The Antimicrobial Peptide Histatin-5 Causes a Spatially Restricted Disruption on the Candida albicans Surface, Allowing Rapid Entry of the Peptide into the Cytoplasm
Antimicrobial peptides play an important role in host defense against microbial pathogens. Their high cationic charge and strong amphipathic structure allow them to bind to the anionic microbial cell membrane and disrupt the membrane bilayer by forming pores or channels. In contrast to the classical pore-forming peptides, studies on histatin-5 (Hst-5) have suggested that the peptide is transported into the cytoplasm of Candida albicans in a non-lytic manner, and cytoplasmic Hst-5 exerts its candicidal activities on various intracellular targets, consistent with its weak amphipathic structure. To understand how Hst-5 is internalized, we investigated the localization of FITC-conjugated Hst-5. We find that Hst-5 is internalized into the vacuole through receptor-mediated endocytosis at low extracellular Hst-5 concentrations, whereas under higher physiological concentrations, Hst-5 is translocated into the cytoplasm through a mechanism that requires a high cationic charge on Hst-5. At intermediate concentrations, two cell populations with distinct Hst-5 localizations were observed. By cell sorting, we show that cells with vacuolar localization of Hst-5 survived, while none of the cells with cytoplasmic Hst-5 formed colonies. Surprisingly, extracellular Hst-5, upon cell surface binding, induces a perturbation on the cell surface, as visualized by an immediate and rapid internalization of Hst-5 and propidium iodide or rhodamine B into the cytoplasm from the site using time-lapse microscopy, and a concurrent rapid expansion of the vacuole. Thus, the formation of a spatially restricted site in the plasma membrane causes the initial injury to C. albicans and offers a mechanism for its internalization into the cytoplasm. Our study suggests that, unlike classical channel-forming antimicrobial peptides, action of Hst-5 requires an energized membrane and causes localized disruptions on the plasma membrane of the yeast. This mechanism of cell membrane disruption may provide species-specific killing with minimal damage to microflora and the host and may be used by many other antimicrobial peptides.
References
[1]
Kwon-Chung KJ, Bennett JE (1992) Medical mycology. Philadelphia: Lea & Febiger.
[2]
vanderSpek JC, Wyandt HE, Skare JC, Milunsky A, Oppenheim FG, et al. (1989) Localization of the genes for histatins to human chromosome 4q13 and tissue distribution of the mRNAs. Am J Hum Genet 45: 381–387.
[3]
Raj PA, Edgerton M, Levine MJ (1990) Salivary histatin 5: dependence of sequence, chain length, and helical conformation for candidacidal activity. J Biol Chem 265: 3898–3905.
[4]
Xu T, Levitz SM, Diamond RD, Oppenheim FG (1991) Anticandidal activity of major human salivary histatins. Infect Immun 59: 2549–2554.
[5]
Raj PA, Marcus E, Sukumaran DK (1998) Structure of human salivary histatin 5 in aqueous and nonaqueous solutions. Biopolymers 45: 51–67.
[6]
Helmerhorst EJ, van't Hoff W, Breeuwer P, Veerman EC, Abee T, et al. (2001) Characterization of histatin 5 with respect to amphipathicity, hydrophobicity, and effects on cell and mitochondrial membrane integrity excludes a candidacidal mechanism of pore formation. J Biol Chem 276: 5643–5649.
[7]
Oren Z, Shai Y (1998) Mode of action of linear amphipathic alpha-helical antimicrobial peptides. Biopolymers 47: 451–463.
[8]
Lear JD, Wasserman ZR, DeGrado WF (1988) Synthetic amphiphilic peptide models for protein ion channels. Science 240: 1177–1181.
[9]
Den Hertog AL, Wong Fong Sang HW, Kraayenhof R, Bolscher JG, Van't Hof W, et al. (2004) Interactions of histatin 5 and histatin 5-derived peptides with liposome membranes: surface effects, translocation and permeabilization. Biochem J 379: 665–672.
[10]
Helmerhorst EJ, Breeuwer P, van't Hoff W, Walgreen-Weterings E, Oomen LC, et al. (1999) The cellular target of histatin 5 on Candida albicans is the energized mitochondrion. J Biol Chem 274: 7286–7291.
[11]
Helmerhorst EJ, Troxler RF, Oppenheim FG (2001) The human salivary peptide histatin 5 exerts its antifungal activity through the formation of reactive oxygen species. Proc Natl Acad Sci U S A 98: 14637–14642.
[12]
Koshlukova SE, Lloyd TL, Araujo MW, Edgerton M (1999) Salivary histatin 5 induces non-lytic release of ATP from Candida albicans leading to cell death. J Biol Chem 274: 18872–18879.
[13]
Xu Y, Ambudkar I, Yamagishi H, Swaim W, Walsh TJ, et al. (1999) Histatin 3-mediated killing of Candida albicans: effect of extracellular salt concentration on binding and internalization. Antimicrob Agents Chemother 43: 2256–2262.
[14]
Baev D, Li XS, Dong J, Keng P, Edgerton M (2002) Human salivary histatin 5 causes disordered volume regulation and cell cycle arrest in Candida albicans. Infect Immun 70: 4777–4784.
[15]
Vylkova S, Jang WS, Li W, Nayyar N, Edgerton M (2007) Histatin 5 initiates osmotic stress response in Candida albicans via activation of the Hog1 mitogen-activated protein kinase pathway. Eukaryot Cell 6: 1876–1888.
[16]
Ruissen AL, Groenink J, Helmerhorst EJ, Walgreen-Weterings E, Van't Hof W, et al. (2001) Effects of histatin 5 and derived peptides on Candida albicans. Biochem J 356: 361–368.
[17]
Veerman EC, Valentijn-Benz M, Nazmi K, Ruissen AL, Walgreen-Weterings E, et al. (2007) Energy depletion protects Candida albicans against antimicrobial peptides by rigidifying its cell membrane. J Biol Chem 282: 18831–18841.
[18]
Gyurko C, Lendenmann U, Troxler RF, Oppenheim FG (2000) Candida albicans mutants deficient in respiration are resistant to the small cationic salivary antimicrobial peptide histatin 5. Antimicrob Agents Chemother 44: 348–354.
[19]
Raj PA, Soni SD, Levine MJ (1994) Membrane-induced helical conformation of an active candidacidal fragment of salivary histatins. J Biol Chem 269: 9610–9619.
[20]
Li XS, Reddy MS, Baev D, Edgerton M (2003) Candida albicans Ssa1/2p is the cell envelope binding protein for human salivary histatin 5. J Biol Chem 278: 28553–28561.
[21]
Riezman H, Munn A, Geli MI, Hicke L (1996) Actin-, myosin- and ubiquitin-dependent endocytosis. Experientia 52: 1033–1041.
[22]
Geli MI, Riezman H (1998) Endocytic internalization in yeast and animal cells: similar and different. J Cell Sci 111(Pt 8): 1031–1037.
[23]
Mukhopadhyay D, Riezman H (2007) Proteasome-independent functions of ubiquitin in endocytosis and signaling. Science 315: 201–205.
[24]
Duchardt F, Fotin-Mleczek M, Schwarz H, Fischer R, Brock R (2007) A comprehensive model for the cellular uptake of cationic cell-penetrating peptides. Traffic 8: 848–866.
[25]
Rajarao GK, Nekhotiaeva N, Good L (2002) Peptide-mediated delivery of green fluorescent protein into yeasts and bacteria. FEMS Microbiol Lett 215: 267–272.
[26]
Vylkova S, Sun JN, Edgerton M (2007) The role of released ATP in killing Candida albicans and other extracellular microbial pathogens by cationic peptides. Purinergic Signal 3: 91–97.
[27]
Gyurko C, Lendenmann U, Helmerhorst EJ, Troxler RF, Oppenheim FG (2001) Killing of Candida albicans by histatin 5: cellular uptake and energy requirement. Antonie Van Leeuwenhoek 79: 297–309.
[28]
den Hertog AL, van Marle J, van Veen HA, Van't Hof W, Bolscher JG, et al. (2005) Candidacidal effects of two antimicrobial peptides: histatin 5 causes small membrane defects, but LL-37 causes massive disruption of the cell membrane. Biochem J 388: 689–695.
[29]
Staub O, Rotin D (2006) Role of ubiquitylation in cellular membrane transport. Physiol Rev 86: 669–707.
[30]
Gill DJ, Teo H, Sun J, Perisic O, Veprintsev DB, et al. (2007) Structural insight into the ESCRT-I/-II link and its role in MVB trafficking. Embo J 26: 600–612.
[31]
Odorizzi G, Katzmann DJ, Babst M, Audhya A, Emr SD (2003) Bro1 is an endosome-associated protein that functions in the MVB pathway in Saccharomyces cerevisiae. J Cell Sci 116: 1893–1903.
[32]
Geli MI, Riezman H (1996) Role of type I myosins in receptor-mediated endocytosis in yeast. Science 272: 533–535.
[33]
Oberholzer U, Nantel A, Berman J, Whiteway M (2006) Transcript profiles of Candida albicans cortical actin patch mutants reflect their cellular defects: contribution of the Hog1p and Mkc1p signaling pathways. Eukaryot Cell 5: 1252–1265.
[34]
Yeaman MR, Yount NY (2003) Mechanisms of antimicrobial peptide action and resistance. Pharmacol Rev 55: 27–55.
[35]
Rothstein DM, Spacciapoli P, Tran LT, Xu T, Roberts FD, et al. (2001) Anticandida activity is retained in P-113, a 12-amino-acid fragment of histatin 5. Antimicrob Agents Chemother 45: 1367–1373.
[36]
Helmerhorst EJ, Van't Hof W, Veerman EC, Simoons-Smit I, Nieuw Amerongen AV (1997) Synthetic histatin analogues with broad-spectrum antimicrobial activity. Biochem J 326(Pt 1): 39–45.
[37]
Tsai H, Raj PA, Bobek LA (1996) Candidacidal activity of recombinant human salivary histatin-5 and variants. Infect Immun 64: 5000–5007.
[38]
Galgut PN (2001) The relevance of pH to gingivitis and periodontitis. J Int Acad Periodontol 3: 61–67.
[39]
Smith RM, Baibakov B, Lambert NA, Vogel SS (2002) Low pH inhibits compensatory endocytosis at a step between depolarization and calcium influx. Traffic 3: 397–406.
[40]
Nikawa H, Jin C, Fukushima H, Makihira S, Hamada T (2001) Antifungal activity of histatin-5 against non-albicans Candida species. Oral Microbiol Immunol 16: 250–252.
[41]
Helmerhorst EJ, Venuleo C, Beri A, Oppenheim FG (2005) Candida glabrata is unusual with respect to its resistance to cationic antifungal proteins. Yeast 22: 705–714.
[42]
De Smet K, Reekmans R, Contreras R (2004) Role of oxidative phosphorylation in histatin 5-induced cell death in Saccharomyces cerevisiae. Biotechnol Lett 26: 1781–1785.
[43]
Isola R, Isola M, Conti G, Lantini MS, Riva A (2007) Histatin-induced alterations in Candida albicans: a microscopic and submicroscopic comparison. Microsc Res Tech 70: 607–616.
[44]
Auger P, Marquis G, Dallaire L (1979) Viability assessment by dye exclusion. A fluorescent method for fungal cells. Arch Dermatol 115: 1195–1196.
[45]
Darzynkiewicz Z, Traganos F, Staiano-Coico L, Kapuscinski J, Melamed MR (1982) Interactions of Rhodamine 123 with Living Cells Studied by Flow Cytometry. Cancer Res 42: 799–806.
[46]
Driscoll J, Duan C, Zuo Y, Xu T, Troxler R, et al. (1996) Candidacidal activity of human salivary histatin recombinant variants produced by site-directed mutagenesis. Gene 177: 29–34.
[47]
Alvarez FJ, Douglas LM, Konopka JB (2007) Sterol-rich plasma membrane domains in fungi. Eukaryot Cell 6: 755–763.
[48]
Zheng XD, Wang YM, Wang Y (2003) CaSPA2 is important for polarity establishment and maintenance in Candida albicans. Mol Microbiol 49: 1391–1405.
[49]
Koshlukova SE, Araujo MW, Baev D, Edgerton M (2000) Released ATP is an extracellular cytotoxic mediator in salivary histatin 5-induced killing of Candida albicans. Infect Immun 68: 6848–6856.
[50]
Baev D, Li X, Edgerton M (2001) Genetically engineered human salivary histatin genes are functional in Candida albicans: development of a new system for studying histatin candidacidal activity. Microbiology 147: 3323–3334.
[51]
Dathe M, Nikolenko H, Meyer J, Beyermann M, Bienert M (2001) Optimization of the antimicrobial activity of magainin peptides by modification of charge. FEBS Lett 501: 146–150.
[52]
Matsuzaki K, Sugishita K, Fujii N, Miyajima K (1995) Molecular basis for membrane selectivity of an antimicrobial peptide, magainin 2. Biochemistry 34: 3423–3429.
[53]
Yeaman MR, Bayer AS, Koo SP, Foss W, Sullam PM (1998) Platelet microbicidal proteins and neutrophil defensin disrupt the Staphylococcus aureus cytoplasmic membrane by distinct mechanisms of action. J Clin Invest 101: 178–187.
[54]
Vacata V, Kotyk A, Sigler K (1981) Membrane potentials in yeast cells measured by direct and indirect methods. Biochim Biophys Acta 643: 265–268.
[55]
Prasad R, Hofer M (1986) Tetraphenylphosphonium is an indicator of negative membrane potential in Candida albicans. Biochim Biophys Acta 861: 377–380.
[56]
Hohmann S (2002) Osmotic stress signaling and osmoadaptation in yeasts. Microbiol Mol Biol Rev 66: 300–372.
[57]
Coote PJ, Holyoak CD, Bracey D, Ferdinando DP, Pearce JA (1998) Inhibitory action of a truncated derivative of the amphibian skin peptide dermaseptin s3 on Saccharomyces cerevisiae. Antimicrob Agents Chemother 42: 2160–2170.
[58]
Petruzzelli R, Clementi ME, Marini S, Coletta M, Di Stasio E, et al. (2003) Respiratory inhibition of isolated mammalian mitochondria by salivary antifungal peptide histatin-5. Biochem Biophys Res Commun 311: 1034–1040.
[59]
Luque-Ortega JR, Van't Hof W, Veerman EC, Saugar JM, Rivas L (2008) Human antimicrobial peptide histatin 5 is a cell-penetrating peptide targeting mitochondrial ATP synthesis in Leishmania. Faseb J.
[60]
Balguerie A, Bagnat M, Bonneu M, Aigle M, Breton AM (2002) Rvs161p and sphingolipids are required for actin repolarization following salt stress. Eukaryot Cell 1: 1021–1031.
[61]
Alonso-Monge R, Navarro-Garcia F, Molero G, Diez-Orejas R, Gustin M, et al. (1999) Role of the mitogen-activated protein kinase Hog1p in morphogenesis and virulence of Candida albicans. J Bacteriol 181: 3058–3068.
[62]
Alonso-Monge R, Navarro-Garcia F, Roman E, Negredo AI, Eisman B, et al. (2003) The Hog1 mitogen-activated protein kinase is essential in the oxidative stress response and chlamydospore formation in Candida albicans. Eukaryot Cell 2: 351–361.
[63]
San Jose C, Alonso R, Perez-Diaz RM, Pla J, Nombela C (1996) The mitogen-activated protein kinase homolog HOG1 gene controls glycerol accumulation in the pathogenic fungus Candida albicans. Journal of Bacteriology 178: 5850–5852.
[64]
Reiser V, Raitt DC, Saito H (2003) Yeast osmosensor Sln1 and plant cytokinin receptor Cre1 respond to changes in turgor pressure. J Cell Biol 161: 1035–1040.
[65]
van't Hoff W, Reijnders I, Helmerhorst E, Walgreen-Weterings E, Simoons-Smit I, et al. (2000) Synergistic effects of low doses of histatin 5 and its analogues on amphotericin B anti-mycotic activity. Antonie Van Leeuwenhoek 78: 163–169.
[66]
Tanida T, Okamoto T, Ueta E, Yamamoto T, Osaki T (2006) Antimicrobial peptides enhance the candidacidal activity of antifungal drugs by promoting the efflux of ATP from Candida cells. J Antimicrob Chemother 57: 94–103.
[67]
Xu W, Smith FJ Jr, Subaran R, Mitchell AP (2004) Multivesicular body-ESCRT components function in pH response regulation in Saccharomyces cerevisiae and Candida albicans. Mol Biol Cell 15: 5528–5537.
[68]
Oberholzer U, Marcil A, Leberer E, Thomas DY, Whiteway M (2002) Myosin I is required for hypha formation in Candida albicans. Eukaryot Cell 1: 213–228.
[69]
Fidel PL Jr, Cutright JL, Tait L, Sobel JD (1996) A murine model of Candida glabrata vaginitis. J Infect Dis 173: 425–431.
[70]
Colman-Lerner A, Gordon A, Serra E, Chin T, Resnekov O, et al. (2005) Regulated cell-to-cell variation in a cell-fate decision system. Nature 437: 699–706.
