Molecular imaging of isolated peptidoglycan sacculi identifies the mechanism of action of cell wall-inhibiting antibiotics

Antibiotic resistance of pathogenic bacteria needs to be urgently addressed by the development of new antibacterial entities. Although the prokaryotic cell wall comprises a valuable target for this purpose, development of novel cell wall-active antibiotics is mostly missing today. This is mainly caused by hinderances in the assessment of isolated enzymes of the co-dependent murein synthesis machineries, e.g. the elongasome and divisome. We therefore present imaging methodologies to evaluate inhibitors of bacterial cell wall synthesis by high-resolution atomic force microscopy on isolated Escherichia coli murein sacculi. With the ability to elucidate the peptidoglycan ultrastructure of E. coli individuals, unprecedented molecular insights into the mechanisms of antibiotics were established. The nanoscopic impairments introduced by ampicillin, amoxicillin and fosfomycin were not only identified by AFM but readily correlated to their known mechanism of action. These valuable in vitro capabilities will facilitate the identification and evaluation of new antibiotic leads in the future.