Michaela Wenzel

Daptomycin inhibits cell envelope synthesis by interfering with fluid membrane microdomains

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. 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.

Analysis of the Mechanism of Action of Potent Antibacterial Hetero-tri-organometallic Compounds: A Structurally New Class of Antibiotics

Two hetero-tri-organometallic compounds with potent activity against Gram-positive bacteria including multi-resistant Staphylococcus aureus (MRSA) were identified. The compounds consist of a peptide nucleic acid backbone with an alkyne side chain, substituted with a cymantrene, a (dipicolyl)Re(CO)3 moiety, and either a ferrocene (FcPNA) or a ruthenocene (RcPNA). Comparative proteomic analysis indicates the bacterial membrane as antibiotic target structure. FcPNA accumulation in the membrane was confirmed by manganese tracing with atomic absorption spectroscopy. Both organometallics disturbed several essential cellular processes taking place at the membrane such as respiration and cell wall biosynthesis, suggesting that the compounds affect membrane architecture. Correlating with enhanced antibacterial activity, oxidative stress was induced only by the ferrocene-substituted compound. The organometallics described here target the cytoplasmic membrane, a clinically proven antibacterial target structure, feature a bactericidal but non-bacteriolytic mode of action and limited cytotoxicity within the limits of solubility. Thus, FcPNA represents a promising lead structure for the development of a new synthetic class of antibiotics.

Proteomic Response of Bacillus subtilis to Lantibiotics Reflects Differences in Interaction with the Cytoplasmic Membrane

ABSTRACT Mersacidin, gallidermin, and nisin are lantibiotics, antimicrobial peptides containing lanthionine. They show potent antibacterial activity. All three interfere with cell wall biosynthesis by binding lipid II, but they display different levels of interaction with the cytoplasmic membrane. On one end of the spectrum, mersacidin interferes with cell wall biosynthesis by binding lipid II without integrating into bacterial membranes. On the other end of the spectrum, nisin readily integrates into membranes, where it forms large pores. It destroys the membrane potential and causes leakage of nutrients and ions. Gallidermin, in an intermediate position, also readily integrates into membranes. However, pore formation occurs only in some bacteria and depends on membrane composition. In this study, we investigated the impact of nisin, gallidermin, and mersacidin on cell wall integrity, membrane pore formation, and membrane depolarization in Bacillus subtilis. The impact of the lantibiotics on the cell envelope was correlated to the proteomic response they elicit in B. subtilis. By drawing on a proteomic response library, including other envelope-targeting antibiotics such as bacitracin, vancomycin, gramicidin S, or valinomycin, YtrE could be identified as the most reliable marker protein for interfering with membrane-bound steps of cell wall biosynthesis. NadE and PspA were identified as markers for antibiotics interacting with the cytoplasmic membrane.

The Lantibiotic NAI-107 Binds to Bactoprenol-bound Cell Wall Precursors and Impairs Membrane Functions

Background: NAI-107 is a potent lantibiotic with an unknown mode of action. Results: NAI-107 targets bactoprenol-bound cell envelope precursors, e.g. lipid II, and in addition affects the bacterial membrane. Conclusion: Cell wall biosynthesis is blocked by sequestration of lipid II and functional disorganization of the cell wall machinery. Significance: The dual mechanism of action may explain the potency of NAI-107 and related lantibiotics. The lantibiotic NAI-107 is active against Gram-positive bacteria including vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus. To identify the molecular basis of its potency, we studied the mode of action in a series of whole cell and in vitro assays and analyzed structural features by nuclear magnetic resonance (NMR). The lantibiotic efficiently interfered with late stages of cell wall biosynthesis and induced accumulation of the soluble peptidoglycan precursor UDP-N-acetylmuramic acid-pentapeptide (UDP-MurNAc-pentapeptide) in the cytoplasm. Using membrane preparations and a complete cascade of purified, recombinant late stage peptidoglycan biosynthetic enzymes (MraY, MurG, FemX, PBP2) and their respective purified substrates, we showed that NAI-107 forms complexes with bactoprenol-pyrophosphate-coupled precursors of the bacterial cell wall. Titration experiments indicate that first a 1:1 stoichiometric complex occurs, which then transforms into a 2:1 (peptide: lipid II) complex, when excess peptide is added. Furthermore, lipid II and related molecules obviously could not serve as anchor molecules for the formation of defined and stable nisin-like pores, however, slow membrane depolarization was observed after NAI-107 treatment, which could contribute to killing of the bacterial cell.

Proteomic signature of fatty acid biosynthesis inhibition available for in vivo mechanism of action studies

ABSTRACT Fatty acid biosynthesis is a promising novel antibiotic target. Two inhibitors of fatty acid biosynthesis, platencin and platensimycin, were recently discovered and their molecular targets identified. Numerous structure-activity relationship studies for both platencin and platensimycin are currently being undertaken. We established a proteomic signature for fatty acid biosynthesis inhibition in Bacillus subtilis using platencin, platensimycin, cerulenin, and triclosan. The induced proteins, FabHA, FabHB, FabF, FabI, PlsX, and PanB, are enzymes involved in fatty acid biosynthesis and thus linked directly to the target pathway. The proteomic signature can now be used to assess the in vivo mechanisms of action of compounds derived from structure-activity relationship programs, as demonstrated for the platensimycin-inspired chromium bioorganometallic PM47. It will further serve as a reference signature for structurally novel natural and synthetic antimicrobial compounds with unknown mechanisms of action. In summary, we described a proteomic signature in B. subtilis consisting of six upregulated proteins that is diagnostic of fatty acid biosynthesis inhibition and thus can be applied to advance antibacterial drug discovery programs.

Modulating the activity of short arginine-tryptophan containing antibacterial peptides with N-terminal metallocenoyl groups

Summary A series of small synthetic arginine and tryptophan containing peptides was prepared and analyzed for their antibacterial activity. The effect of N-terminal substitution with metallocenoyl groups such as ferrocene (FcCO) and ruthenocene (RcCO) was investigated. Antibacterial activity in different media, growth inhibition, and killing kinetics of the most active peptides were determined. The toxicity of selected derivatives was determined against erythrocytes and three human cancer cell lines. It was shown that the replacement of an N-terminal arginine residue with a metallocenoyl moiety modulates the activity of WRWRW-peptides against Gram-positive and Gram-negative bacteria. MIC values of 2–6 µM for RcCO-W(RW)2 and 1–11 µM for (RW)3 were determined. Interestingly, W(RW)2-peptides derivatized with ferrocene were significantly less active than those derivatized with ruthenocene which have similar structural but different electronic properties, suggesting a major influence of the latter. The high activities observed for the RcCO-W(RW)2- and (RW)3-peptides led to an investigation of the origin of activity of these peptides using several important activity-related parameters. Firstly, killing kinetics of the RcCO-W(RW)2-peptide versus killing kinetics of the (RW)3 derivative showed faster reduction of the colony forming units for the RcCO-W(RW)2-peptide, although MIC values indicated higher activity for the (RW)3-peptide. This was confirmed by growth inhibition studies. Secondly, hemolysis studies revealed that both peptides did not lead to significant destruction of erythrocytes, even up to 500 µg/mL for (RW)3 and 250 µg/mL for RcCO-W(RW)2. In addition, toxicity against three human cancer cell lines (HepG2, HT29, MCF7) showed that the (RW)3-peptide had an IC50 value of ~140 µM and the RcW(RW)2 one of ~90 µM, indicating a potentially interesting therapeutic window. Both the killing kinetics and growth inhibition studies presented in this work point to a membrane-based mode of action for these two peptides, each having different kinetic parameters.

Proteomic signatures in antibiotic research

Antibiotics disturb the physiological homeostasis of bacterial cells by interfering with essential cellular functions or structures. The bacterial proteome adjusts quickly in response to antibiotic challenge. This physiological response is specifically tailored to overcome the inflicted damage and, thus, closely linked to the antibiotic target and mechanism of action. In a way, the proteome mirrors the antibiotic insult. This connection can be exploited to guide the development of novel antibiotics. By using structurally different antibiotics, which cause the same physiological disturbance, proteomic signatures diagnostic of the mechanism of action can be defined. These proteomic signatures inform about mechanism‐related differential protein expression as well as about protein modifications. This review recapitulates how antibiotic proteomic signatures are established and highlights areas of antibiotic research benefiting most from proteomic signatures. Antibacterial research programs designed to structurally advance existing antibiotic classes profit from rapid in vivo mechanism of action confirmation. What is more, a comprehensive reference compendium of antibiotic proteomic signatures allows rapid mechanism of action identification of those structurally novel compounds that inhibit known targets. Finally, novel proteomic response profiles indicate unprecedented mechanisms. Here, the proteome profile provides evidence on the nature of the antibiotic‐caused physiological disturbance leading to testable hypotheses on the mechanism of action.

Influence of lipidation on the mode of action of a small RW-rich antimicrobial peptide.

Antimicrobial peptides are a potent class of antibiotics. In the Gram-positive model organism Bacillus subtilis the synthetic peptide RWRWRW-NH2 integrates into the bacterial membrane and delocalizes essential peripheral membrane proteins involved in cell wall biosynthesis and respiration. A lysine residue has been added to the hexapeptide core structure, either C or N-terminally. Lipidation of the lysine residues by a C8-acyl chain significantly improved antibacterial activity against both Gram-positive and Gram-negative bacteria. Here, we report a comparative proteomic study in B. subtilis on the mechanism of action of the lipidated and non-lipidated peptides. All derivatives depolarized the bacterial membrane without forming pores and all affected cell wall integrity. Proteomic profiling of the bacterial stress responses to the small RW-rich antimicrobial peptides was reflective of non-disruptive membrane integration. Overall, our results indicate that antimicrobial peptides can be derivatized with lipid chains enhancing antibacterial activity without significantly altering the mechanism of action. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

Antimicrobial Peptides from the Aurein Family Form Ion-Selective Pores in Bacillus subtilis

The mechanism of action of aurein 2.2 and aurein 2.3, antimicrobial peptides from the frog Litoria aurea, was investigated. Proteomic profiling of the Bacillus subtilis stress response indicates that the cell envelope is the main target for both aureins. Upon treatment, the cytoplasmic membrane depolarizes and cellular ATP levels decrease. Global element analysis shows that intracellular concentrations of certain metal ions (potassium, magnesium, iron, and manganese) strongly decrease. Selective translocation of some ions over others was demonstrated in vitro. The same set of ions also leaks from B. subtilis cells treated with sublethal concentrations of gramicidin S, MP196, and nisin. Aureins do not permeabilize the cell membrane for propidium iodide thus excluding formation of large, unspecific pores. Our data suggest that the aureins acts by forming small pores thereby causing membrane depolarization, and by triggering the release of certain metal ions thus disturbing cellular ion homeostasis.

Extracting iron and manganese from bacteria with ionophores - a mechanism against competitors characterized by increased potency in environments low in micronutrients.

To maintain their metal ion homeostasis, bacteria critically depend on membrane integrity and controlled ion translocation. Terrestrial Streptomyces species undermine the function of the cytoplasmic membrane as diffusion barrier for metal cations in competitors using ionophores. Although the properties of the divalent cation ionophores calcimycin and ionomycin have been characterized to some extent in vitro, their effects on bacterial ion homeostasis, the factors leading to bacterial cell death, and their ecological role are poorly understood. To gain insight into their antibacterial mechanism, we determined the metal ion composition of the soil bacterium Bacillus subtilis after treatment with calcimycin and ionomycin. Within 15 min the cells lost approximately half of their cellular iron and manganese content whereas calcium levels increased. The proteomic response of B. subtilis provided evidence that disturbance of metal cation homeostasis is accompanied by intracellular oxidative stress, which was confirmed with a ROS‐specific fluorescent probe. B. subtilis showed enhanced sensitivity to the ionophores in medium lacking iron or manganese. Furthermore, in the presence of ionophores bacteria were sensitive to high calcium levels. These findings suggest that divalent cation ionophores are particularly effective against competing microorganisms in soils rich in available calcium and low in available iron and manganese.