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.

Short antimicrobial peptides rich in arginine (R) and tryptophan (W) interact with membranes. To learn how this interaction leads to bacterial death, we characterized the effects of the minimal pharmacophore RWRWRW-NH₂. A ruthenium-substituted derivative of this peptide localized to the membrane in vivo, and the peptide also integrated readily into mixed phospholipid bilayers that resemble Gram-positive membranes. Proteome and Western blot analyses showed that integration of the peptide caused delocalization of peripheral membrane proteins essential for respiration and cell-wall biosynthesis, limiting cellular energy and undermining cell-wall integrity. This delocalization phenomenon also was observed with the cyclic peptide gramicidin S, indicating the generality of the mechanism. Exogenous glutamate increases tolerance to the peptide, indicating that osmotic destabilization also contributes to antibacterial efficacy. Bacillus subtilis responds to peptide stress by releasing osmoprotective amino acids, in part via mechanosensitive channels. This response is triggered by membrane-targeting bacteriolytic peptides of different structural classes as well as by hypoosmotic conditions. Significance Multidrug-resistant bacteria present an acute problem to medicine, generating interest in novel antimicrobial strategies. Antimicrobial peptides currently are being investigated, both as antibiotics and as immunomodulatory agents. Many antimicrobial peptides interact with the bacterial membrane, a previously underexplored antibiotic target. We present a system-based study of the mode of action of small cationic peptides and the mechanisms that bacteria use to defend against them. We show that peptide integration into the membrane causes delocalization of essential peripheral membrane proteins. This delocalization impacts on two cellular processes, namely respiration and cell-wall biosynthesis. We describe a bacterial survival strategy in which mechanosensitive channels in the bacterial membrane establish osmoprotection against membrane-targeting bacteriolytic peptides. Understanding the peptides9 mode of action and bacterial survival strategies opens up new avenues for devising peptide-based antibacterial strategies.

We have used proteomic technology to elucidate the complex cellular responses of Bacillus subtilis to antimicrobial compounds belonging to classical and emerging antibiotic classes. We established on two-dimensional gels a comprehensive database of cytoplasmic proteins with pIs covering a range of 4 to 7 that were synthesized during treatment with antibiotics or agents known to cause generalized cell damage. Although each antibiotic showed an individual protein expression profile, overlaps in the expression of marker proteins reflected similarities in molecular drug mechanisms, suggesting that novel compounds with unknown mechanisms of action may be classified. Indeed, one such substance, a structurally novel protein synthesis inhibitor (BAY 50-2369), could be classified as a peptidyltransferase inhibitor. These results suggest that this technique gives new insights into the bacterial response toward classical antibiotics and hints at modes of action of novel compounds. Such a method should prove useful in the process of antibiotic drug discovery.

Cold atmospheric-pressure plasmas are currently in use in medicine as surgical tools and are being evaluated for new applications, including wound treatment and cosmetic care. The disinfecting properties of plasmas are of particular interest, given the threat of antibiotic resistance to modern medicine. Plasma effluents comprise (V)UV photons and various reactive particles, such as accelerated ions and radicals, that modify biomolecules; however, a full understanding of the molecular mechanisms that underlie plasma-based disinfection has been lacking. Here, we investigate the antibacterial mechanisms of plasma, including the separate, additive and synergistic effects of plasma-generated (V)UV photons and particles at the cellular and molecular levels. Using scanning electron microscopy, we show that plasma-emitted particles cause physical damage to the cell envelope, whereas UV radiation does not. The lethal effects of the plasma effluent exceed the zone of physical damage. We demonstrate that both plasma-generated particles and (V)UV photons modify DNA nucleobases. The particles also induce breaks in the DNA backbone. The plasma effluent, and particularly the plasma-generated particles, also rapidly inactivate proteins in the cellular milieu. Thus, in addition to physical damage to the cellular envelope, modifications to DNA and proteins contribute to the bactericidal properties of cold atmospheric-pressure plasma.

Allicin (diallyl thiosulfinate) from garlic is a highly potent natural antimicrobial substance. It inhibits growth of a variety of microorganisms, among them antibiotic-resistant strains. However, the precise mode of action of allicin is unknown. Here, we show that growth inhibition of Escherichia coli during allicin exposure coincides with a depletion of the glutathione pool and S-allylmercapto modification of proteins, resulting in overall decreased total sulfhydryl levels. This is accompanied by the induction of the oxidative and heat stress response. We identified and quantified the allicin-induced modification S-allylmercaptocysteine for a set of cytoplasmic proteins by using a combination of label-free mass spectrometry and differential isotope-coded affinity tag labeling of reduced and oxidized thiol residues. Activity of isocitrate lyase AceA, an S-allylmercapto-modified candidate protein, is largely inhibited by allicin treatment in vivo. Allicin-induced protein modifications trigger protein aggregation, which largely stabilizes RpoH and thereby induces the heat stress response. At sublethal concentrations, the heat stress response is crucial to overcome allicin stress. Our results indicate that the mode of action of allicin is a combination of a decrease of glutathione levels, unfolding stress, and inactivation of crucial metabolic enzymes through S-allylmercapto modification of cysteines.