Cell wall inhibition in L-forms or via β-lactam antibiotics induces reactive oxygen-mediated bacterial killing through increased glycolytic flux

The peptidoglycan (PG) cell wall is an essential structure for the growth of most bacteria. However, many are capable of switching into a wall-deficient L-form state, which is resistant to antibiotics that target cell wall synthesis, under osmoprotective conditions, including host environments. L-form cells might have an important role in chronic or recurrent infections. Crucially, the cellular pathways involved in switching to and from the L-form state are still poorly understood. This work shows that the lack of cell wall or blocking its synthesis by β-lactam antibiotics, results in an increased flux through glycolysis. This leads to the production of reactive oxygen species (ROS) from the respiratory chain (RC), which prevents L-form growth. Compensation for the metabolic imbalance by slowing down glycolysis, activating gluconeogenesis, or depleting oxygen, enables L-form growth in Bacillus subtilis, Listeria monocytogenes and Staphylococcus aureus. These effects do not occur in Enterococcus faecium, which lacks the RC pathway. Our results collectively show that when cell wall synthesis is blocked under aerobic and glycolytic conditions the perturbation of cellular metabolism causes cell death. We provide a mechanistic framework for many anecdotal descriptions of the optimal conditions for L-form growth and non-lytic killing by β-lactam antibiotics.