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 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.antibiotics | daptomycin | membrane potential | cell wall biosynthesis | Bacillus subtilis

Daptomycin–Phosphatidylglycerol Domains in Lipid Membranes

“…Caution should to be exercised when interpreting bright spots in fluorescence microscopy images (22). This is because the shape of a high-intensity fluorescent area does not faithfully reflect the shape of the structure from which that emission arises.…”

“…The clustering of membrane proteins appears to be related to the lipid distribution. Müller et al (22) proposed that daptomycin partitions to regions of the membrane rich in fluid lipids and coclusters with these lipids, causing them to coalesce into large patches. When labeled with fluorophores, those clusters are visible as highly fluorescent patches, coincident with fluorescently labeled daptomycin.…”

An Acyl-Linked Dimer of Daptomycin Is Strongly Inhibited by the Bacterial Cell Wall

“…Permeabilization of bacterial cell membranes, while not confirmed by (16), has been observed repeatedly before…”

“…It may be noted that increasing calcium concentrations to unphysiologically high concentrations increases the activity of daptomycin on vegetative Bacillus subtilis cells, and that again this increase is more marked with the dimer than the monomer (see Table 2). Since calcium should mask negative charges in both the cell wall and on daptomycin, this observation suggests that electrostatic repulsion plays an important role in impeding the permeation of daptomycin across the cell wall.The small discrepancy that remains-the dimer at least matches the monomer's activity on liposomes, but it is somewhat less active on L-forms-may leave some scope for a possible contribution of mechanisms other than membrane permeabilization to the antibacterial effect of daptomycin, possibly the inhibition of some biosynthetic pathway (16,24,25) that may be disrupted or diminished in the dimer. However, the antibacterial activity of daptomycin varies with the length of the fatty acyl residue (26), and accordingly the replacement of daptomycin's native decanoyl residue by octadecanedioate to effect dimerization might also account for the observed difference in activity.…”

“…Several investigators have suggested that the bactericidal effect of daptomycin may not be due to membrane permeabilization alone but also involve the direct inhibition of biosynthetic pathways (13)(14)(15)(16). Such a mechanism would require a specific interaction between daptomycin and a bacterial macromolecule or, possibly, certain type of membrane lipid domain (16,17).…”