title: Salivary histatin 5 internalization by translocation, but not endocytosis, is required for fungicidal activity in Candida albicans.
abstract: Salivary histatin 5 (Hst 5) is a cationic salivary protein with high fungicidal activity against Candida albicans. Binding to the cell wall followed by intracellular translocation is required for killing; however, specific binding components and critical toxic events are not understood. In this study, laminarin (beta-1,3-glucan) but not sialic acid, mannan or pustulan mediated Hst 5 binding to C. albicans, and was disassociated by 100 mM NaCl. Time-lapse confocal microscopy revealed a dose-dependent rate of cytosolic uptake of Hst 5 that invariably preceded propidium iodide (PI) entry, demonstrating that translocation itself does not disrupt membrane integrity. Cell toxicity was manifest by vacuolar expansion followed by PI entrance; however, loss of endocytotic vacuolar trafficking of Hst 5 did not reduce killing. Extracellular NaCl (100 mM), but not sorbitol, prevented vacuolar expansion and PI entry in cells already containing cytosolic Hst 5, thus showing a critical role for ionic balance in Hst 5 toxicity. Hst 5 uptake, but not cell wall binding, was blocked by pretreatment with azide or carbonyl cyanide m-chlorophenylhydrazone; however, 10% of de-energized cells had membrane disruption. Thus, Hst 5 is capable of heterogeneous intracellular entry routes, but only direct cytosolic translocation causes cell death as a result of ionic efflux.
authors.affiliation: Department of Oral Biology, School of Dental Medicine, State University of New York at Buffalo, Buffalo, NY 14214, USA.
authors.forename: Woong Sik
authors.initials: WS
authors.lastname: Jang
authors.forename: Jashanjot Singh
authors.initials: JS
authors.lastname: Bajwa
authors.forename: Jianing N
authors.initials: JN
authors.lastname: Sun
authors.forename: Mira
authors.initials: M
authors.lastname: Edgerton
mesh_terms: D002176:Candida albicans / Q000378:metabolism*; D002473:Cell Wall / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004705:Endocytosis; D054808:Histatins / Q000378:metabolism*; D006801:Humans; D011419:Propidium / Q000494:pharmacology; D021381:Protein Transport; D012965:Sodium Chloride / Q000494:pharmacology; D013012:Sorbitol / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism*; D047071:beta-Glucans / Q000378:metabolism*
chemical_list: C068075:HTN3 protein, human; D054808:Histatins; D047071:beta-Glucans; D011419:Propidium; D012965:Sodium Chloride; D013012:Sorbitol; C033363:beta-1,3-glucan