Histidine Position Regulates Polybia-MP1 Antimicrobial Activity in E. Coli Membranes

Antimicrobial peptides (AMPs) represent a promising alternative to conventional antibiotics against multidrug-resistant pathogens. The amphipathic peptide Polybia-MP1, a derivative of Polybia paulista wasp venom, exhibits potent antibacterial activity. However, optimizing its cell selectivity, toxicity profile, and membrane-interaction mechanisms remains a crucial challenge. Histidine residues can act as pH-sensitive molecular switches due to their imidazole side chains, altering local peptide charge distribution and protonation state, which in turn modulates peptide-lipid membrane binding, insertion depth, and pore formation. In this study, we evaluated the positional influence of histidine (H) substitution on MP1 activity by comparing it with newly designed analogs containing H at distinct positions along the sequence. Minimal Inhibitory Concentration assays against E. coli ATCC 25922 established comparative antibacterial potency, while hemolytic assays using human erythrocytes assessed cytotoxicity, biocompatibility, and therapeutic selectivity. To elucidate membrane interaction dynamics, inner membrane permeability was monitored E. coli cells in vitro using Sytox Green uptake assays. Direct visualization of GUVs via phase-contrast microscopy allowed real-time observation of morphological disruptions, pore induction, and vesicle leakage induced by peptides. Surface charge dynamics and peptide structuration upon binding to membrane surface were determined using Zeta Potential and Circular Dichroism assays on LUVs, respectively. Our results demonstrate that strategic histidine residues modulate electrostatic interaction with anionic bacterial membranes and alters lipid packing, thereby tuning the pH-dependent activity of the peptides. Notably, the analogue in which all lysine residues were replaced by histidines exhibited reduced antimicrobial activity against E. coli, indicating that histidine incorporation alone is insufficient to maintain optimal activity and highlighting the importance of balancing permanent and pH-dependent positive charges. Overall, these findings provide structural-activity insights for the rational design of histidine-modified AMPs with enhanced antimicrobial efficacy against Gram-negative bacteria while minimizing hemolytic activity. This work was supported by São Paulo Research Foundation (DSA: 2025/09473-6 and 2022/07231-7; MPSC: 2025/16298-6; JEFS: 2026/02264-5; KMFM: 2026/03774-7; TLAL: 2024/10235-0).