Dissecting the cellular effects of antimicrobial agents: The spatio-temporal impact of peptidoglycan synthesis inhibiting antibiotics in <em>Staphylococcus aureus</em> and mechanism of action elucidation of epilancin A37

Abstract

The increasing threat of antibiotic resistance requires innovation in antibiotic research and development. However, the knowledge of how antimicrobial agents kill bacteria is still limited, even for clinically relevant antibiotics. Bacterial cell wall biosynthesis is the target of many important antibiotics. Its spatiotemporal organization is closely coordinated with cell division. However, the roles of the cell wall biosynthesis machinery (CWBM) and peptidoglycan synthesis (PGS) within cell division are not fully understood. Even less is known about the impact of antibiotics on the coordination of these two essential processes. <br /> In this work, the cellular effects of clinically used PGS-targeting antibiotics on <em>Staphylococcus aureus</em> were investigated to construct a model of how PGS inhibition impacts the spatio-temporal organization of the CWBM and cell division. Blocking the ultimate PGS substrate lipid II with the glycopeptide antibiotics vancomycin or telavancin caused a complete inhibition of septum constriction. The beta-lactam oxacillin stopped cell division by preventing recruitment of the major peptidoglycan synthase PBP2 to the septum. Accordingly, this work identifies cell division as a main cellular target of PGS-targeting antibiotics. It further provides evidence that PGS is the essential driving force of septum constriction throughout cell division of <em>S. aureus</em> and reveals PBP2 as being crucial for septum closure. Inhibition of PGS was found to ultimately cause total arrest of <em>S. aureus</em> cell division. This newly established framework was subsequently used to investigate the cellular effects of the PGS-inhibiting natural product moenomycin and the small synthetic molecule DCAP on the CWBM and cell division. Moenomycin was found to impair cell splitting independent from its PGS-inhibiting action, indicating an additional target of moenomycin, which is involved in cell separation. DCAP was found to interfere with septum formation and CWBM organization, suggesting a PGS-inhibiting mechanism of action. Additionally, the impact of several PGS-targeting antibiotics on the transmembrane potential was investigated and the interaction of the <em>S. aureus</em> heme-transporter and virulence factor IsdF with the fluid membrane microdomain scaffold protein FloA was examined. <br /> Similar to the knowledge gap for how antibiotics kill pathogens, not much is known about the mechanisms of action and ecological roles of the many antimicrobial agents produced by members of the human microbiome, despite its clearly established role in health and infection. In this work, cellular effects of the epilancin A37 from a human nasal <em>Staphylococcus epidermidis</em> isolate were investigated using the model organism <em>Corynebacterium glutamicum</em>. Staphylococci and corynebacteria constitute key genera of the nasal microbiome, and production of A37 was found to convey an advantage to <em>S. epidermidis</em> in this inter-species competition. A37 was found to enter the corynebacterial cytoplasm without impairing the cell membrane in a partially transmembrane dependent manner. Upon cytoplasmic accumulation, A37 was found to induce the formation of intracellular membrane vesicles, which were found to be linked to antibacterial activity. Thus, this work provides evidence for a microbiome shaping effect of epilancin A37 and reveals that A37 kills corynebacteria with an intricate and unique mechanism of action.