Single-Molecule-Level Quantification Based on Atomic Force Microscopy Data Reveals the Interaction between Melittin and Lipopolysaccharide in Gram-Negative Bacteria

The interaction forces and mechanical properties of the interaction between melittin (Mel) and lipopolysaccharide (LPS) are considered to be crucial driving forces for Mel when killing Gram-negative bacteria (GNB). However, how their interaction forces perform at the single-molecule level and the dissociation kinetic characteristics of the Mel/LPS complex remain poorly understood. In this study, the single-molecule-level interaction forces between Mel and LPSs from E. coli K−12, O55:B5, O111:B4, and O128:B12 were explored using atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS). AFM-based dynamic force spectroscopy (DFS) and an advanced analytical model were employed to investigate the kinetic characteristics of the Mel/LPS complex dissociation. The results indicated that Mel could interact with both rough (R)-form LPS (E. coli K−12) and smooth (S)-form LPSs (E. coli O55:B5, O111:B4, and O128:B12). The S-form LPS showed a more robust interaction with Mel than the R-form LPS, and a slight difference existed in the interaction forces between Mel and the diverse S-form LPS. Mel interactions with the S-form LPSs showed greater specific and non-specific interaction forces than the R-form LPS (p < 0.05), as determined by AFM-based SMFS. However, there was no significant difference in the specific and non-specific interaction forces among the three samples of S-form LPSs (p > 0.05), indicating that the variability in the O-antigen did not affect the interaction between Mel and LPSs. The DFS result showed that the Mel/S-form LPS complexes had a lower dissociation rate constant, a shorter energy barrier width, a longer bond lifetime, and a higher energy barrier height, demonstrating that Mel interacted with S-form LPS to form more stable complexes. This research enhances the existing knowledge of the interaction micromechanics and kinetic characteristics of Mel and LPS at the single-molecule level. Our research may help with the design and evaluation of new anti-GNB drugs.