In-situ study of Bacterial Cell Wall Disruption

Antimicrobial resistance is a growing global threat, projected to cause 10 million deaths annually by 2050. To develop better treatments, it is essential to understand how antibiotics interact with bacterial cells, particularly their cell walls. In gram-positive bacteria like Staphylococcus aureus, survival depends on the structural integrity of the peptidoglycan (PG) layer. ß-lactam antibiotics target and disrupt PG synthesis, but their effectiveness drops in stationary phase, when cells undergo significant physiological changes in response to nutrient limitation. Two main mechanisms may explain this reduced antibiotic susceptibility. First, nutrient stress may trigger the Stringent Response, a signalling cascade regulated by (pp)pGpp and c-diAMP, which downregulates PG synthesis and other growth-related processes. Second, WalKR, essential for regulating PG hydrolases may be suppressed during stationary phase, reducing PG turnover, leading to a thickened cell walls. Building on previous beamtime, where we characterised PG integrity and cell volume changes across bacterial growth stages, we now propose in-situ measurements on live S. aureus cells. We will monitor PG layer changes in real-time comparing exponential and stationary phases, untreated and antibiotic-treated cells, and mutants lacking either WalKR or the Stringent Response. These experiments will reveal how bacterial cell walls adapt to antibiotics and stress, guiding future strategies to combat AMR.