Salivary histatin 5 internalization by translocation, but not endocytosis, is required for fungicidal activity in Candida albicans.
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.
Jang
Woong Sik
WS
Department of Oral Biology, School of Dental Medicine, State University of New York at Buffalo, Buffalo, NY 14214, USA.
Bajwa
Jashanjot Singh
JS
Sun
Jianing N
JN
Edgerton
Mira
M
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*
C068075:HTN3 protein, human; D054808:Histatins; D047071:beta-Glucans; D011419:Propidium; D012965:Sodium Chloride; D013012:Sorbitol; C033363:beta-1,3-glucan