title: Daptomycin inhibits cell envelope synthesis by interfering with fluid membrane microdomains.
abstract: Daptomycin is a highly efficient last-resort antibiotic that targets the bacterial cell membrane. Despite its clinical importance, the exact mechanism by which daptomycin kills bacteria is not fully understood. Different experiments have led to different models, including (i) blockage of cell wall synthesis, (ii) membrane pore formation, and (iii) the generation of altered membrane curvature leading to aberrant recruitment of proteins. To determine which model is correct, we carried out a comprehensive mode-of-action study using the model organismBacillus subtilisand different assays, including proteomics, ionomics, and fluorescence light microscopy. We found that daptomycin causes a gradual decrease in membrane potential but does not form discrete membrane pores. Although we found no evidence for altered membrane curvature, we confirmed that daptomycin inhibits cell wall synthesis. Interestingly, using different fluorescent lipid probes, we showed that binding of daptomycin led to a drastic rearrangement of fluid lipid domains, affecting overall membrane fluidity. Importantly, these changes resulted in the rapid detachment of the membrane-associated lipid II synthase MurG and the phospholipid synthase PlsX. Both proteins preferentially colocalize with fluid membrane microdomains. Delocalization of these proteins presumably is a key reason why daptomycin blocks cell wall synthesis. Finally, clustering of fluid lipids by daptomycin likely causes hydrophobic mismatches between fluid and more rigid membrane areas. This mismatch can facilitate proton leakage and may explain the gradual membrane depolarization observed with daptomycin. Targeting of fluid lipid domains has not been described before for antibiotics and adds another dimension to our understanding of membrane-active antibiotics.
authors: Institute of Pharmaceutical Microbiology, University of Bonn, 53115 Bonn, Germany.|German Centre for Infection Research, 53127 Bonn, Germany.
authors: Anna
authors: A
authors: Müller
authors: Bacterial Cell Biology, Swammerdam Institute for Life Sciences, University of Amsterdam, 1081 HZ Amsterdam, The Netherlands.
authors: Michaela
authors: M
authors: Wenzel
authors: Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne NE2 4AX, United Kingdom.
authors: Henrik
authors: H
authors: Strahl
authors: Institute of Pharmaceutical Microbiology, University of Bonn, 53115 Bonn, Germany.|German Centre for Infection Research, 53127 Bonn, Germany.
authors: Fabian
authors: F
authors: Grein
authors: Bacterial Cell Biology, Swammerdam Institute for Life Sciences, University of Amsterdam, 1081 HZ Amsterdam, The Netherlands.
authors: Terrens N V
authors: TNV
authors: Saaki
authors: Applied Microbiology, Ruhr University Bochum, 44801 Bochum, Germany.
authors: Bastian
authors: B
authors: Kohl
authors: Bacterial Cell Biology, Swammerdam Institute for Life Sciences, University of Amsterdam, 1081 HZ Amsterdam, The Netherlands.
authors: Tjalling
authors: T
authors: Siersma
authors: Applied Microbiology, Ruhr University Bochum, 44801 Bochum, Germany.
authors: Julia E
authors: JE
authors: Bandow
authors: German Centre for Infection Research, 53127 Bonn, Germany.|Institute of Medical Microbiology, Immunology and Parasitology, University of Bonn, 53115 Bonn, Germany.
authors: Hans-Georg
authors: HG
authors: Sahl
authors: Institute of Pharmaceutical Microbiology, University of Bonn, 53115 Bonn, Germany; l.w.hamoen@uva.nl tanja@mibi03.meb.uni-bonn.de.|German Centre for Infection Research, 53127 Bonn, Germany.
authors: Tanja
authors: T
authors: Schneider
authors: Bacterial Cell Biology, Swammerdam Institute for Life Sciences, University of Amsterdam, 1081 HZ Amsterdam, The Netherlands; l.w.hamoen@uva.nl tanja@mibi03.meb.uni-bonn.de.
authors: Leendert W
authors: LW
authors: Hamoen
keywords: Bacillus subtilis; antibiotics; cell wall biosynthesis; daptomycin; membrane potential