Polymyxin B lethality requires energy-dependent outer membrane disruption

Abstract Polymyxin antibiotics target lipopolysaccharide (LPS) in both membranes of the bacterial cell envelope, leading to bacterial killing through a mechanism that remains poorly understood. Here, we demonstrate that metabolic activity is essential for polymyxin lethality and leverage this insight to determine its mode of action. Polymyxin B (PmB) efficiently killed exponential phase E. coli but was unable to eliminate stationary phase cells unless a carbon source was available. Antibiotic lethality correlated with surface protrusions, LPS loss, and a significant reduction in outer membrane (OM) barrier function, processes that required LPS synthesis and transport. While the energy-dependent OM disruption was not directly lethal, it facilitated PmB access to the inner membrane (IM), which the antibiotic permeabilised in an energy-independent manner, leading to cell death. Finally, we show that the polymyxin resistance determinant MCR-1 prevents PmB-mediated LPS loss and OM protrusions and thereby renders the antibiotic ineffective.