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• W2010999630 abstract "Salivary histatins are potent in vitro antifungal proteins and have promise as therapeutic agents against oral candidiasis. We performed pharmacological studies directed at understanding the biochemical basis of Hst 5 candidacidal activity. Three inhibitors of mitochondrial metabolism: carbonyl cyanidep-chlorophenylhydrazone, dinitrophenol, and azide inhibited Hst 5 killing of Candida albicans, while not inhibiting cellular ATP production. In contrast, Hst 5 caused a drastic reduction of C. albicans intracellular ATP content, which was a result of an efflux of ATP. Carbonyl cyanidep-chlorophenylhydrazone, dinitrophenol, and azide inhibited Hst 5-induced ATP efflux, thus establishing a correlation between ATP release and cell killing. Furthermore, C. albicans cells were respiring and had polarized membranes at least 80 min after ATP release, thus implying a non-lytic exit of cellular ATP in response to Hst 5. Based on evidence that transmembrane ATP efflux can occur in the absence of cytolysis through a channel-like pathway and that released ATP can act as a cytotoxic mediator by binding to membrane purinergic receptors, we evaluated whether extracellular ATP released by Hst 5 may have further functional role in cell killing. Consistent with this hypothesis, purinergic agonists BzATP and adenosine 5′O-(thiotriphosphate) induced loss of C. albicans cell viability and purinergic antagonists prevented Hst 5 killing. Salivary histatins are potent in vitro antifungal proteins and have promise as therapeutic agents against oral candidiasis. We performed pharmacological studies directed at understanding the biochemical basis of Hst 5 candidacidal activity. Three inhibitors of mitochondrial metabolism: carbonyl cyanidep-chlorophenylhydrazone, dinitrophenol, and azide inhibited Hst 5 killing of Candida albicans, while not inhibiting cellular ATP production. In contrast, Hst 5 caused a drastic reduction of C. albicans intracellular ATP content, which was a result of an efflux of ATP. Carbonyl cyanidep-chlorophenylhydrazone, dinitrophenol, and azide inhibited Hst 5-induced ATP efflux, thus establishing a correlation between ATP release and cell killing. Furthermore, C. albicans cells were respiring and had polarized membranes at least 80 min after ATP release, thus implying a non-lytic exit of cellular ATP in response to Hst 5. Based on evidence that transmembrane ATP efflux can occur in the absence of cytolysis through a channel-like pathway and that released ATP can act as a cytotoxic mediator by binding to membrane purinergic receptors, we evaluated whether extracellular ATP released by Hst 5 may have further functional role in cell killing. Consistent with this hypothesis, purinergic agonists BzATP and adenosine 5′O-(thiotriphosphate) induced loss of C. albicans cell viability and purinergic antagonists prevented Hst 5 killing. histatins histatin 5 carbonyl cyanidem-chlorophenylhydrazone dinitrophenol 3′-O-(4-benzoylbenzoyl)-ATP pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid 3,3′-dipentyloxacarbocyanine iodide relative light units adenosine 5′-O-(thiotriphosphate) Oral candidiasis is a superficial mucosal infection in whichCandida albicans is the predominant species isolated from infected areas (1Cannon R.D. Holmes A.R. Mason A.B. Monk B. J. Dent. Res. 1995; 74: 1152-1161Crossref PubMed Scopus (248) Google Scholar). Pathogenicity of C. albicans is postulated to involve adhesion to host cells and filamentous growth (2Gale C. Bendel C. McClellan M. Hauser M. Becker J. Berman J. Hostetter M. Science. 1998; 27: 1355-1358Crossref Scopus (306) Google Scholar). Incidence of oropharyngeal candidiasis has risen dramatically in the past 20 years due to increased antibiotic and pharmaceutic drug use and longer survival of people with compromised immune systems including patients on cancer chemotherapy, diabetics, and premature infants. Oropharyngeal candidiasis is so frequently associated with AIDS that it is a criterion for staging progression of disease (3Coleman D.C. Bennett D.E. Sullivan D.J. Gallagher P.J. Henman M.C. Shanley D.B. Russell R.J. Crit. Rev. Microbiol. 1993; 19: 61-82Crossref PubMed Scopus (122) Google Scholar). Increased use of current antifungal agents to treat candidiasis in late stage AIDS and cancer patients has resulted in emergence of Candidal species with antifungal drug resistance, especially to azole-based drugs. Even with aggressive treatment with the available antifungal drugs, morbidity from C. albicans infections in immunocompromised patients is high. These facts point to a pressing need for new antifungal agents and improved drug therapies.Innate host defense systems include a wide variety of small (3000–5000 Da) cationic proteins with potent antibacterial and antifungal activity (4Gabay J.E. Science. 1994; 264: 373Crossref PubMed Scopus (109) Google Scholar). Most locations that are in contact with indigenous microorganisms directly express or posses cells that can produce antimicrobial proteins. Despite structural variations, virtually all antimicrobial proteins carry a net positive charge.Histatins (Hsts)1 are structurally related histidine-rich basic proteins of acinar cell origin expressed only in humans and higher subhuman primates (5Oppenheim F.G. Xu T. McMillian M. Levitz S. Diamond R. Offner G. Troxler R. J. Biol. Chem. 1988; 263: 7472-7477Abstract Full Text PDF PubMed Google Scholar, 6Zuo Y. Xu T. Troxler R. Li J. Driscoll J. Oppenheim F. Gene (Amst.). 1995; 161: 87-91Crossref PubMed Scopus (17) Google Scholar). Salivary Hsts possess in vitro candidacidal and candidastatic activities (5Oppenheim F.G. Xu T. McMillian M. Levitz S. Diamond R. Offner G. Troxler R. J. Biol. Chem. 1988; 263: 7472-7477Abstract Full Text PDF PubMed Google Scholar, 7Santarpia III, R. Brant E. Lal M. Brasseur M. Hong A. Pollock J.J. Arch. Oral Biol. 1988; 33: 567-573Crossref PubMed Scopus (27) Google Scholar), and to a lesser degree bactericidal properties (8MacKay B. Denepitiya L. Ianoco V.J. Krost S. Pollock J. Infect. Immun. 1984; 44: 695-701Crossref PubMed Google Scholar). Hst 1 and Hst 3 are the full-length proteins (38 and 32 amino acids, respectively) encoded by two closely linked and related genes, HIS1 and HIS2 (9Sabatini L.M. Azen E.A. Biochem. Biophys. Res. Commun. 1989; 160: 495-502Crossref PubMed Scopus (72) Google Scholar), while Hst 2 and Hsts 4–12 are generated by proteolytic cleavage during secretion (10Perinpanayagam H.E.R. VanWuyckhuyse B.C. Ji Z.S. Tabak L.A. J. Dent. Res. 1995; 74: 345-350Crossref PubMed Scopus (58) Google Scholar). In vitro, Hst 5 (24 amino acids) is the most potent candidacidal member of the family that kills yeast and filamentous forms of Candida species at physiological concentrations (15–50 μm) (11Xu T. Levitz M. Diamond R. Oppenheim F. Infect. Immun. 1991; 70: 2549-2554Crossref Google Scholar, 12Raj P.A. Edgerton M. Levine M.J. J. Biol. Chem. 1990; 265: 3898-3905Abstract Full Text PDF PubMed Google Scholar, 13Oppenheim F. Yang Y. Diamond R. Hyslop D. Offner G. Troxler R. J. Biol. Chem. 1986; 261: 1177-1182Abstract Full Text PDF PubMed Google Scholar). Salivary Hsts have potential as therapeutic agents in patients with oral candidiasis, being potent antifungal agents, while non-toxic to humans.Hsts have similar size and net positive charge as other naturally occurring antimicrobial proteins; however, they possess unique structural features such as high histidine content and lack of disulfide bonds. Extensive structural and conformational analysis of Hst 5 revealed that the weak amphipathic character of the helical structure precludes spontaneous insertion into microbial membranes and direct formation of pores or ion channels across the membrane (12Raj P.A. Edgerton M. Levine M.J. J. Biol. Chem. 1990; 265: 3898-3905Abstract Full Text PDF PubMed Google Scholar, 14Raj P.A. Marcus E. Sukumaran D. Biopolymers. 1998; 45: 51-67Crossref PubMed Scopus (79) Google Scholar). Furthermore, Hst 5 variants with reduced killing ability exhibited similar helical content to Hst 5, suggesting the α-helical conformation alone is not solely responsible for optimal candidacidal activity (15Tsai H. Raj P.A. Bobek L.A. Infect. Immun. 1996; 64: 5000-5007Crossref PubMed Google Scholar). In contrast to the wealth of knowledge obtained on the primary structure and conformation of Hst 5, very little is known about its mode of action. Previous studies on the biochemical mechanism of Hsts activity have revealed three major findings: 1) K+ was rapidly released following exposure of C. albicans cells to a mixture of purified Hsts (16Pollock J. Denepitiya L. MacKay B. Ionoco V. Infect. Immun. 1984; 44: 702-707Crossref PubMed Google Scholar); 2) Mg2+ and Ca2+ inhibited Hst 5-induced killing of C. albicans (11Xu T. Levitz M. Diamond R. Oppenheim F. Infect. Immun. 1991; 70: 2549-2554Crossref Google Scholar); 3) exposure of C. albicans cells for 1 h to Hst 1 did not alter membrane permeability to the vital dyes methylene blue and acridine orange (13Oppenheim F. Yang Y. Diamond R. Hyslop D. Offner G. Troxler R. J. Biol. Chem. 1986; 261: 1177-1182Abstract Full Text PDF PubMed Google Scholar). Although altered yeast membrane permeability would readily account for the observed release of K+, Hst 5 direct channel-forming properties or Hst-induced nonspecific permeabilization of C. albicans cell membrane is not supported by the structural studies (14Raj P.A. Marcus E. Sukumaran D. Biopolymers. 1998; 45: 51-67Crossref PubMed Scopus (79) Google Scholar), early dye exclusion assays (13Oppenheim F. Yang Y. Diamond R. Hyslop D. Offner G. Troxler R. J. Biol. Chem. 1986; 261: 1177-1182Abstract Full Text PDF PubMed Google Scholar), or Hsts' inability to lyse lipid membranes (17Edgerton M. Koshlukova S. Lo T. Chrzan B. Straubinger R. Raj P.A. J. Biol. Chem. 1998; 273: 20438-20447Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). We have recently reported the presence of functional C. albicans-binding sites for salivary Hsts and a 67-kDa yeast Hst 5-binding protein. This finding provided a new insight into the potential mechanism of killing and suggested a basis for the selectivity of Hst 5 yeast killing and lack of toxicity to human host cells (17Edgerton M. Koshlukova S. Lo T. Chrzan B. Straubinger R. Raj P.A. J. Biol. Chem. 1998; 273: 20438-20447Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar).The physiological mechanism of action of a number of cationic host defense proteins including bactenecins (18Raj P.A. Marcus M. Edgerton M. Biochemistry. 1996; 35: 4314-4325Crossref PubMed Scopus (49) Google Scholar) defensins (19Kagan B. Selsted M. Ganz T. Lehrer R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 210-214Crossref PubMed Scopus (445) Google Scholar, 20Lehrer R.I. Lichtenstein A.K. Ganz T. Rev. immunol. 1993; 5: 97-103Google Scholar), cecropins (21Boman H.G. Cell. 1991; 65: 205-207Abstract Full Text PDF PubMed Scopus (535) Google Scholar), and maganins (22Zasloff M. Curr. Opin. Immunol. 1992; 4: 3-8Crossref PubMed Scopus (328) Google Scholar) is thought to be related to their abilities to disrupt lipid packing or form ion-permeable channels (14Raj P.A. Marcus E. Sukumaran D. Biopolymers. 1998; 45: 51-67Crossref PubMed Scopus (79) Google Scholar,15Tsai H. Raj P.A. Bobek L.A. Infect. Immun. 1996; 64: 5000-5007Crossref PubMed Google Scholar). The currently used drugs in treatment of candidiasis, polyene antimycotics and azole derivatives have also been reported to alter yeast membrane permeability. Polyene antimycotics complex with ergosterol of the plasma membrane resulting in release of cellular K+ (23Prasad R. Prasad R. Candida albicans: Cellular and Molecular Biology. Springer-Verlag, Berlin1991: 215-252Google Scholar, 24Beggs W.G. Antimicrob. Agents Chemother. 1994; 38: 363-364Crossref PubMed Scopus (44) Google Scholar). The azole-based drugs inhibit the biosynthesis of ergosterol (23Prasad R. Prasad R. Candida albicans: Cellular and Molecular Biology. Springer-Verlag, Berlin1991: 215-252Google Scholar, 25Vanden Bossche H. Willemsens G. Marichal P. CRC Crit. Rev. Microbiol. 1987; 15: 57-76Crossref Scopus (123) Google Scholar) and like the polyene antibiotics and some antimicrobial proteins, induced release of K+ and 260 nm absorbing materials from C. albicans cells (26Uno J. Shigematsu M. Arai T. Antimicrob. Agents Chemother. 1982; 21: 912-918Crossref PubMed Scopus (68) Google Scholar). The ultraviolet absorbing materials could represent ATP since release of cellular ATP was detected following 10 min of C. albicanstreatment with ketoconazole and other azole derivatives (27Ansehn S. Nilsson L. Antimicrob. Agents Chemother. 1984; 26: 22-25Crossref PubMed Scopus (50) Google Scholar). Cell lysis is one route by which ATP could be released from the cells. However, release of cytosolic ATP via plasma membrane channels in the absence of cytolysis has also been described, and members of the ATP-binding cassette (ABC) protein family were putatively associated with conductive ATP transport (28Schwiebert E. Egan M. Guggino W. Methods Enzymol. 1998; 292: 664-675Crossref PubMed Scopus (9) Google Scholar). Furthermore, released ATP can function outside of the cell via activating membrane purinergic receptors of the P2X family to cause increased K+ and Cl− membrane permeability and even cell lysis (29Wang Y. Roman R. Lidofsky S. Fitz J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12020-12025Crossref PubMed Scopus (299) Google Scholar, 30Surprenant A. Rassendren F. Kawashima R. North R. Buell G. Science. 1996; 272: 735-738Crossref PubMed Scopus (1487) Google Scholar, 31Di Virgilio F. Immunol. Today. 1995; 16: 524-528Abstract Full Text PDF PubMed Scopus (349) Google Scholar).Hst 5 has been characterized at structural and conformational levels with the aim of producing more effective fungicidal protein. However, despite extensive efforts, this goal has not been achieved in large part because of lack of understanding of the cellular target and mechanism of action of Hsts. We examined the biochemical mechanism of Hst 5 yeast killing by performing pharmacological assays and functional studies on C. albicans cells. We showed that Hst 5 induced efflux of cellular ATP that correlated with cell death. Furthermore, the detected extracellular ATP did not represent leakage from lysed cells, but rather appeared to be released from intact and metabolically active cells. We conclude that, under the experimental paradigms used, extracellular ATP released by Hst 5 can activate putative purinergic receptors on C. albicans to induce cell death.DISCUSSIONThis is the first study to describe ATP efflux from C. albicans in response to Hst 5 that occurs in the absence of cytolysis and correlates with cell death. We report here that Hst 5-induced killing of C. albicans is initiated by a release of intracellular ATP while yeast cells are metabolically active and have polarized membranes. Furthermore, we provide evidence for the presence of putative purinergic-like receptors on these cells. Extracellular ATP, released from C. albicans by Hst 5, may activate membrane ATP receptors to cause cell death.We began the characterization of the mechanism of Hst 5 candidacidal activity by testing pharmacological agents for effects on Hst 5-induced killing of C. albicans. Of many agents tested, the proton ionophore CCCP partially inhibited Hst 5-induced killing of C. albicans, while not affecting the viability of control cells. The effect of CCCP was specific to the cells and was not due to interaction and/or inactivation of Hst 5, since increased survival was observed when cells were first preincubated with CCCP, then washed free of drug before exposure to Hst 5 (data not shown). Uncouplers like CCCP dissipate the electrochemical gradient (Fig. 6) resulting in a decline in proton-coupled ATP synthesis, acidification of cytoplasm, inhibition of active transport of nutrients, and export of toxic drugs (35Noshiro A. Rurwin C. Laux M. Nicolay K. Scheffers W.A. Holzer H. J. Biol. Chem. 1987; 262: 14154-14157Abstract Full Text PDF PubMed Google Scholar, 39Clark F. Parkinson T. Hitchcock C. Gow N. Antimicrob. Agents Chemother. 1996; 40: 419-425Crossref PubMed Google Scholar,40Serrano R. Eur. J. Biochem. 1977; 80: 97-102Crossref PubMed Scopus (157) Google Scholar). Therefore, the observed inhibition of Hst 5 cell killing by CCCP can be due to the inhibition of Hst 5 initial interaction with the cell, e.g. binding or transport, or to the inhibition of an essential step in the intracellular cascade leading to cell death. The proton ionophore DNP and azide at concentrations higher than 1 mm have also been reported to function as uncouplers inC. albicans (37Olson V. Hansing R. McClary D. Can. J. Microbiol. 1976; 23: 166-174Crossref Scopus (12) Google Scholar) and both agents protected these cells from Hst 5 killing (Fig. 3, Table I). Interestingly, the same three agents (CCCP, DNP, and azide) were shown to inhibit human neutrophil defensin-1 killing of C. albicans (43Lehrer R. Ganz T. Szklarek D. Selested M. J. Clin. Invest. 1988; 81: 1829-1835Crossref PubMed Scopus (178) Google Scholar). The abilities of these drugs to reduce human neutrophil defensin-1 cytotoxic action were tentatively ascribed to their effects on mitochondrial metabolism. However, our ATP measurements revealed that ATP content in the presence of CCCP and azide was the same as in the control cells and DNP caused only a 2-fold reduction of the intracellular ATP level (Fig. 4). Therefore, the protective effects of these agents to C. albicans against Hst 5 killing cannot be explained by depletion of cellular ATP.A striking finding of this study is that Hst 5 itself induced a drastic reduction of intracellular ATP (Fig. 4), which coincided with the 180-fold increase of the extracellular ATP (Fig. 5). ATP release occurred in 10 min (the earliest time investigated) following exposure to Hst 5 and the extracellular ATP level remained unchanged or slightly increased for up to 1.5 h. Most importantly, studies using inhibitors of Hst 5 activity suggested a relationship between ATP release and killing of C. albicans. Preincubation of the cells with concentrations of CCCP, DNP, and azide that inhibited Hst 5-induced killing by 50, 69, and 82%, also inhibited Hst 5-induced ATP release by 87, 69, and 97%, respectively (Table II). Although the exact mechanism by which CCCP, DNP, and azide induce protection is currently unclear, the use of these agents was crucial in establishing a correlation between ATP release and cell killing.Several observations indicate that Hst 5-induced ATP release fromC. albicans represents an efflux from structurally intact and metabolically active cells. First, flow cytometry experiments, using membrane potential sensitive dye showed that C. albicans cell membranes remained polarized after 1.5 h of exposure to Hst 5 (Fig. 6). Second, released ATP was not hydrolysed extracellularly for approximately 80 min (from 10 min to 1.5 h incubation of the cells with Hst 5) at 37 °C, suggesting that the cell membrane was intact and no intracellular ATPases were present to rapidly degrade ATP. This is consistent with the finding that C. albicans cells were not stained by methylene blue and acridine orange after 1 h of exposure to Hst 1 (13Oppenheim F. Yang Y. Diamond R. Hyslop D. Offner G. Troxler R. J. Biol. Chem. 1986; 261: 1177-1182Abstract Full Text PDF PubMed Google Scholar). Finally, C. albicans cells treated with Hst 5 for either 10 min (ATP release) or 1.5 h (complete cell killing measured by inability to form colonies) were consuming oxygen at rates similar to untreated cells, indicating active respiration (Table III). Although the concentration of ATP in the cells is an indicator of viability and ATP is rapidly degraded when cells die, actively metabolizing organisms with inhibited DNA replication have also been described (44Abbott A. Odds F. J. Antimicrob. Chemother. 1989; 24: 905-919Crossref PubMed Scopus (5) Google Scholar). Perhaps, the loss of cellular ATP, caused by Hst 5, forces the cell to maintain the proton gradient across the membranes at the expense of all of the newly synthesized ATP. Whether cells maintain their integrity, but have no energy to replicate, or Hst 5-induced ATP release initiates processes leading to later loss of cell structure will require further study. However, the effect of Hst 5 on cell viability, at least during the 1.5 h of treatment, is not a result of a destructive action.The findings described here suggest a specific cellular mechanism for exit of ATP in response to Hst 5. The known mechanisms of specific ATP release include: 1) facilitated transport of ATP through a transporter down a favorable concentration gradient; 2) conductive transport through an ATP-specific or anion channel; and 3) release of ATP by exocytosis (28Schwiebert E. Egan M. Guggino W. Methods Enzymol. 1998; 292: 664-675Crossref PubMed Scopus (9) Google Scholar). Although little is known about ATP-specific channels, members of ATP-binding cassette (ABC) proteins, such as P-glycoprotein (45Roman R.M. Wang Y. Lidofsky S.D. Feranchak A.P. Lomri N. Scharschmidt B.F. Fitz J.G. J. Biol. Chem. 1997; 272: 21970-21976Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar) and cystic fibrosis transmembrane conductance regulator (46Schwiebert E. Egan M. Hwang T.E. Fulmer S.B. Allen S. Cutting G. Guggino W. Cell. 1995; 81: 1063-1073Abstract Full Text PDF PubMed Scopus (593) Google Scholar), have been implicated in conductive transport of ATP. ABC transporters which carry out transport of a wide range of substrates or function as efflux pumps have been identified in Saccharomyces cerevisiae and resistance to azole antifungal agents in C. albicans has been shown to be mediated by the ABC transporters CDR1 and CDR2 (47Prasad R. Wergifosse P. Goffeau A. Balzi E. Curr. Genet. 1995; 27: 320-329Crossref PubMed Scopus (398) Google Scholar,48Sanglard D. Ischer F. Monod M. Bille J. Microbiology. 1997; 143: 405-416Crossref PubMed Scopus (495) Google Scholar). Furthermore, S. cerevisiae released ATP in response to the toxic H+/K+ ionophore nigericin, while the cell membrane permeability was not altered (41Boyum R. Guidotti G. Microbiology. 1997; 143: 1901-1908Crossref PubMed Scopus (35) Google Scholar) and a plasma membrane ATP-specific transporter was purified from Aspergillus nigerthat was activated by the lipid-reactive antibiotic mycobacillin (49Chowdhury B. Bose S. Bhaduri S. Bose S. Eur. J. Biochem. 1997; 247: 673-680Crossref PubMed Scopus (5) Google Scholar).Extracellular ATP can cause striking changes in membrane permeability through activating membrane receptors for extracellular nucleotides (purinoceptors, P2) expressed on susceptible cells (50Burnstock G. CIBA Found. Symp. 1996; 198: 1-28PubMed Google Scholar). Among the P2 receptors, P2Y are G-protein-coupled receptors and P2X are ligand-gated channels more selectively localized on excitable cells (31Di Virgilio F. Immunol. Today. 1995; 16: 524-528Abstract Full Text PDF PubMed Scopus (349) Google Scholar, 51Dubyak G. Moatassim C. Am. J. Physiol. 1993; 265: C577-C606Crossref PubMed Google Scholar). By contrast, P2Z (P2X7) receptors operate as ATP-gated pores and cells expressing these receptors are killed when stimulated with agonists, ATP, or ATP analogues (30Surprenant A. Rassendren F. Kawashima R. North R. Buell G. Science. 1996; 272: 735-738Crossref PubMed Scopus (1487) Google Scholar, 31Di Virgilio F. Immunol. Today. 1995; 16: 524-528Abstract Full Text PDF PubMed Scopus (349) Google Scholar, 52Hickman S.E. Semrad C. Silverstein S. CIBA Found. Symp. 1996; 198: 71-90PubMed Google Scholar). ATP binding, but not hydrolysis is needed for receptor activation. We showed here that stimulation of C. albicans cells with the purinergic agonists BzATP or ATPγS resulted in 72 and 57% loss of cell viability, respectively; and purinergic antagonists suramin and PPAD prevented Hst 5 killing (Table IV), thus, providing evidence for the presence of Candidal purinergic-like receptors. The P2Z/P2X7 receptor is selectively activated by ATP in its fully dissociated tetra-anionic form (ATP4-) and extracellular Ca2+ and Mg2+ prevent channel opening and cell lysis (30Surprenant A. Rassendren F. Kawashima R. North R. Buell G. Science. 1996; 272: 735-738Crossref PubMed Scopus (1487) Google Scholar, 31Di Virgilio F. Immunol. Today. 1995; 16: 524-528Abstract Full Text PDF PubMed Scopus (349) Google Scholar, 53Wiley J.S. Gargett C. Zhang W. Snook M. Jamieson G. Am. J. Physiol. 1998; 275: C1224-C1231Crossref PubMed Google Scholar). In this context, it is of interest to note that Ca2+ and Mg2+ have been reported to abolish Hst 5 killing C. albicans (11Xu T. Levitz M. Diamond R. Oppenheim F. Infect. Immun. 1991; 70: 2549-2554Crossref Google Scholar).Activation of P2Z/P2X7 receptors requires relatively high doses of extracellular ATP, from 100 μm to 1 mm. The most likely source of this amount of ATP is release from stressed or injured cells, intracellular concentration of ATP is 5–10 mm for higher eukaryotic cells (31Di Virgilio F. Immunol. Today. 1995; 16: 524-528Abstract Full Text PDF PubMed Scopus (349) Google Scholar) and about 1 mm for yeast cells (35Noshiro A. Rurwin C. Laux M. Nicolay K. Scheffers W.A. Holzer H. J. Biol. Chem. 1987; 262: 14154-14157Abstract Full Text PDF PubMed Google Scholar). Our data showed that Hst 5 induced release of more than half of the reported C. albicansintracellular ATP, a sufficient amount to activate P2Z/P2X7 receptors. ATP-pulsed cells expressing these receptors appear to initially maintain their integrity and physiological function, yet cytolysis occurs 10 to 12 h after receptor stimulation (31Di Virgilio F. Immunol. Today. 1995; 16: 524-528Abstract Full Text PDF PubMed Scopus (349) Google Scholar, 54Ferrari D. Chiozzi P. Falzoni S. DalSusino M. Collo G. Buell G. DiVirgilo F. Neuropharmacology. 1997; 36: 1295-1301Crossref PubMed Scopus (242) Google Scholar). Similarly, C. albicans cells were actively respiring and had polarized membranes after 1.5 h treatment with Hst 5, but could not subsequently form colonies after 24 h. Admittedly, purinergic-like receptors have not been described on yeast and, P2Z/P2X7 cytotoxic receptors have been found mainly on immune and inflammatory cells. However, C. albicans is known to possess complement receptors, CR2- and CR3-like proteins for C3d and iC3b, that are mostly expressed on the B lymphocytes and phagocytic cells (55Hostetter M. Clin. Microbiol. Rev. 1994; 7: 29-42Crossref PubMed Scopus (194) Google Scholar, 56Calderone R. Braun P. Microbiol. Rev. 1991; 55: 1-20Crossref PubMed Google Scholar).Our findings provide support for the concept that extracellular ATP plays a role in cell killing and raise the possibility of a novel mechanism for Hst 5 cytotoxic action that include: (a) killing of C. albicans is initiated with release of cellular ATP; (b) ATP release is via non-lytic, perhaps, conductive pathway; (c) extracellular ATP, released in response to Hst 5, may activate putative purinergic receptors on C. albicansto ultimately induce cell death.Characterization of many other important aspects of Hst 5 cytotoxic action will require further study. It is currently unknown whether Hst 5 induces efflux of cellular ATP through binding to a cell surface component or needs to be transported into the cells. Furthermore, it is yet unanswered how ATP is released from the cells and whether ATP-binding cassette proteins are involved in conductive transport of ATP. We have previously reported the presence of C. albicansbinding sites for salivary Hsts and a 67-kDa yeast Hst 5-binding protein (17Edgerton M. Koshlukova S. Lo T. Chrzan B. Straubinger R. Raj P.A. J. Biol. Chem. 1998; 273: 20438-20447Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). This protein is of similar size to the purified plasma membrane ATP transporter from A. niger (49Chowdhury B. Bose S. Bhaduri S. Bose S. Eur. J. Biochem. 1997; 247: 673-680Crossref PubMed Scopus (5) Google Scholar) and may function as an ATP channel activated upon Hst 5 binding. Purification and identification of the Hst 5-binding protein would be an important step in understanding the killing mechanism. Exploring the protective effects of the uncouplers to C. albicans cells against Hst 5 killing will aid in understanding the biochemical mechanism of Hst 5 candidacidal activity. Finally, the molecular identity of ATP receptors involved in Hst 5 killing of C. albicans is crucial in understanding the cytotoxic mechanism. The mechanism described here for Hst 5-induced yeast killing has not been evaluated for other antifungal agents and antimicrobial proteins. Consequently, it will be important to determine whether it represents a common antifungal mechanism or it is unique to Hst 5.In conclusion, major disadvantages of currently used antifungal drugs to treat candidiasis are their toxicity and the development of resistant yeast strains. In contrast, salivary Hsts are nontoxic to humans and yet potent candidacidal agents even with drug-resistant strains. While the therapeutic potential of Hsts is becoming apparent, particularly against azole-resistant strains, the exact mechanism of cell killing must be elucidated to fully utilize them as therapeutic agents. Oral candidiasis is a superficial mucosal infection in whichCandida albicans is the predominant species isolated from infected areas (1Cannon R.D. Holmes A.R. Mason A.B. Monk B. J. Dent. Res. 1995; 74: 1152-1161Crossref PubMed Scopus (248) Google Scholar). Pathogenicity of C. albicans is postulated to involve adhesion to host cells and filamentous growth (2Gale C. Bendel C. McClellan M. Hauser M. Becker J. Berman J. Hostetter M. Science. 1998; 27: 1355-1358Crossref Scopus (306) Google Scholar). Incidence of oropharyngeal candidiasis has risen dramati" @default.
• W2010999630 created "2016-06-24" @default.
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• W2010999630 date "1999-07-01" @default.
• W2010999630 modified "2023-10-15" @default.
• W2010999630 title "Salivary Histatin 5 Induces Non-lytic Release of ATP fromCandida albicans Leading to Cell Death" @default.
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