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Chapter 13 : Salivary Histatins: Structure, Function, and Mechanisms of Antifungal Activity
Category: Fungi and Fungal Pathogenesis; Clinical Microbiology
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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.
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
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
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
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
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