The bacterial cytoplasmic membrane is a compelling target for novel antibiotics due to its essential nature and relative genetic stability. Agents disrupting membrane integrity are less prone to developing conventional resistance, remain effective against metabolically dormant persister cells and biofilms, and employ rapid, diverse bactericidal mechanisms. Natural products have consistently proven to be a fertile ground for drug discovery, yielding unique molecular scaffolds with therapeutic potential (Butler et al., J. Nat. Prod., 2025). A prominent class among them is natural antimicrobial peptides (AMPs), which often operate via membrane-associated mechanisms. Within this promising landscape, our research focuses on the gausemycins—a unique family of Ca²⁺-dependent (glyco)lipopeptide antibiotics that exhibit activity against Gram-positive pathogens (Tyurin et al., Angew. Chem. 2021; Kravchenko et al., J. Nat. Prod. 2024). However, the precise details of their membrane interaction and subsequent bactericidal mechanism remain to be fully elucidated. This study aims to define these molecular details and evaluate their specific interplay with key membrane components.

Our approach combines kinetic analysis of bactericidal activity with studies using model lipid membranes to define the membrane-disrupting properties of gausemycins. To validate the membrane as the primary target and probe resistance pathways, we characterized spontaneous resistant mutants and assessed activity against bacterial strains with engineered membrane alterations, including Bacillus subtilis overexpressing the ugtP gene and clinically relevant daptomycin-resistant strains with lysyl-phosphatidylglycerol-enriched membranes. Finally, fluorescence microscopy was utilized to directly visualize the cellular localization of gausemycin, providing direct evidence for its membrane-targeting mechanism.

This work unravels the mechanism of gausemycin action, positioning it as a distinct class of membrane-targeting antibiotics. The established combination of membrane association and bactericidal efficacy highlights their potential as a promising new class of antibiotics.

This work was supported by the Russian Science Foundation (project № 25-14-00281).