Membrane depolarisation induced by cell wall-targeting antibiotics triggers bactericidal activity independent of cell lysis

Abstract

Antibiotics that target different steps of cell wall synthesis are generally thought to induce cell lysis as the shared bactericidal mode of action. This lysis process is catalysed by autolysins, which, upon inhibition of synthesis, degrade peptidoglycan until the cell wall is no longer able to withstand the turgor pressure. However, lysis is insufficient to explain the entirety of the bacterial killing observed, especially in Gram-positive bacteria which have a thicker cell wall. Preliminary research leading to my thesis project found heterogeneous depolarisation of the cytoplasmic membrane that precedes cell lysis in Bacillus subtilis upon addition of cell wall targeting antibiotics vancomycin, penicillin G and D-cycloserine. Following this discovery, my research focused on different aspects of this phenomenon, including how well it is conserved in different bacteria, what is the underlying mechanism, and how much it contributes to the bactericidal activity. To answer these questions, the mode of action of antibiotics was evaluated on a single-cell level using predominantly fluorescence microscopic techniques. I found that cell wall-targeting antibiotics induce membrane depolarisation before lysis in the Grampositive Firmicutes B. subtilis and Staphylococcus aureus, but only rarely in the Gram-negative Proteobacterium Escherichia coli. The collected evidence suggests that the observed membrane depolarisation is not triggered by an arrest of peptidoglycan synthesis per se, but rather via an indirect process linked to the continuing accumulation of biomass within a cell envelope unable to expand. In addition, using fluorescence reporters and mass spectrometry, I show that the membrane depolarisation induced by cell wall-targeting antibiotics leads to energy starvation and production of reactive oxygen species (ROS), which is accompanied by protein, lipid and DNA damage. These findings highlight the complexity of the cellular processes underpinning the bactericidal activity of antibiotics and the ability of cell wall-targeting antibiotics to disturb several biological pathways at the same time.