Ramoplanin at Bactericidal Concentrations Induces Bacterial Membrane Depolarization in Staphylococcus aureus

Abstract

Ramoplanin is an actinomycetes-derived antibiotic with broad-spectrum activity against Gram-positive bacteria that has been evaluated in clinical trials for the treatment of gastrointestinal vancomycin-resistant enterococci (VRE) and Clostridium difficile infections. Recent studies have proposed that ramoplanin binds to bacterial membranes as a C2 symmetrical dimer that can sequester Lipid II, which causes inhibition of cell wall peptidoglycan biosynthesis and cell death. In this study, ramoplanin was shown to bind to anionic and zwitterionic membrane mimetics with a higher affinity for anionic membranes and to induce membrane depolarization of methicillin-susceptible Staphylococcus aureus (MSSA) ATCC 25923 at concentrations at or above the minimal bactericidal concentration (MBC). The ultrastructural effects of ramoplanin on S. aureus were also examined by transmission electron microscopy (TEM), and this showed dramatic changes to bacterial cell morphology. The correlation observed between membrane depolarization and bacterial cell viability suggests that this mechanism may contribute to the bactericidal activity of ramoplanin.

Morphological and ultrastructural changes in bacterial cells as an indicator of antibacterial mechanism of action

“…Thickening is most pronounced at the septum because this is where new wall synthesis is most vigorous [190]. Septal thickening has also been observed in bacteria treated with low concentrations of the peptidoglycan synthesis inhibitors vancomycin (0.5xMIC for 2-3 hours) [153] and ramoplanin (1xMIC for 3 hours) [165]. Higher concentrations, at least in the case of vancomycin (≥10xMIC), do not have this effect [165,190].…”

“…Fig. 3) has been observed in bacteria treated with inhibitors of DNA synthesis (ciprofloxacin [83] and norfloxacin [48]), RNA synthesis (rifampicin [83]), protein synthesis (amikacin [200] and quinupristin/dalfopristin [203]), and peptidoglycan synthesis (ramoplanin [165] and several β-lactams [55,83,141]). Bacteria with a collapsed or deflated appearance (Suppl.…”

“…Antibiotics eliciting these effects include the cytoplasmic membrane disruptor daptomycin [164] and the peptidoglycan synthesis inhibitors penicillin [69] and ramoplanin [165]. That these agents disrupt septum formation is not surprising given cell division requires both Z-ring formation at the cytoplasmic membrane, and septal peptidoglycan synthesis [166].…”

“…Lastly, 'ghost cells', a term used to describe lysed bacteria devoid or near-devoid of cytoplasm (Fig. 12), have been observed following treatment with inhibitors of DNA synthesis (ciprofloxacin [52] and norfloxacin [48]), RNA synthesis (bicyclomycin [143]), protein synthesis (clarithromycin [196], erythromycin [83,141], gentamicin [181], and quinupristin/dalfopristin [203]), and peptidoglycan and arabinogalactan synthesis (ramoplanin [165], several β-lactams [55,78,83,120,141], and ethambutol [196]). Like the separation of cell envelope layers (Fig.…”

“…Peptidoglycan synthesis inhibitors cause thickening predominantly at the septal wall [69, 125,127,148,151,153,155,165,190], and the thickened septa lack a central dense layer [125,127,153,158] (Fig. 4).…”