Substitutions in PBP2b from β-lactam resistant Streptococcus pneumoniae have different effects on enzymatic activity and drug reactivity

Pneumococcus resists β-lactams by expressing variants of its target enzymes, the penicillin-binding proteins (PBPs), with many amino acid substitutions. Up to 10% of the sequence can be modified. These altered PBPs have a much reduced reactivity with the drugs but retain their physiological activity of cross-linking the peptidoglycan, the major constituent of the bacterial cell wall. However, as β-lactams are chemical and structural mimics of the natural substrate, resistance mediated by altered PBPs raises the following paradox: how PBPs that react poorly with the drugs maintain a sufficient level of activity with the physiological substrate? This question is addressed for the first time in this study, which compares the peptidoglycan cross-linking activity of PBP2b from susceptible and resistant strains with their inhibition by different β-lactams. Unexpectedly, the enzymatic activity of the variants did not correlate with their antibiotic reactivity. This finding indicates that some of the numerous amino acid substitutions were selected to restore a viable level of enzymatic activity by a compensatory molecular mechanism

Real time monitoring of peptidoglycan synthesis by membrane-reconstituted penicillin binding proteins

Peptidoglycan is an essential component of the bacterial cell envelope that surrounds the cytoplasmic membrane to protect the cell from osmotic lysis. Important antibiotics such as β-lactams and glycopeptides target peptidoglycan biosynthesis. Class A penicillin-binding proteins (PBPs) are bifunctional membrane-bound peptidoglycan synthases that polymerize glycan chains and connect adjacent stem peptides by transpeptidation. How these enzymes work in their physiological membrane environment is poorly understood. Here, we developed a novel Förster resonance energy transfer-based assay to follow in real time both reactions of class A PBPs reconstituted in liposomes or supported lipid bilayers and applied this assay with PBP1B homologues from Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii in the presence or absence of their cognate lipoprotein activator. Our assay will allow unravelling the mechanisms of peptidoglycan synthesis in a lipid-bilayer environment and can be further developed to be used for high-throughput screening for new antimicrobials

Brevibacillin 2V, a Novel Antimicrobial Lipopeptide With an Exceptionally Low Hemolytic Activity

Bacterial non-ribosomally produced peptides (NRPs) form a rich source of antibiotics, including more than 20 of these antibiotics that are used in the clinic, such as penicillin G, colistin, vancomycin, and chloramphenicol. Here we report the identification, purification, and characterization of a novel NRP, i.e., brevibacillin 2V (lipo-tridecapeptide), from Brevibacillus laterosporus DSM 25. Brevibacillin 2V has a strong antimicrobial activity against Gram-positive bacterial pathogens (minimum inhibitory concentration = 2 mg/L), including difficult-to-treat antibiotic-resistant Enterococcus faecium, Enterococcus faecalis, and Staphylococcus aureus. Notably, brevibacillin 2V has a much lower hemolytic activity (HC(50) > 128 mg/L) and cytotoxicity (CC(50) = 45.49 ± 0.24 mg/L) to eukaryotic cells than previously reported NRPs of the lipo-tridecapeptide family, including other brevibacillins, which makes it a promising candidate for antibiotic development. In addition, our results demonstrate that brevibacillins display a synergistic action with established antibiotics against Gram-negative bacterial pathogens. Probably due to the presence of non-canonical amino acids and D-amino acids, brevibacillin 2V showed good stability in human plasma. Thus, we identified and characterized a novel and promising antimicrobial candidate (brevibacillin 2V) with low hemolytic activity and cytotoxicity, which can be used either on its own or as a template for further total synthesis and modification

Brevibacillin 2V Exerts Its Bactericidal Activity via Binding to Lipid II and Permeabilizing Cellular Membranes

Lipo-tridecapeptides, a class of bacterial non-ribosomally produced peptides, show strong antimicrobial activity against Gram-positive pathogens, including antibiotic-resistant Staphylococcus aureus and Enterococcus spp. However, many of these lipo-tridecapeptides have shown high hemolytic activity and cytotoxicity, which has limited their potential to be developed into antibiotics. Recently, we reported a novel antimicrobial lipo-tridecapeptide, brevibacillin 2V, which showed no hemolytic activity against human red blood cells at a high concentration of 128 mg/L, opposite to other brevibacillins and lipo-tridecapeptides. In addition, brevibacillin 2V showed much lower cytotoxicity than the other members of the brevibacillin family. In this study, we set out to elucidate the antimicrobial mode of action of brevibacillin 2V. The results show that brevibacillin 2V acts as bactericidal antimicrobial agent against S. aureus (MRSA). Further studies show that brevibacillin 2V exerts its bactericidal activity by binding to the bacterial cell wall synthesis precursor Lipid II and permeabilizing the bacterial membrane. Combined solid-state NMR, circular dichroism, and isothermal titration calorimetry assays indicate that brevibacillin 2V binds to the GlcNAc-MurNAc moiety and/or the pentapeptide of Lipid II. This study provides an insight into the antimicrobial mode of action of brevibacillin 2V. As brevibacillin 2V is a novel and promising antibiotic candidate with low hemolytic activity and cytotoxicity, the here-elucidated mode of action will help further studies to develop it as an alternative antimicrobial agent

Analyzing mechanisms of action of antimicrobial peptides on bacterial membranes requires multiple complimentary assays and different bacterial strains

Antimicrobial peptides (AMPs) commonly target bacterial membranes and show broad-spectrum activity against microorganisms. In this research we used three AMPs (nisin, epilancin 15×, [R4L10]-teixobactin) and tested their membrane effects towards three strains (Staphylococcus simulans, Micrococcus flavus, Bacillus megaterium) in relation with their antibacterial activity. We describe fluorescence and luminescence-based assays to measure effects on membrane potential, intracellular pH, membrane permeabilization and intracellular ATP levels. The results show that our control peptide, nisin, performed mostly as expected in view of its targeted pore-forming activity, with fast killing kinetics that coincided with severe membrane permeabilization in all three strains. However, the mechanisms of action of both Epilancin 15× as well as [R4L10]-teixobactin appeared to depend strongly on the bacterium tested. In certain specific combinations of assay, peptide and bacterium, deviations from the general picture were observed. This was even the case for nisin, indicating the importance of using multiple assays and bacteria for mode of action studies to be able to draw proper conclusions on the mode of action of AMPs

Enhanced Membrane Pore Formation through High-Affinity Targeted Antimicrobial Peptides

Many cationic antimicrobial peptides (AMPs) target the unique lipid composition of the prokaryotic cell membrane. However, the micromolar activities common for these peptides are considered weak in comparison to nisin, which follows a targeted, pore-forming mode of action. Here we show that AMPs can be modified with a high-affinity targeting module, which enables membrane permeabilization at low concentration. Magainin 2 and a truncated peptide analog were conjugated to vancomycin using click chemistry, and could be directed towards specific membrane embedded receptors both in model membrane systems and whole cells. Compared with untargeted vesicles, a gain in permeabilization efficacy of two orders of magnitude was reached with large unilamellar vesicles that included lipid II, the target of vancomycin. The truncated vancomycin-peptide conjugate showed an increased activity against vancomycin resistant Enterococci, whereas the full-length conjugate was more active against a targeted eukaryotic cell model: lipid II containing erythrocytes. This study highlights that AMPs can be made more selective and more potent against biological membranes that contain structures that can be targeted

Teixobactin analogues reveal enduracididine to be non-essential for highly potent antibacterial activity and lipid II binding

Abstract. Teixobactin is a highly promising antibacterial depsipeptide consisting of four D-amino acids and a rare L-allo-enduracididine amino acid. L-allo-enduracididine is reported to be important for the highly potent antibacterial activity of teixobactin. However, it is also a key limiting factor in the development of potent teixobactin analogues due to several synthetic challenges such as it is not commercially available, requires a multistep synthesis, long and repititive couplings (16-30 hours). Due to all these challenges, the total synthesis of teixobactin is laborious and low yielding (3.3%). In this work, we have identified a unique design and developed a rapid synthesis (10 min μwave assisted coupling per amino acid, 30 min cyclisation) of several highly potent analogues of teixobactin with yields of 10-24% by replacing the L-allo-enduracididine with commercially available non-polar residues such as leucine and isoleucine. Most importantly, the Leu10-teixobactin and Ile10-teixobactin analogues have shown highly potent antibacterial activity against a broader panel of MRSA and Enterococcus faecalis (VRE). Time-kill kinetics data indicate that both these compounds are superior to vancomycin against MRSA (16 times more potent). Furthermore, these synthetic analogues displayed identical antibacterial activity to natural teixobactin (MIC 0.25 μg/ml) against MRSA ATCC 33591 despite their simpler design and ease of synthesis. Detailed NMR analyses have provided us with further insight into the 3D structures of these important analogues. We have confirmed lipid II binding and measured the binding affinities of individual amino acid residues of Ala10-teixobactin towards geranyl pyrophosphate (a lipid II mimic) by NMR to understand the nature and strength of binding interactions of the amino acid residues. An antagonization assay further confirms a lipid II mediated mode of action. Contrary to current understanding, we have shown that a cationic amino acid at position 10 is not essential for target (lipid II) binding and potent antibacterial activity of teixobactin. We thus provide strong evidence contrary to the many assumptions made about the mechanism of action of this exciting new antibiotic. Introduction of a non-cationic residue at position 10 allows for tremendous diversification in terms of the design and synthesis of highly potent teixobactin analogues and lays the foundations for the development of teixobactin analogues as new drug-like molecules to target MRSA and Mycobacterium tuberculosis

Specific Lipid Studies in Complex Membranes by Solid-State NMR Spectroscopy

Specific interactions with phospholipids are often critical for the function of proteins or drugs, but studying these interactions at high resolution remains difficult, especially in complex membranes that mimic biological conditions. In principle, molecular interactions with phospholipids could be directly probed by solid-state NMR (ssNMR). However, due to the challenge to detect specific lipids in mixed liposomes and limited spectral sensitivity, ssNMR studies of specific lipids in complex membranes are scarce. Here, by using purified biological 13C,15N-labeled phospholipids, we show that we can selectively detect traces of specific lipids in complex membranes. In combination with 1H-detected ssNMR, we show that our approach provides unprecedented high-resolution insights into the mechanisms of drugs that target specific lipids. This broadly applicable approach opens new opportunities for the molecular characterization of specific lipid interactions with proteins or drugs in complex fluid membranes

Формування органів управління кінематографа в Україні (1919)

У статті розглядається процес створення та діяльність органів управління кінематографа в перші роки функціонування радянської влади в Україні.В статье рассматривается процесс создания и деятельность органов управления кинематографа в первые годы функционирования советской власти в Украине.The process of creating and functioning of the cinematography authorities in the early years of the Soviet Union in Ukraine is examined in the article