Heteroaggregation of Antimicrobial Peptides LL-37 and HNP-1 Drives Cooperative Neutralization of Cytotoxicity

Synergistic bactericidal effects between antimicrobial peptides (AMPs) have been widely studied, yet their cooperative roles in reducing host-cell cytotoxicity remain poorly understood. Inspired by the cross-seeding observed in α-synuclein and β-amyloid fibril formation, we report that the cooperative reduction of cytotoxicity by two major AMPs, human cathelicidin (LL-37) and human α-defensin-1 (HNP-1), is governed by their heteroaggregation states. Through the integration of dynamic light scattering (DLS), MTT assays, Fluo-3 calcium imaging, live/dead staining, apoptosis/necrosis assays, ROS detection (DCFH-DA), and both optical and atomic force microscopy, we demonstrate that the neutralization of MDCK-I cytotoxicity occurs only at a stoichiometric ratio of HNP-1 to LL-37 of approximately 0.025. Furthermore, the observed loss of function results from mutual antagonism: HNP-1 suppresses LL-37-induced necrosis, while LL-37 reduces the level of HNP-1-induced apoptosis. These findings highlight peptide aggregation as a critical determinant of AMP structure–function relationships.