BPS2026 – Unraveling thermodynamic and kinetic determinants of antimicrobial peptide activity and selectivity

Biophysical studies of antimicrobial peptide (AMP) interactions with model membranes are essential for modelling pore formation mechanisms. However, quantitative data on the interaction of AMPs with live cells are limited. Therefore, we developed assays enabling thermodynamic characterization of AMP interactions with live bacterial cells. Our studies clarified several essential aspects, e.g., the affinity of AMP binding to live bacterial cells and the number of bound peptide molecules required to kill a single bacterium. Despite elucidating several key aspects of AMP function, our findings raised several new questions. For example, we often observed a concentration range in which some bacteria are killed while others survive. This interval, often called the “mutant selection window” (MSW), may allow resistance to develop. Our results indicate that the equilibrium of water/membrane partition for an AMP plays a central role in the heterogeneous response. In liposomes, the MSW is essentially abolished, which may be explained via thermodynamic insights. In bacterial suspensions, binding heterogeneity is strongly influenced by phenotypic or genotypic differences. Nevertheless, a complete understanding of AMP selectivity requires studies under more physiologically relevant conditions, involving two distinct cell populations (bacteria and host cells) or membrane composition. Unlike model membranes, our previous data indicate that selectivity between cells cannot be accounted for by thermodynamics alone, indicating that kinetics play a role too. Accordingly, the kinetics of AMP-cell interactions was investigated. By employing Gram-negative bacteria, we demonstrated that AMPs bind to bacterial membranes within less than a second, perturb them within minutes, and induce bacterial death before inner-membrane permeabilization occurs. Thus, outer-membrane permeabilization is the critical step in bacterial killing. Ultimately, AMPs become localized within partially lysed cells. Therefore, integrating equilibrium and analysis of kinetics provides a comprehensive view of AMP activity and cell selectivity.