao R. Requirement for ergosterol in V-ATPase function underlies antifungal activity of azole drugs. PLoS Pathog 2010; 6:e1000939. 5. Jin H, McCaffery JM, Grote E. Ergosterol promotes pheromone signaling and plasma membrane fusion in mating yeast. J Cell Biol 2008; 180:813-26. 6. Bagnat M, Keranen S, Shevchenko A, Simons K. Lipid rafts function in biosynthetic delivery of proteins to the cell surface in yeast. Proc Natl Acad Sci USA 2000; 97:3254-9. 7. Umebayashi K, Nakano A. Ergosterol is required for targeting of tryptophan permease to the yeast plasma membrane. J Cell Biol 2003; 161:1117-31. 8. Proszynski TJ, Klemm RW, Gravert M, Hsu PP, Gloor Y, Wagner J, et al. A genomewide visual screen reveals a role for sphingolipids and ergosterol in cell surface delivery in yeast. Proc Natl Acad Sci USA 2005; 102:17981-6. 4 9. Heese-Peck A, Pichler H, Zanolari B, Watanabe R, Daum G, Riezman H. Multiple functions of sterols in yeast endocytosis. Mol Biol Cell 2002; 13:2664-80. 10.Kato M, Wickner W. Ergosterol is required for the Sec18/ATP-dependent priming step of homotypic vacuole fusion. EMBO J 2001; 20:4035-40. 11. Altmann K, Westermann B. Role of essential genes in mitochondrial morphogenesis in Saccharomyces cerevisiae. Mol Biol Cell 2005; 16:5410-7. 12. Crowley JH, Tove S, Parks LW. A calcium-dependent ergosterol mutant of Saccharomyces cerevisiae. Curr Genet 1998; 34:93-9. 13.Welihinda AA, Beavis AD, Trumbly RJ. Mutations in LIS1 (ERG6) gene confer increased sodium and lithium uptake in Saccharomyces cerevisiae. Biochim Biophys Acta 1994; 1193:107-17. 14. Gupta SS, Ton VK, Beaudry V, Rulli S, Cunningham K, Rao R. Antifungal activity of amiodarone is mediated by disruption of calcium homeostasis. J Biol Chem 2003; 278:28831-9. 15. Lafourcade C, Sobo K, Kieffer-Jaquinod S, Garin J, van der Goot FG. Regulation of the V-ATPase along the endocytic pathway occurs through reversible subunit association and membrane localization. PLoS One 2008; 3:e2758. 16. Yoshinaka K, Kumanogoh H, Nakamura S, Maekawa S. Identification of VATPase as a major component in the raft fraction prepared from the synaptic plasma membrane and the synaptic vesicle of rat brain. Neurosci Lett 2004; 363:168-72. 17. Ryu J, Kim H, Chang EJ, Kim HJ, Lee Y, Kim HH. Proteomic analysis of osteoclast lipid rafts: the role of the integrity of lipid rafts on V-ATPase activity in osteoclasts. J Bone Miner Metab 2010; 28:410-7. 18.Sautin YY, Lu M, Gaugler A, Zhang L, Gluck SL. Phosphatidylinositol 3-kinase-mediated effects of glucose on vacuolar H+-ATPase assembly, translocation, and acidification of intracellular compartments in renal epithelial cells. Mol Cell Biol 2005; 25:575-89. 19.Chen S, Tarsio M, Kane PM, Greenberg ML. Cardiolipin mediates crosstalk between mitochondria and the vacuole. Mol Biol Cell 2008; 19:5047-58. 20.Chung JH, Lester RL, Dickson RC. Sphingolipid requirement for generation of a functional v1 component of the vacuolar ATPase. J Biol Chem 2003; 278:28872-81. 21.Kane PM. The where, when, and how of organelle acidification by the yeast vacuolar H+-ATPase. Microbiol Mol Biol Rev 2006; 70:177-91. 22.Nishi T, Forgac M. The vacuolar (H+)-ATPases-nature’s most versatile proton pumps. Nat Rev Mol Cell Biol 2002; 3:94-103. 23. Gkantiragas I, Brugger B, Stuven E, Kaloyanova D, Li XY, Lohr K, et al. Sphingomyelin-enriched microdomains at the Golgi complex. Mol Biol Cell 2001; 12:1819-33. 24. Frohner IE, Bourgeois C, Yatsyk K, Majer O, Kuchler K. Candida albicans cell surface superoxide dismutases degrade host-derived reactive oxygen species to escape innate immune surveillance. Mol Microbiol 2009; 71:240-52. Virulence 25.Paraje MG, Correa SG, Renna MS, Theumer M, Sotomayor CE. Candida albicans-secreted lipase induces injury and steatosis in immune and parenchymal cells. Can J Microbiol 2008; 54:647-59. 26.Poltermann S, Nguyen M, Gunther J, Wendland J, Hartl A, Kunkel W, et al. The putative vacuolar ATPase subunit Vma7p of Candida albicans is involved in vacuole acidification, hyphal development and virulence. Microbiology 2005; 151:1645-55. 27. Jia N, Arthington-Skaggs B, Lee W, Pierson CA, Lees ND, Eckstein J, et al. Candida albicans sterol C-14 reductase, encoded by the ERG24 gene, as a potential antifungal target site. Antimicrob Agents Chemother 2002; 46:947-57. 28.Chau AS, Gurnani M, Hawkinson R, Laverdiere M, Cacciapuoti A, McNicholas PM. Inactivation of sterol Delta5,6-desaturase attenuates virulence in Candida albicans. Antimicrob Agents Chemother 2005; 49:3646-51. 29. Ha KC, White TC. Effects of azole antifungal drugs on the transition from yeast cells to hyphae in susceptible and resistant isolates of the pathogenic yeast Candida albicans. Antimicrob Agents Chemother 1999; 43:763-8. 30.Bastidas RJ, Heitman J. Trimorphic stepping stones pave the way to fungal virulence. Proc Natl Acad Sci USA 2009; 106:351-2. 31.Dalle F, Wachtler B, L’Ollivier C, Holland G, Bannert N, Wilson D, et al. Cellular interactions of Candida albicans with human oral epithelial cells and enterocytes. Cell Microbiol 2010; 12:248-71. 32. Fernandez-Arenas E, Bleck CK, Nombela C, Gil C, Griffiths G, Diez-Orejas R. Candida albicans actively modulates intracellular membrane trafficking in mouse macrophage phagosomes. Cell Microbiol 2009; 11:560-89. 33. Maresova L, Muend S, Zhang YQ, Sychrova H, Rao R. Membrane hyperpolarization drives cation influx and fungicidal activity of amiodarone. J Biol Chem 2009; 284:2795-802. 34.Muend S, Rao R. Fungicidal activity of amiodarone is tightly coupled to calcium influx. FEMS Yeast Res 2008; 8:425-31. 35. Gamarra S, Rocha EM, Zhang YQ, Park S, Rao R, Perlin DS. Mechanism of the Synergistic Effect of Amiodarone and Fluconazole in Candida albicans. Antimicrob Agents Chemother 2010; In press. 36. Guo Q, Sun S, Yu J, Li Y, Cao L. Synergistic activity of azoles with amiodarone against clinically resistant Candida albicans tested by chequerboard and timekill methods. J Med Microbiol 2008; 57:457-62. Volume 1 Issue 6