Lateral Membrane Heterogeneity Shapes Antimicrobial Association and Convergent Mechanical Responses

Abstract

Bacterial membranes are laterally heterogeneous, creating local physicochemical environments that can shape interactions with membrane-active antimicrobials. However, whether this spatial organization only affects local drug association or propagates into collective membrane responses remains unclear. Here we show that lateral membrane heterogeneity shapes antimicrobial association while distinct molecular responses converge on a common loss of membrane mechanical stability. Combining coarse-grained molecular dynamics, giant unilamellar vesicle fluctuation spectroscopy and microfluidic trapping, we follow octenidine, chlorhexidine and polymyxin B from molecular association to vesicle-scale behaviour. All three compounds preferentially associate with domain-associated membrane environments, but differ in how this preference changes with loading and in their effects on lipid mobility. Despite these differences, all three shift the membrane toward a mechanically softer state, while increasing exposure accelerates loss of vesicle stability under flow. These findings identify lateral membrane heterogeneity as an active determinant of antimicrobial response and provide a physical framework for understanding membrane-targeting activity beyond bulk composition and membrane-averaged charge.