Differential membrane lipid disruption by lipopeptide antibiotics, colistin and turnercyclamycins.

Lipopeptide natural products are essential agents against multidrug-resistant bacteria, but their clinical utility is often constrained by toxicity and resistance. Here, we compare the mechanisms of action of two superficially similar lipopeptide antibiotics: colistin, a last-line treatment for Gram-negative infections, and turnercyclamycins, a new class active against certain colistin-resistant strains. Both antibiotics require lipopolysaccharide (LPS) biosynthesis, even when LPS transport to the outer membrane (OM) is impaired. Colistin rapidly disrupts both the OM and the cytoplasmic membrane (CM), causing swift bacterial death. Turnercyclamycins, by contrast, act independently of the CM, with delayed OM disruption. Unlike colistin, which binds LPS directly to damage membranes, turnercyclamycins show no measurable LPS binding by calorimetry. Instead, their activity is modulated by different phospholipids, as confirmed by phospholipidomic profiling on whole cells, which identifies alterations in bacterial lipid biosynthesis and membrane homeostasis. These findings support a mechanistically distinct mode of action for turnercyclamycins, which we propose to correlate with their different pharmacological properties and potential therapeutic applications. Our results highlight how subtle structural differences between lipopeptides can lead to major functional divergence, offering a framework for the rational design of next-generation antibiotics with improved safety and efficacy profiles.