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 (β-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.

How Does It Kill?: Understanding the Candidacidal Mechanism of Salivary Histatin 5

“…Vacuolar localization of Hst 5 occurred in Hst 5-treated C. albicans cells and this localization was reduced in endocytic mutants (28,29), indicating a role for the endocytic pathway in uptake. However, endocytic uptake is insignificant in terms of Hst 5 toxicity, since cells defective in the endocytic pathway or cells lacking vacuoles had no differences in Hst 5 susceptibilities compared to the wild-type (WT) cells (28,29).…”

“…Vacuolar localization of Hst 5 occurred in Hst 5-treated C. albicans cells and this localization was reduced in endocytic mutants (28,29), indicating a role for the endocytic pathway in uptake. However, endocytic uptake is insignificant in terms of Hst 5 toxicity, since cells defective in the endocytic pathway or cells lacking vacuoles had no differences in Hst 5 susceptibilities compared to the wild-type (WT) cells (28,29). In contrast, C. albicans cells lacking genes encoding Rvs161 and Rvs167 BinAmphiphysin-Rvs homology domain proteins (that were shown to play a significant role in endocytosis) exhibited resistance toward Hst 5, leading the authors to conclude that endocytosis may play some role in the candidacidal mechanism of Hst 5 (41).…”

“…Interestingly, energy-depleted cells had longer cell wall retention times, and a small number of such cells had simultaneous uptake of Hst 5 and propidium iodine (PI) (indicative of membrane permeabilization or pore formation) (29). This led to two important observations: Hst 5 uptake is energy dependent, and more importantly, Hst 5 may indeed have direct membrane effects, albeit when applied to cells at sufficiently higher concentrations (greater than the maximal physiological concentration of 30 M) over a longer time span.…”

“…Hst 5 uptake and killing are lower in C. albicans ssa2⌬/⌬ cells (35), showing that binding to the cell wall is an important step in allowing for localized retention before energy-dependent uptake. However, surface binding, unlike uptake, is energy independent, since energy-depleted cells (cells treated with sodium azide, which reduces ATP synthesis) still had abundant Hst 5 binding but not intracellular uptake (29).…”

“…Thus, uptake of Hst 5 may involve all three uptake pathways (transporter-mediated uptake, direct transfer across the membrane, and receptor-mediated endocytosis). The actual uptake potentially is a combination of one or more these processes, the choice of which is seemingly dependent on Hst 5 concentration (28,29).…”