Human Histatin 5 Exerts Anti-Trypanosomal Activity Against Trypanosoma cruzi and Induces Ultrastructural Damage, Apoptosis-like Cell Death, and Oxidative/Nitrosative Stress

Chagas disease, caused by Trypanosoma cruzi, remains a neglected tropical disease of relevance due to its chronic complications and limited therapeutic options. Current treatments, mainly benznidazole and nifurtimox, are limited by toxicity, adverse effects, and reduced efficacy in chronic infection. Therefore, antimicrobial peptides (AMPs) have emerged as promising candidates for identifying new antiparasitic strategies because of their ability to affect multiple cellular targets. In this study, we evaluated the anti-trypanosomal activity, host-cell cytotoxicity, and cellular alterations induced by the human salivary peptide Histatin 5 (Hist 5) against T. cruzi epimastigotes. The reversibility of the antiparasitic effect was assessed through washout assays after 48 h of exposure. Cytotoxicity was evaluated in Vero cells and RAW264.7 macrophages, whereas parasite susceptibility was determined using dose–response curves. The reversibility of the antiparasitic effect was assessed through washout assays after 48 h of exposure. Ultrastructural alterations induced by Hist 5 were analyzed by transmission electron microscopy. Cell death-associated events were assessed using Annexin V/PI staining and TUNEL assays to detect phosphatidylserine externalization and DNA fragmentation, respectively. Reactive oxygen species and nitric oxide production were quantified using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) fluorescence and the Griess reaction. Hist 5 showed limited cytotoxicity toward mammalian cells and reduced parasite viability in a time- and concentration-dependent manner. Following peptide removal, parasite growth recovered only partially, indicating that the antiparasitic effect was not completely reversible. Hist 5 also induced marked ultrastructural damage, apoptosis-like cell death, increased intracellular ROS levels, and enhanced NO production. These findings suggest that Hist 5 affects T. cruzi epimastigotes through multiple cellular alterations while exerting limited effects on host-cell viability.