Daptomycin-Mediated Reorganization of Membrane Architecture Causes Mislocalization of Essential Cell Division Proteins

Abstract

bDaptomycin is a lipopeptide antibiotic used clinically for the treatment of certain types of Gram-positive infections, including those caused by methicillin-resistant Staphylococcus aureus (MRSA). Details of the mechanism of action of daptomycin continue to be elucidated, particularly the question of whether daptomycin acts on the cell membrane, the cell wall, or both. Here, we use fluorescence microscopy to directly visualize the interaction of daptomycin with the model Gram-positive bacterium Bacillus subtilis. We show that the first observable cellular effects are the formation of membrane distortions (patches of membrane) that precede cell death by more than 30 min. Membrane patches are able to recruit the essential cell division protein DivIVA. Recruitment of DivIVA correlates with membrane defects and changes in cell morphology, suggesting a localized alteration in the activity of enzymes involved in cell wall synthesis that could account for previously described effects of daptomycin on cell wall morphology and septation. Membrane defects colocalize with fluorescently labeled daptomycin, DivIVA, and fluorescent reporters of peptidoglycan biogenesis (Bocillin FL and BODIPY FL-vancomycin), suggesting that daptomycin plays a direct role in these events. Our results support a mechanism for daptomycin with a primary effect on cell membranes that in turn redirects the localization of proteins involved in cell division and cell wall synthesis, causing dramatic cell wall and membrane defects, which may ultimately lead to a breach in the cell membrane and cell death. These results help resolve the longstanding questions regarding the mechanism of action of this important class of antibiotics.

The action mechanism of daptomycin

“…64,72 The remainder of the cell surface is non-uniformly labelled, in a pattern that has been described as spiral-shaped 64 or patchy. 72 Daptomycin-rich membrane areas also bind labelled vancomycin and penicillin derivatives, suggesting that daptomycin, too, may bind to one of the substrates or enzymes involved in peptidoglycan synthesis.…”

“…64,72 The remainder of the cell surface is non-uniformly labelled, in a pattern that has been described as spiral-shaped 64 or patchy. 72 Daptomycin-rich membrane areas also bind labelled vancomycin and penicillin derivatives, suggesting that daptomycin, too, may bind to one of the substrates or enzymes involved in peptidoglycan synthesis. 72 An alternative interpretation is based on membrane curvature; daptomycin is proposed to prefer "negative curvature," or concave membrane surfaces in the case of the septum, and "positive curvature," or convex surfaces, in case of the forespore membrane.…”

“…72 Daptomycin-rich membrane areas also bind labelled vancomycin and penicillin derivatives, suggesting that daptomycin, too, may bind to one of the substrates or enzymes involved in peptidoglycan synthesis. 72 An alternative interpretation is based on membrane curvature; daptomycin is proposed to prefer "negative curvature," or concave membrane surfaces in the case of the septum, and "positive curvature," or convex surfaces, in case of the forespore membrane. 72 It should be noted, however, that in the latter study it was not ascertained how the bound daptomycin was distributed between the inner and the outer membrane leaflets.…”

“…72 An alternative interpretation is based on membrane curvature; daptomycin is proposed to prefer "negative curvature," or concave membrane surfaces in the case of the septum, and "positive curvature," or convex surfaces, in case of the forespore membrane. 72 It should be noted, however, that in the latter study it was not ascertained how the bound daptomycin was distributed between the inner and the outer membrane leaflets. The outer leaflet is concave where the inner one is convex, and vice versa; therefore, any curvature preference that daptomycin may have cannot be diagnosed with certainty from the reported findings.…”

A High‐Resolution Crystal Structure that Reveals Molecular Details of Target Recognition by the Calcium‐Dependent Lipopeptide Antibiotic Laspartomycin C

“…While a variety of antibacterial mechanisms have been ascribed to the various CDAs, an atomic‐level understanding of the recognition of their bacterial target(s) remains elusive. At present, daptomycin is the only clinically used CDA, and its mode of action is the topic of ongoing investigation 6, 7, 8, 9. By comparison, the structurally similar CDAs laspartomycin C, friulimycin B, tsushimycin, and amphomycin have more clearly understood antibacterial mechanisms 10, 11, 12, 13, 14.…”