Lysis-independent bacterial killing by cell wall-targeting antibiotics

Antibiotics targeting bacterial cell wall synthesis are generally assumed to induce cell lysis as their core antibacterial mechanism. This process is catalysed by cell wall autolysins that degrade peptidoglycan in an uncontrolled manner upon inhibition of cell wall synthesis. The resulting degradation weakens the cell wall sacculus, ultimately leading to cell lysis. However, lysis alone cannot explain the rapid killing observed in Gram-positive bacteria, which often die much faster than they lyse.

To investigate the mechanisms behind this unexpectedly rapid killing, we analysed the mode of action of cell wall antibiotics using microscopic, single-cell techniques. Our experiments with Bacillus subtilis and Staphylococcus aureus revealed that inhibition of cell wall synthesis triggers depolarisation of the cytoplasmic membrane, which precedes and is independent of the lysis process.

Using various fluorescence reporters and cellular assays, we found that depolarisation induced by cell wall antibiotics leads to energy starvation, extensive disturbances in cellular organisation, and production of reactive oxygen species (ROS) that leads to protein and DNA damage. Thus, rather than being a simple lytic process, the bactericidal activity of cell wall-targeting antibiotics is a complex cellular phenomenon that integrates autolysis with severe disturbances and damage triggered by membrane depolarisation.