Characterization of Histatin 5 with Respect to Amphipathicity, Hydrophobicity, and Effects on Cell and Mitochondrial Membrane Integrity Excludes a Candidacidal Mechanism of Pore Formation

Abstract

Histatin 5 is a 24-residue peptide from human saliva with antifungal properties. We recently demonstrated that histatin 5 translocates across the yeast membrane and targets to the mitochondria, suggesting an unusual antifungal mechanism (Helmerhorst, E. J., Breeuwer, P., van't Hof, W., Walgreen-Weterings, E., Oomen, L. C. J. M., Veerman, E. C. I., Nieuw Amerongen, A. V., and Abee, T. The results obtained show that the amphipathic analogs exhibited a high fungicidal activity, a high propensity to form an ␣-helix, dissipated the cytoplasmic transmembrane potential, and uncoupled the respiration of isolated mitochondria, similar to the pore-forming peptide PGLa (Peptide with N-terminal Glycine and C-terminal Leucine-amide). In contrast, histatin 5 and dh-5 showed fewer or none of these features. The difference in these functional characteristics between histatin 5 and dh-5 on the one hand and dhvar1, dhvar4, and PGLa on the other hand correlated well with their predicted affinity for membranes based on hydrophobicity/amphipathicity analysis. These data indicate that the salivary protein histatin 5 exerts its antifungal function through a mechanism other than pore formation.

Respiratory inhibition of isolated mammalian mitochondria by salivary antifungal peptide histatin-5

“…In summary, our results indicate that not only mitochondria from C. albicans [16,17], but also mammalian mitochondria may be targets of histatin-5 toxic action. This finding supports the hypothesis that binding of histatin-5 to fungal cell membrane and its internalisation and transport represent critical events in the entire killing process [38,39] mediated by histatin-5.…”

“…First, Helmerhorst et al suggested that histatin-5, after its entrance in the yeast cell, reaches the mitochondria and inhibits the complexes I and/or III of the respiratory chain. However, the amplitude of the dissipation of the transmembrane potential (DW) is quite different in mammalian and in C. albicans mitochondria, probably due to differences in the method of membrane potential measurements, and even to the features of the cardiac mitochondria system [16,17].…”

“…Biochemical and physiological data evidence that histatin-5 causes structural changes in the cell wall, membrane [13] and it induces release of potassium and ATP [14,15]. Earlier reports have suggested that the cytotoxic activity of histatin-5 may be related to the structural features of the C-terminal fragment of the entire peptide, which in a hydrophobic environment has the propensity to assume a non-amphipathic a-helical structure [16]. Although several biochemical studies have described the toxicity of this peptide in Candida albicans cells, the real mechanism underlying the toxicity has not yet been established.…”

CCL28 Has Dual Roles in Mucosal Immunity as a Chemokine with Broad-Spectrum Antimicrobial Activity

“…5), we originally expected that the antimicrobial mechanism of CCL28 was similar to that of histatin-5, which selectively binds to the mitochondrial membrane after internalization into yeast cells (34,35). However, we now consider this unlikely because of the following observations: 1) CCL28 shows a much broader spectrum of antimicrobial activity than does histatin-5, which is mainly effective against Candida species (26), 2) CCL28 induces a rapid membrane permeability in target microbes, in contrast with the reported relatively slow antimicrobial effect of histatin-5 (34), and 3) FITClabeled CCL28 mainly stained cell surface of C. albicans (data not shown), in contrast with the reported association of histatin-5 to mitochondria membrane within yeast cells (25).…”

The TRK1 Potassium Transporter Is the Critical Effector for Killing of Candida albicans by the Cationic Protein, Histatin 5

“…Hst 5 has been shown not to act like conventional poreforming cationic peptides (8,9). Instead, it acts via a multistep process that includes initial binding of extracellular Hst 5 to a Candida surface protein Ssa1/2p (10), followed by subsequent entry of Hst 5 into the cytoplasm (11,12).…”