Salivary Histatins: Structure, Function, and Mechanisms of Antifungal Activity

Woong Sik Jang; Mira Edgerton

doi:10.1128/9781555817176.ch13

Chapter 13 : Salivary Histatins: Structure, Function, and Mechanisms of Antifungal Activity

Authors: Woong Sik Jang¹, Mira Edgerton²

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Affiliations: 1: Department of Oral Biology, School of Dental Medicine, State University of New York at Buffalo, Buffalo, NY 14214; 2: Department of Oral Biology, School of Dental Medicine, State University of New York at Buffalo, Buffalo, NY 14214

Content Type: Monograph

Publication Year: 2012

Category: Fungi and Fungal Pathogenesis; Clinical Microbiology

Book DOI: 10.1128/9781555817176

Chapter DOI: 10.1128/9781555817176.ch13

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Abstract:

Immunohistochemical studies identified serous cells of the glandular acini as the cells responsible for production of salivary histatins. Histatins exhibit fungicidal activity against several Candida species, Aspergillus fumigatus, some strains of Saccharomyces cerevisiae, and Cryptococcus neoformans. Studies of levels of salivary histatins in vivo show large intersubject variation in both the concentrations of histatins and their rates of degradation. The total concentration of histatins in whole saliva is balanced between secretion of new proteins and removal of “older” proteins by degradation. Endocytosis was initially suggested as a means of histatin cellular entry based upon the observation that bafilomycin, an inhibitor of endosomal acidification, significantly decreased antifungal activity. Confocal imaging of C. albicans cells showed that some histatin 5 was localized to the vacuole but that cells containing only vacuolar histatin were viable. The cell wall of C. albicans is a thick multilayered structure of glucans, chitin, and mannoproteins that protects cells from osmotic stress and maintains structural integrity. Animal and human clinical studies to evaluate histatins as topical agents in prevention of gingivitis reported therapeutic efficacy without adverse side effects. The major requirements for effective use of salt-insensitive fungicidal peptides are selective and specific binding and uptake by candidal cells, efficacy at low concentrations that allow rapid eradication of yeast pathogens within the ionic strength of saliva, and minimal fungal resistance.

Citation: Jang W, Edgerton M. 2012. Salivary Histatins: Structure, Function, and Mechanisms of Antifungal Activity, p 185-194. In Calderone R, Clancy C (ed), Candida and Candidiasis, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817176.ch13

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Programmed Cell Death: 0.4706052
Cell Wall Proteins: 0.45211276
Basic Amino Acids: 0.4511504
Cell Wall Components: 0.41661718
Transmission Electron Microscopy: 0.41661718

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Salivary Histatins: Structure, Function, and Mechanisms of Antifungal Activity

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FIGURE 1

Structure and functional domains of histatins. (Top) Primary structure of major histatins 1, 3, and 5. Domains involved in antifungal activity (AF) and wound healing (WH) are bracketed. Shaded amino acids are crucial for antifungal activity. Sulfated (S) and phosphorylated (P) residues are indicated. (Bottom) Cleavage sites that generate other histatin family members (H2, histatin 2; H4, histatin 4; H5, histatin 5; H6, histatin 6; H7, histatin 7; H8, histatin 8) are shown with inverted triangles. doi:10.1128/9781555817176.ch13.f1

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FIGURE 1

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FIGURE 2

Histatin uptake by C. albicans is energy dependent. Fluorescein isothiocyanate-labeled histatin 5 (F-Hst 5) added to cells (control) is rapidly translocated to the cytosol, while pretreatment with the protonophore CCCP substantially inhibits cytosolic translocation, although cell wall binding of histatin 5 remains intact. doi:10.1128/9781555817176.ch13.f2

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FIGURE 2

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FIGURE 3

Model of histatin binding and uptake by C. albicans. Both cell wall polysaccharides and Ssa2 proteins play an important role in initial capture and binding of extracellular histatins. These proteins are facilitators that transfer cell wall-bound histatins (and other cationic peptides) to the actual importer mechanism involving active transport via Dur permeases and energy dependent endocytosis. doi:10.1128/9781555817176.ch13.f3

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FIGURE 3

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FIGURE 4

Histatin intracellular trafficking utilizes two pathways in C. albicans. Two distinct routes of intracellular trafficking of histatin 5 are observed. Endocytotic trafficking directly to the vacuole is slower (top), while cytoplasm-to-vacuole trafficking is more rapid (bottom). Histatin 5 is labeled with fluorescein isothiocyanate for visualization using confocal microscopy ( 39 ). doi:10.1128/9781555817176.ch13.f4

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FIGURE 4

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FIGURE 5

Model for histatin toxicity in C. albicans. After intracellular entry, histatin causes loss of intracellular ions and nucleotides that are dependent on the presence of Trk1 proteins. Efflux of ions causes osmotic stress-like conditions sensed by the membrane sensors Sho1, Sln1, and Msb2, which initiate signaling by the C. albicans Hog1 MAP kinase (or high-osmolarity glycerol) pathway, to allow adaptation to osmotic and oxidative stresses induced by histatins. doi:10.1128/9781555817176.ch13.f5

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FIGURE 5

References

/content/book/10.1128/9781555817176.ch13

1. Ahmad, M.,, M. Piludu,, F. G. Oppenheim,, E. J. Helmerhorst, and, A. R. Hand. 2004. Immunocytochemical localization of histatins in human salivary glands. J. Histochem. Cytochem. 52: 361– 370.

2. Atkinson, J. C.,, C. Yeh,, F. G. Oppenheim,, D. Bermudez,, B. J. Baum, and, P. C. Fox. 1990. Elevation of salivary antimicrobial proteins following HIV-1 infection. J. Acquir. Immune Defic. Syndr. 3: 41– 48.

3. Baev, D.,, X. Li, and, M. Edgerton. 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.

4. Baev, D.,, X. S. Li,, J. Dong,, P. Keng, and, M. Edgerton. 2002. Human salivary histatin 5 causes disordered volume regulation and cell cycle arrest in Candida albicans. Infect. Immun. 70: 4777– 4784.

5. Baev, D.,, A. Rivetta,, X. S. Li,, S. Vylkova,, E. Bashi,, C. L. Slayman, and, M. Edgerton. 2003. Killing of Candida albicans by human salivary histatin 5 is modulated, but not determined, by the potassium channel TOK1. Infect. Immun. 71: 3251– 3260.

6. Baev, D.,, A. Rivetta,, S. Vylkova,, J. N. Sun,, G. F. Zeng,, C. L. Slayman, and, M. Edgerton. 2004. The TRK1 potassium transporter is the critical effector for killing of Candida albicans by the cationic protein, Histatin 5. J. Biol. Chem. 279: 55060– 55072.

7. Bercier, J. G.,, I. Al-Hashimi,, N. Haghighat,, T. D. Rees, and, F. G. Oppenheim. 1999. Salivary histatins in patients with recurrent oral candidiasis. J. Oral Pathol. Med. 28: 26– 29.

8. Brewer, D.,, H. Hunter, and, G. Lajoie. 1998. NMR studies of the antimicrobial salivary peptides histatin 3 and histatin 5 in aqueous and nonaqueous solutions. Biochem. Cell Biol. 76: 247– 256.

9. Brewer, D.,, and G. Lajoie. 2002. Structure-based design of potent histatin analogues. Biochemistry 41: 5526– 5536.

10. Bromuro, C.,, R. La Valle,, S. Sandini,, F. Urbani,, C. M. Ausiello,, L. Morelli,, C. Fe d’Ostiani,, L. Romani, and, A. Cassone. 1998. A 70-kilodalton recombinant heat shock protein of Candida albicans is highly immunogenic and enhances systemic murine candidiasis. Infect. Immun. 66: 2154– 2162.

11. Campese, M.,, X. Sun,, J. A. Bosch,, F. G. Oppenheim, and, E. J. Helmerhorst. 2009. Concentration and fate of histatins and acidic proline-rich proteins in the oral environment. Arch. Oral Biol. 54: 345– 353.

12. Castagnola, M.,, R. Inzitari,, D. V. Rossetti,, C. Olmi,, T. Cabras,, V. Piras,, P. Nicolussi,, M. T. Sanna,, M. Pellegrini,, B. Giardina, and, I. Messana. 2004. A cascade of 24 histatins (histatin 3 fragments) in human saliva. Suggestions for a pre-secretory sequential cleavage pathway. J. Biol. Chem. 279: 41436– 41443.

13. Chadha, S.,, J. S. Bajwa,, J. N. Sun,, J. Paluszynski,, W. S. Jang, and, M. Edgerton. 2010. Spermidine plasma membrane transporter Dur31 is involved in the translocation of Histatin 5 in C. albicans. 10th ASM Conf. Candida Candidiasis, abstr. 100B, p. 96.

14. Craig, E. A.,, B. D. Gambill, and, R. J. Nelson. 1993. Heat shock proteins: molecular chaperones of protein biogenesis. Microbiol. Rev. 57: 402– 414.

15. den Hertog, A. L.,, J. van Marle,, H. A. van Veen,, W. Van’t Hof,, J. G. Bolscher,, E. C. Veerman, and, A. V. Nieuw Amerongen. 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.

16. Den Hertog, A. L.,, H. W. Wong Fong Sang,, R. Kraayenhof,, J. G. Bolscher,, W. Van’t Hof,, E. C. Veerman, and, A. V. Nieuw Amerongen. 2004. Interactions of histatin 5 and histatin 5-derived peptides with liposome membranes: surface effects, translocation and permeabilization. Biochem. J. 379: 665– 672.

17. Denny, P.,, F. K. Hagen,, M. Hardt,, L. Liao,, W. Yan,, M. Arellanno,, S. Bassilian,, G. S. Bedi,, P. Boontheung,, D. Cociorva,, C. M. Delahunty,, T. Denny,, J. Dunsmore,, K. F. Faull,, J. Gilligan,, M. Gonzalez-Begne,, F. Halgand,, S. C. Hall,, X. Han,, B. Henson,, J. Hewel,, S. Hu,, S. Jeffrey,, J. Jiang,, J. A. Loo,, R. R. Ogorzalek Loo,, D. Malamud,, J. E. Melvin,, O. Miroshnychenko,, M. Navazesh,, R. Niles,, S. K. Park,, A. Prakobphol,, P. Ramachandran,, M. Rich-ert,, S. Robinson,, M. Sondej,, P. Souda,, M. A. Sullivan,, J. Takashima,, S. Than,, J. Wang,, J. P. Whitelegge,, H. E. Witkowska,, L. Wolinsky,, Y. Xie,, T. Xu,, W. Yu,, J. Ytterberg,, D. T. Wong,, J. R. Yates III, and, S. J. Fisher. 2008. The proteomes of human parotid and submandibular/ sublingual gland salivas collected as the ductal secretions. J. Proteome Res. 7: 1994– 2006.

18. Dong, J.,, S. Vylkova,, X. S. Li, and, M. Edgerton. 2003. Calcium blocks fungicidal activity of human salivary histatin 5 through disruption of binding with Candida albicans. J. Dent. Res. 82: 748– 752.

19. Douglas, L. M.,, S. W. Martin, and, J. B. Konopka. 2009. BAR domain proteins Rvs161 and Rvs167 contribute to Candida albicans endocytosis, morphogenesis, and virulence. Infect. Immun. 77: 4150– 4160.

20. Driscoll, J.,, Y. Zuo,, T. Xu,, J. R. Choi,, R. F. Troxler, and, F. G. Oppenheim. 1995. Functional comparison of native and recombinant human salivary histatin 1. J. Dent. Res. 74: 1837– 1844.

21. Edgerton, M.,, S. E. Koshlukova,, T. E. Lo,, B. G. Chrzan,, R. M. Straubinger, and, P. A. Raj. 1998. Candidacidal activity of salivary histatins. Identification of a histatin 5-binding protein on Candida albicans. J. Biol. Chem. 273: 20438– 20447.

22. Eroles, P.,, M. Sentandreu,, M. V. Elorza,, and R. Sentandreu. 1997. The highly immunogenic enolase and Hsp70p are adventitious Candida albicans cell wall proteins. Microbiology 143( Pt. 2) : 313– 320.

23. Fitzgerald-Hughes, D. H.,, D. C. Coleman, and, B. C. O’Connell. 2007. Differentially expressed proteins in derivatives of Candida albicans displaying a stable histatin 3-resistant phenotype. Antimicrob. Agents Chemother. 51: 2793– 2800.

24. Flaherty, K. M.,, C. DeLuca-Flaherty, and, D. B. McKay. 1990. Three-dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein. Nature 346: 623– 628.

25. Giacometti, A.,, O. Cirioni,, W. Kamysz,, G. D’Amato,, C. Silvestri,, M. S. Del Prete,, A. Licci,, A. Riva,, J. Lukasiak, and, G. Scalise. 2005. In vitro activity of the histatin derivative P-113 against multidrug-resistant pathogens responsible for pneumonia in immunocompromised patients. Antimicrob. Agents Chemother. 49: 1249– 1252.

26. Gyurko, C.,, U. Lendenmann,, R. F. Troxler, and, F. G. Oppenheim. 2000. Candida albicans mutants deficient in respiration are resistant to the small cationic salivary antimicrobial peptide histatin 5. Antimicrob. Agents Chemother. 44: 348– 354.

27. Hardt, M.,, L. R. Thomas,, S. E. Dixon,, G. Newport,, N. Agabian,, A. Prakobphol,, S. C. Hall,, H. E. Witkowska, and, S. J. Fisher. 2005. Toward defining the human parotid gland salivary proteome and peptidome: identification and characterization using 2D SDS-PAGE, ultrafiltration, HPLC, and mass spectrometry. Biochemistry 44: 2885– 2899.

28. Helmerhorst, E. J.,, A. S. Alagl,, W. L. Siqueira, and, F. G. Oppenheim. 2006. Oral fluid proteolytic effects on histatin 5 structure and function. Arch. Oral Biol. 51: 1061– 1070.

29. Helmerhorst, E. J.,, P. Breeuwer,, W. van’t Hof,, E. Walgreen-Weterings,, L. C. Oomen,, E. C. Veerman,, A. V. Amerongen, and, T. Abee. 1999. The cellular target of histatin 5 on Candida albicans is the energized mitochondrion. J. Biol. Chem. 274: 7286– 7291.

30. Helmerhorst, E. J.,, B. Flora,, R. F. Troxler, and, F. G. Oppenheim. 2004. Dialysis unmasks the fungicidal properties of glandular salivary secretions. Infect. Immun. 72: 2703– 2709.

31. Helmerhorst, E. J.,, and F. G. Oppenheim. 2007. Saliva: a dynamic proteome. J. Dent. Res. 86: 680– 693.

32. Helmerhorst, E. J.,, I. M. Reijnders,, W. van’t Hof,, I. Si-moons-Smit,, E. C. Veerman, and, A. V. Amerongen. 1999. Amphotericin B- and fluconazole-resistant Candida spp., Aspergillus fumigatus, and other newly emerging pathogenic fungi are susceptible to basic antifungal peptides. Antimicrob. Agents Chemother. 43: 702– 704.

33. Helmerhorst, E. J.,, R. F. Troxler, and, F. G. Oppenheim. 2001. The human salivary peptide histatin 5 exerts its antifungal activity through the formation of reactive oxygen species. Proc. Natl. Acad. Sci. USA 98: 14637– 14642.

34. Helmerhorst, E. J.,, C. Venuleo,, A. Beri, and, F. G. Oppenheim. 2005. Candida glabrata is unusual with respect to its resistance to cationic antifungal proteins. Yeast 22: 705– 714.

35. Helmerhorst, E. J.,, C. Venuleo,, D. Sanglard, and, F. G. Oppenheim. 2006. Roles of cellular respiration, CgCDR1, and CgCDR2 in Candida glabrata resistance to histatin 5. Antimicrob. Agents Chemother. 50: 1100– 1103.

36. Imamura, Y.,, Y. Fujigaki,, Y. Oomori,, K. Ouryouji,, S. Yanagisawa,, H. Miyazawa, and, P. L. Wang. 2009. Transcriptional regulation of the salivary histatin gene: finding of a strong positive regulatory element and its binding protein. J. Biochem. 145: 279– 288.

37. Isola, R.,, M. Isola,, G. Conti,, M. S. Lantini, and, A. Riva. 2007. Histatin-induced alterations in Candida albicans: a microscopic and submicroscopic comparison. Microsc. Res. Tech. 70: 607– 616.

38. Jainkittivong, A.,, D. A. Johnson, and, C. K. Yeh. 1998. The relationship between salivary histatin levels and oral yeast carriage. Oral Microbiol. Immunol. 13: 181– 187.

39. Jang, W. S.,, J. S. Bajwa,, J. N. Sun, and, M. Edgerton. 2010. Salivary histatin 5 internalization by translocation, but not endocytosis, is required for fungicidal activity in Candida albicans. Mol. Microbiol. 77: 354– 370.

40. Jang, W. S.,, X. S. Li,, J. N. Sun, and, M. Edgerton. 2008. The P-113 fragment of histatin 5 requires a specific peptide sequence for intracellular translocation in Candida albicans, which is independent of cell wall binding. Antimicrob. Agents Chemother. 52: 497– 504.

41. Kavanagh, K.,, and S. Dowd. 2004. Histatins: antimicrobial peptides with therapeutic potential. J. Pharm. Pharmacol. 56: 285– 289.

42. Konopka, K.,, B. Dorocka-Bobkowska,, S. Gebremedhin, and, N. Duzgunes. 2010. Susceptibility of Candida biofilms to histatin 5 and fluconazole. Antonie van Leeuwenhoek 97: 413– 417.

43. Koshlukova, S. E.,, T. L. Lloyd,, M. W. Araujo, and, M. Edgerton. 1999. Salivary histatin 5 induces non-lytic release of ATP from Candida albicans leading to cell death. J. Biol. Chem. 274: 18872– 18879.

44. La Valle, R.,, C. Bromuro,, L. Ranucci,, H. M. Muller,, A. Crisanti, and, A. Cassone. 1995. Molecular cloning and expression of a 70-kilodalton heat shock protein of Candida albicans. Infect. Immun. 63: 4039– 4045.

45. Li, X. S.,, M. S. Reddy,, D. Baev, and, M. Edgerton. 2003. Candida albicans Ssa1/2p is the cell envelope binding protein for human salivary histatin 5. J. Biol. Chem. 278: 28553– 28561.

46. Li, X. S.,, J. N. Sun,, K. Okamoto-Shibayama, and, M. Edgerton. 2006. Candida albicans cell wall Ssa proteins bind and facilitate import of salivary histatin 5 required for toxicity. J. Biol. Chem. 281: 22453– 22463.

47. Lis, M.,, and L. A. Bobek. 2008. Proteomic and metabolic characterization of a Candida albicans mutant resistant to the antimicrobial peptide MUC7 12-mer. FEMS Immunol. Med. Microbiol. 54: 80– 91.

48. Lopez-Ribot, J. L.,, H. M. Alloush,, B. J. Masten, and, W. L. Chaffin. 1996. Evidence for presence in the cell wall of Candida albicans of a protein related to the hsp70 family. Infect. Immun. 64: 3333– 3340.

49. Luque-Ortega, J. R.,, W. van’t Hof,, E. C. Veerman,, J. M. Saugar, and, L. Rivas. 2008. Human antimicrobial peptide histatin 5 is a cell-penetrating peptide targeting mitochondrial ATP synthesis in Leishmania. FASEB J. 22: 1817– 1828.

50. Martinez, J. P.,, M. L. Gil,, J. L. Lopez-Ribot, and, W. L. Chaffin. 1998. Serologic response to cell wall mannoproteins and proteins of Candida albicans. Clin. Microbiol. Rev. 11: 121– 141.

51. Meiller, T. F.,, B. Hube,, L. Schild,, M. E. Shirtliff,, M. A. Scheper,, R. Winkler,, A. Ton, and, M. A. Jabra-Rizk. 2009. A novel immune evasion strategy of Candida albicans: proteolytic cleavage of a salivary antimicrobial peptide. PLoS One 4: e5039.

52. Messana, I.,, T. Cabras,, E. Pisano,, M. T. Sanna,, A. Olianas,, B. Manconi,, M. Pellegrini,, G. Paludetti,, E. Scarano,, A. Fiorita,, S. Agostino,, A. M. Contucci,, L. Calo,, P. M. Picciotti,, A. Manni,, A. Bennick,, A. Vitali,, C. Fanali,, R. Inzitari, and, M. Castagnola. 2008. Trafficking and postsecretory events responsible for the formation of secreted human salivary peptides: a proteomics approach. Mol. Cell. Proteomics 7: 911– 926.

53. Mochon, A. B.,, and H. Liu. 2008. The antimicrobial peptide histatin-5 causes a spatially restricted disruption on the Candida albicans surface, allowing rapid entry of the peptide into the cytoplasm. PLoS Pathog. 4: e1000190.

54. O’Brien-Simpson, N. M.,, S. G. Dashper, and, E. C. Reynolds. 1998. Histatin 5 is a substrate and not an inhibitor of the Arg- and Lys-specific proteinases of Porphyromonas gingivalis. Biochem. Biophys. Res. Commun. 250: 474– 478.

55. Oppenheim, F. G.,, T. Xu,, F. M. McMillian,, S. M. Levitz,, R. D. Diamond,, G. D. Offner, and, R. F. Troxler. 1988. Histatins, a novel family of histidine-rich proteins in human parotid secretion. Isolation, characterization, primary structure, and fungistatic effects on Candida albicans. J. Biol. Chem. 263: 7472– 7477.

56. Oppenheim, F. G.,, Y. C. Yang,, R. D. Diamond,, D. Hyslop,, G. D. Offner, and, R. F. Troxler. 1986. The primary structure and functional characterization of the neutral histidine-rich polypeptide from human parotid secretion. J. Biol. Chem. 261: 1177– 1182.

57. Paquette, D. W.,, D. M. Simpson,, P. Friden,, V. Braman, and, R. C. Williams. 2002. Safety and clinical effects of topical histatin gels in humans with experimental gingivitis. J. Clin. Periodontol. 29: 1051– 1058.

58. Piludu, M.,, M. S. Lantini,, M. Cossu,, M. Piras,, F. G. Oppenheim,, E. J. Helmerhorst,, W. Siqueira, and, A. R. Hand. 2006. Salivary histatins in human deep posterior lingual glands (of von Ebner). Arch. Oral Biol. 51: 967– 973.

59. Pusateri, C. R.,, E. A. Monaco, and, M. Edgerton. 2009. Sensitivity of Candida albicans biofilm cells grown on denture acrylic to antifungal proteins and chlorhexidine. Arch. Oral Biol. 54: 588– 594.

60. Raj, P. A.,, M. Edgerton, and, M. J. Levine. 1990. Salivary histatin 5: dependence of sequence, chain length, and helical conformation for candidacidal activity. J. Biol. Chem. 265: 3898– 3905.

61. Raj, P. A.,, E. Marcus, and, D. K. Sukumaran. 1998. Structure of human salivary histatin 5 in aqueous and non-aqueous solutions. Biopolymers 45: 51– 67.

62. Rijnkels, M.,, L. Elnitski,, W. Miller, and, J. M. Rosen. 2003. Multispecies comparative analysis of a mammalian-specific genomic domain encoding secretory proteins. Genomics 82: 417– 432.

63. Rothstein, D. M.,, E. J. Helmerhorst,, P. Spacciapoli,, F. G. Oppenheim, and, P. Friden. 2002. Histatin-derived peptides: potential agents to treat localised infections. Expert Opin. Emerg. Drugs 7: 47– 59.

64. Rothstein, D. M.,, P. Spacciapoli,, L. T. Tran,, T. Xu,, F. D. Roberts,, M. Dalla Serra,, D. K Buxton,, F. G. Oppenheim, and, P. Friden. 2001. Anticandidal activity is retained in P-113, a 12-amino-acid fragment of histatin 5. Antimicrob. Agents Chemother. 45: 1367– 1373.

65. Ruissen, A. L.,, J. Groenink,, E. J. Helmerhorst,, E. Walgreen-Weterings,, W. Van’t Hof,, E. C. Veerman, and, A. V. Nieuw Amerongen. 2001. Effects of histatin 5 and derived peptides on Candida albicans. Biochem. J. 356: 361– 368.

66. Sabatini, L. M.,, and E. A. Azen. 1989. Histatins, a family of salivary histidine-rich proteins, are encoded by at least two loci (HIS1 and HIS2). Biochem. Biophys. Res. Commun. 160: 495– 502.

67. Sajjan, U. S.,, L. T. Tran,, N. Sole,, C. Rovaldi,, A. Akiyama,, P. M. Friden,, J. F. Forstner, and, D. M. Rothstein. 2001. P-113D, an antimicrobial peptide active against Pseudomonas aeruginosa, retains activity in the presence of sputum from cystic fibrosis patients. Antimicrob. Agents Chemother. 45: 3437– 3444.

68. Sander, C. S.,, U. C. Hipler,, U. Wollina, and, P. Elsner. 2002. Inhibitory effect of terbinafine on reactive oxygen species (ROS) generation by Candida albicans. Mycoses 45: 152– 155.

69. Shimotoyodome, A.,, H. Kobayashi,, I. Tokimitsu,, T. Matsukubo, and, Y. Takaesu. 2006. Statherin and histatin 1 reduce parotid saliva-promoted Streptococcus mutans strain MT8148 adhesion to hydroxyapatite surfaces. Caries Res. 40: 403– 411.

70. Siqueira, W. L.,, H. C. Margolis,, E. J. Helmerhorst,, F. M. Mendes, and, F. G. Oppenheim. 2010. Evidence of intact histatins in the in vivo acquired enamel pellicle. J. Dent. Res. 29: 29.

71. Situ, H.,, S. V. Balasubramanian, and, L. A. Bobek. 2000. Role of alpha-helical conformation of histatin-5 in candidacidal activity examined by proline variants. Biochim. Biophys. Acta 1475: 377– 382.

72. Sun, J. N.,, W. Li,, W. S. Jang,, N. Nayyar,, M. D. Sutton, and, M. Edgerton. 2008. Uptake of the antifungal cationic peptide Histatin 5 by Candida albicans Ssa2p requires binding to non-conventional sites within the ATPase domain. Mol. Microbiol. 70: 1246– 1260.

73. Sun, X.,, E. Salih,, F. G. Oppenheim, and, E. J. Helmerhorst. 2009. Kinetics of histatin proteolysis in whole saliva and the effect on bioactive domains with metal-binding, antifungal, and wound-healing properties. FASEB J. 23: 2691– 2701.

74. Tam, J. P.,, Y. A. Lu, and, J. L. Yang. 2002. Correlations of cationic charges with salt sensitivity and microbial specificity of cystine-stabilized beta-strand antimicrobial peptides. J. Biol. Chem. 277: 50450– 50456.

75. Torres, S. R.,, A. Garzino-Demo,, T. F. Meiller,, V. Meeks, and, M. A. Jabra-Rizk. 2009. Salivary histatin-5 and oral fungal colonisation in HIV+ individuals. Mycoses 52: 11– 15.

76. Tsai, H.,, and L. A. Bobek. 1997. Studies of the mechanism of human salivary histatin-5 candidacidal activity with histatin-5 variants and azole-sensitive and -resistant Candida species. Antimicrob. Agents Chemother. 41: 2224– 2228.

77. Tsai, H.,, P. A. Raj, and, L. A. Bobek. 1996. Candidacidal activity of recombinant human salivary histatin-5 and variants. Infect. Immun. 64: 5000– 5007.

78. Van Dyke, T.,, D. Paquette,, S. Grossi,, V. Braman,, J. Massaro,, R. D’Agostino,, S. Dibart, and, P. Friden. 2002. Clinical and microbial evaluation of a histatin-containing mouthrinse in humans with experimental gingivitis: a phase-2 multi-center study. J. Clin. Periodontol. 29: 168– 176.

79. Veerman, E. C.,, K. Nazmi,, W. Van’t Hof,, J. G. Bolscher,, A. L. Den Hertog, and, A. V. Nieuw Amerongen. 2004. Reactive oxygen species play no role in the candidacidal activity of the salivary antimicrobial peptide histatin 5. Biochem. J. 381: 447– 452.

80. Veerman, E. C.,, M. Valentijn-Benz,, K. Nazmi,, A. L. Ruissen,, E. Walgreen-Weterings,, J. van Marle,, A. B. Doust,, W. van’t Hof,, J. G. Bolscher, and, A. V. Amerongen. 2007. Energy depletion protects Candida albicans against antimicrobial peptides by rigidifying its cell membrane. J. Biol. Chem. 282: 18831– 18841.

81. Vitorino, R.,, M. J. Calheiros-Lobo,, J. A. Duarte,, P. M. Domingues, and, F. M. Amado. 2008. Peptide profile of human acquired enamel pellicle using MALDI tandem MS. J. Sep. Sci. 31: 523– 537.

82. Vylkova, S.,, W. S. Jang,, W. Li,, N. Nayyar, and, M. Edgerton. 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.

83. Vylkova, S.,, X. S. Li,, J. C. Berner, and, M. Edgerton. 2006. Distinct antifungal mechanisms: beta-defensins require Candida albicans Ssa1 protein, while Trk1p mediates activity of cysteine-free cationic peptides. Antimicrob. Agents Chemother. 50: 324– 331.

84. Wunder, D.,, J. Dong,, D. Baev, and, M. Edgerton. 2004. Human salivary histatin 5 fungicidal action does not induce programmed cell death pathways in Candida albicans. Antimicrob. Agents Chemother. 48: 110– 115.

85. Xu, T.,, S. M. Levitz,, R. D. Diamond, and, F. G. Oppenheim. 1991. Anticandidal activity of major human salivary histatins. Infect. Immun. 59: 2549– 2554.

86. Xu, Y.,, I. Ambudkar,, H. Yamagishi,, W. Swaim,, T. J. Walsh, and, B. C. O’Connell. 1999. Histatin 3-mediated killing of Candida albicans: effect of extracellular salt concentration on binding and internalization. Antimicrob. Agents Chemother. 43: 2256– 2262.

87. Yin, A.,, H. C. Margolis,, J. Grogan,, Y. Yao,, R. F. Troxler, and, F. G. Oppenheim. 2003. Physical parameters of hydroxyapatite adsorption and effect on candidacidal activity of histatins. Arch. Oral Biol. 48: 361– 368.

88. Zhou, X.,, W. Yin,, S. Q. Doi,, S. W. Robinson,, K. Takeyasu, and, X. Fan. 2003. Stimulation of Na,K-ATPase by low potassium requires reactive oxygen species. Am. J. Physiol. Cell Physiol. 285: C319–C326.

89. Zhu, J.,, P. W. Luther,, Q. Leng, and, A. J. Mixson. 2006. Synthetic histidine-rich peptides inhibit Candida species and other fungi in vitro: role of endocytosis and treatment implications. Antimicrob. Agents Chemother. 50: 2797– 2805.