Although nanopores can be used for singlemolecule sequencing of nucleic acids using low-cost portable devices, the characterization of proteins and their modifications has yet to be established.... Show moreAlthough nanopores can be used for singlemolecule sequencing of nucleic acids using low-cost portable devices, the characterization of proteins and their modifications has yet to be established. Here, we show that hydrophilic or glycosylated peptides translocate too quickly across FraC nanopores to be recognized. However, high ionic strengths (i.e., 3 M LiCl) and low pH (i.e., pH 3) together with using a nanopore with a phenylalanine at its constriction allows the recognition of hydrophilic peptides, and to distinguish between mono- and diglycosylated peptides. Using these conditions, we devise a nanopore method to detect, characterize, and quantify posttranslational modifications in generic proteins, which is one of the pressing challenges in proteomic analysis. Show less
AbstractThe continued rise of antibiotic resistance is a global concern that threatens to undermine many aspects of modern medical practice. Key to addressing this threat is the discovery and... Show moreAbstractThe continued rise of antibiotic resistance is a global concern that threatens to undermine many aspects of modern medical practice. Key to addressing this threat is the discovery and development of new antibiotics that operate by unexploited modes of action. The so-called calcium-dependent lipopeptide antibiotics (CDAs) are an important emerging class of natural products that provides a source of new antibiotic agents rich in structural and mechanistic diversity. Notable in this regard is the subset of CDAs comprising the laspartomycins and amphomycins/friulimicins that specifically target the bacterial cell wall precursor undecaprenyl phosphate (C55-P). In this study we describe the design and synthesis of new C55-P-targeting CDAs with structural features drawn from both the laspartomycin and amphomycin/friulimicin classes. Assessment of these lipopeptides revealed previously unknown and surprisingly subtle structural features that are required for antibacterial activity. High-resolution crystal structures further indicate that the amphomycin/friulimicin-like lipopeptides adopt a unique crystal packing that governs their interaction with C55-P and provides an explanation for their antibacterial effect. In addition, live-cell microscopy studies provide further insights into the biological activity of the C55-P targeting CDAs highlighting their unique mechanism of action relative to the clinically used CDA daptomycin. Show less
Multidrug-resistant bacteria pose a serious global health threat as antibiotics are increasingly losing their clinical efficacy. A molecular level understanding of the mechanism of action of... Show moreMultidrug-resistant bacteria pose a serious global health threat as antibiotics are increasingly losing their clinical efficacy. A molecular level understanding of the mechanism of action of antimicrobials plays a key role in developing new agents to combat the threat of antimicrobial resistance. Daptomycin, the only clinically used calcium-dependent lipopeptide antibiotic, selectively disrupts Gram-positive bacterial membranes to illicit its bactericidal effect. In this study, we use isothermal titration calorimetry to further characterize the structural features of the target bacterial phospholipids that drive daptomycin binding. Our studies reveal that daptomycin shows a clear preference for the phosphoglycerol headgroup. Furthermore, unlike other calcium-dependent lipopeptide antibiotics, calcium binding by daptomycin is strongly dependent on the presence of phosphatidylglycerol. These investigations provide new insights into daptomycin's phospholipid specificity and calcium binding behavior. Show less
The growing threat of antibacterial resistance is a global concern. The so-called calcium-dependent lipopeptide antibiotics (CDAs) have emerged as a promising source of new antibiotic agents that... Show moreThe growing threat of antibacterial resistance is a global concern. The so-called calcium-dependent lipopeptide antibiotics (CDAs) have emerged as a promising source of new antibiotic agents that are rich in structural and mechanistic diversity. Over forty unique CDAs have been identified to date and share a number of common features. Recent efforts in our group have provided new mechanistic and structural insights into the laspartomycin family of CDAs. We here describe investigations aimed at probing the role of the three glycine residues found in the laspartomycin peptide macrocycle. In doing so laspartomycin analogues containing the achiral 2-aminoisobutyric acid (AIB) as well as L- or D-alanine in place of glycine were prepared and their antibacterial activities evaluated. Show less
To push back the growing tide of antibacterial resistance the discovery and development of new antibiotics is a must. In recent years the calcium-dependent lipopeptide antibiotics (CDAs) have... Show moreTo push back the growing tide of antibacterial resistance the discovery and development of new antibiotics is a must. In recent years the calcium-dependent lipopeptide antibiotics (CDAs) have emerged as a potential source of new antibacterial agents rich in structural and mechanistic diversity. All CDAs share a common lipidated cyclic peptide motif containing amino acid side chains that specifically chelate calcium. It is only in the calcium bound state that the CDAs achieve their potent antibacterial activities. Interestingly, despite their common structural features, the mechanisms by which different CDAs target bacteria can vary dramatically. This review provides both a historic context for the CDAs while also addressing the state of the art with regards to their discovery, optimization, and antibacterial mechanisms. Show less
The calcium‐dependent antibiotics (CDAs) are an important emerging class of antibiotics. The crystal structure of the CDA laspartomycin C in complex with calcium and the ligand geranyl‐phosphate at... Show moreThe calcium‐dependent antibiotics (CDAs) are an important emerging class of antibiotics. The crystal structure of the CDA laspartomycin C in complex with calcium and the ligand geranyl‐phosphate at a resolution of 1.28 Å is reported. This is the first crystal structure of a CDA bound to its bacterial target. The structure is also the first to be reported for an antibiotic that binds the essential bacterial phospholipid undecaprenyl phosphate (C55‐P). These structural insights are of great value in the design of antibiotics capable of exploiting this unique bacterial target. Show less
Hart, P. 't; Wood, T.M.; Tehrani, K.H.M.E.; Harten, R.M. van; Śleszyńska, M.; Rentero Rebollo, I.; ... ; Martin, N.I. 2017
Creative strategies for identifying new antibiotics are essential to addressing the looming threat of a post-antibiotic era. We here report the use of a targeted peptide phage display screen as a... Show moreCreative strategies for identifying new antibiotics are essential to addressing the looming threat of a post-antibiotic era. We here report the use of a targeted peptide phage display screen as a means of generating novel antimicrobial lipopeptides. Specifically, a library of phage displayed bicyclic peptides was screened against a biomolecular target based on the bacterial cell wall precursor lipid II. In doing so we identified unique lipid II binding peptides that upon lipidation were found to be active against a range of Gram-positive bacteria including clinically relevant strains of vancomycin resistant bacteria. Optimization of the peptide sequence led to variants with enhanced antibacterial activity and reduced hemolytic activity. Biochemical experiments further confirm a lipid II mediated mode of action for these new-to-nature antibacterial lipopeptides. Show less
