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 organism Bacillus subtilis and 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. Significance To date, simple membrane pore formation resulting in cytoplasmic leakage is the prevailing model for how membrane-active antibiotics kill bacteria and also is one of the main explanations for the activity of the membrane-binding antibiotic daptomycin. However, such models, typically derived from model membrane studies, often depict membranes as homogenous lipid bilayers. They do not take into account the complex architecture of biological membranes, with their many different membrane proteins, or the presence of microdomains with different fluidity properties. Here we report that daptomycin perturbs fluid microdomains in bacterial cell membranes, thereby interfering with membrane-bound cell wall and lipid synthesis processes. Our results add a different perspective as to how membrane-active antibiotics can kill bacteria.

The lipopeptide daptomycin is used as an antibiotic to treat severe infections with gram-positive pathogens, such as methicillin resistant Staphylococcus aureus (MRSA) and drug-resistant enterococci. Its precise mechanism of action is incompletely understood, and a specific molecular target has not been identified. Here we show that Ca²⁺-daptomycin specifically interacts with undecaprenyl-coupled cell envelope precursors in the presence of the anionic phospholipid phosphatidylglycerol, forming a tripartite complex. We use microbiological and biochemical assays, in combination with fluorescence and optical sectioning microscopy of intact staphylococcal cells and model membrane systems. Binding primarily occurs at the staphylococcal septum and interrupts cell wall biosynthesis. This is followed by delocalisation of components of the peptidoglycan biosynthesis machinery and massive membrane rearrangements, which may account for the pleiotropic cellular events previously reported. The identification of carrier-bound cell wall precursors as specific targets explains the specificity of daptomycin for bacterial cells. Our work reconciles apparently inconsistent previous results, and supports a concise model for the mode of action of daptomycin.

The rapid rise of multidrug-resistant (MDR) bacteria has once again caused bacterial infections to become a global health concern. Antimicrobial peptides (AMPs), also known as host defense peptides (HDPs), offer a viable solution to these pathogens due to their diverse mechanisms of actions, which include direct killing as well as immunomodulatory properties (e.g., anti-inflammatory activity). HDPs may hence provide a more robust treatment of bacterial infections. In this review, the advent of and the mechanisms that lead to antibiotic resistance will be described. HDP mechanisms of antibacterial and immunomodulatory action will be presented, with specific examples of how the HDP aurein 2.2 and a few of its derivatives, namely peptide 73 and cG4L73, function. Finally, resistance that may arise from a broader use of HDPs in a clinical setting and methods to improve biocompatibility will be briefly discussed.

The Gram-positive cell wall consists of peptidoglycan functionalized with anionic glycopolymers, such as wall teichoic acid and capsular polysaccharide (CP). How the different cell wall polymers are assembled in a coordinated fashion is not fully understood. Here, we reconstitute Staphylococcus aureus CP biosynthesis and elucidate its interplay with the cell wall biosynthetic machinery. We show that the CapAB tyrosine kinase complex controls multiple enzymatic checkpoints through reversible phosphorylation to modulate the consumption of essential precursors that are also used in peptidoglycan biosynthesis. In addition, the CapA1 activator protein interacts with and cleaves lipid-linked CP precursors, releasing the essential lipid carrier undecaprenyl-phosphate. We further provide biochemical evidence that the subsequent attachment of CP is achieved by LcpC, a member of the LytR-CpsA-Psr protein family, using the peptidoglycan precursor native lipid II as acceptor substrate. The Ser/Thr kinase PknB, which can sense cellular lipid II levels, negatively controls CP synthesis. Our work sheds light on the integration of CP biosynthesis into the multi-component Gram-positive cell wall.