Progressive Structuring of a Branched Antimicrobial Peptide on the Path to the Inner Membrane Target

Bai, Yinjuan; Liu, Shouping; Li, Jianguo; Lakshminarayanan, Rajamani; Sarawathi, Padmanabhan; Cl, Tang; Ho, Duncun; Verma, Chandra; Beuerman, Roger W.; Pervushin, Konstantin

doi:10.1074/jbc.m112.363259

Cited by 33 publications

(37 citation statements)

References 81 publications

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“…Thus, when the bacterial outer membrane is perturbed, the dye can partition into the interior of an outer membrane, thereby exhibiting an increase in fluorescence emission intensity 27. 28 A fast increase in NPN fluorescence emission intensity was observed upon addition of Sub3 (Figure 3 C), thus suggesting that it binds promptly to the outer membrane and facilitates the permeation of NPN. A comparison of Sub3 with melittin suggests that Sub3 interacts faster than melittin with the outer membrane but perturbs it to a lesser extent.…”

Section: Resultsmentioning

confidence: 99%

The Antimicrobial Activity of Sub3 is Dependent on Membrane Binding and Cell‐Penetrating Ability

et al. 2013

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Because of their high activity against microorganisms and low cytotoxicity, cationic antimicrobial peptides (AMPs) have been explored as the next generation of antibiotics. Although they have common structural features, the modes of action of AMPs are extensively debated, and a single mechanism does not explain the activity of all AMPs reported so far. Here we investigated the mechanism of action of Sub3, an AMP previously designed and optimised from high-throughput screening with bactenecin as the template. Sub3 has potent activity against Gram-negative and Gram-positive bacteria as well as against fungi, but its mechanism of action has remained elusive. By using AFM imaging, ζ potential, flow cytometry and fluorescence methodologies with model membranes and bacterial cells, we found that, although the mechanism of action involves membrane targeting, Sub3 internalises inside bacteria at lethal concentrations without permeabilising the membrane, thus suggesting that its antimicrobial activity might involve both the membrane and intracellular targets. In addition, we found that Sub3 can be internalised into human cells without being toxic. As some bacteria are able to survive intracellularly and consequently evade host defences and antibiotic treatment, our findings suggest that Sub3 could be useful as an intracellular antimicrobial agent for infections that are notoriously difficult to treat.

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Section: Resultsmentioning

confidence: 99%

The Antimicrobial Activity of Sub3 is Dependent on Membrane Binding and Cell‐Penetrating Ability

et al. 2013

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“…For outer membrane permeability assasys, NPN was added to 2 × 10 6 E. coli ATCC 25922 cells/mL (final NPN concentration of 20 μM) and incubated for 15 min with varying concentrations (64 to 0.015 μg/mL) of lipopeptides with the fluorescence emission intensity recorded (λ exc = 340 nm, λ em = 405 nm) using a Polarstar-Omega (BMG) spectrophotometer to evaluate NPN uptake by the bacterial cells (Bai, et al, 2012; Loh, et al, 1984). For cytoplasmic membrane depolarisation assasys, mid-log phase (OD ~ 0.6) E. coli ATCC 25922 cells were harvested by centrifugation and resuspended in 5 mM HEPES, 20 mM glucose, pH 7.4.…”

Section: Star Methodsmentioning

confidence: 99%

Structure, Function, and Biosynthetic Origin of Octapeptin Antibiotics Active against Extensively Drug-Resistant Gram-Negative Bacteria

Velkov

Gallardo-Godoy

Swarbrick

et al. 2018

Cell Chemical Biology

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Resistance to the last-resort antibiotic colistin is now widespread and new therapeutics are urgently required. We report the first in toto chemical synthesis and pre-clinical evaluation of octapeptins, a class of lipopeptides structurally related to colistin. The octapeptin biosynthetic cluster consisted of three non-ribosomal peptide synthetases (OctA, OctB, and OctC) that produced an amphiphilic antibiotic, octapeptin C4, which was shown to bind to and depolarize membranes. While active against multi-drug resistant (MDR) strains in vitro, octapeptin C4 displayed poor in vivo efficacy, most likely due to high plasma protein binding. Nuclear magnetic resonance solution structures, empirical structure-activity and structure-toxicity models were used to design synthetic octapeptins active against MDR and extensively drug-resistant (XDR) bacteria. The scaffold was then subtly altered to reduce plasma protein binding, while maintaining activity against MDR and XDR bacteria. In vivo efficacy was demonstrated in a murine bacteremia model with a colistin-resistant P. aeruginosa clinical isolate.

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“…Unlike conventional antibiotics, which generally target metabolic enzymes, AMPs act mainly by membrane‐targeting mechanisms and are selective due to the difference in charge of prokaryotic and eukaryotic cell membranes. Furthermore, AMPs have faster antimicrobial activity than conventional antibiotics . Generally speaking, AMPs can be divided into two mechanistic classes: membrane disruptive and non‐membrane disruptive (acting on intracellular targets).…”

Section: Introductionmentioning

confidence: 99%

Large scale ab initio modeling of structurally uncharacterized antimicrobial peptides reveals known and novel folds

et al. 2018

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Antimicrobial resistance within a wide range of infectious agents is a severe and growing public health threat. Antimicrobial peptides (AMPs) are among the leading alternatives to current antibiotics, exhibiting broad spectrum activity. Their activity is determined by numerous properties such as cationic charge, amphipathicity, size, and amino acid composition. Currently, only around 10% of known AMP sequences have experimentally solved structures. To improve our understanding of the AMP structural universe we have carried out large scale ab initio 3D modeling of structurally uncharacterized AMPs that revealed similarities between predicted folds of the modeled sequences and structures of characterized AMPs. Two of the peptides whose models matched known folds are Lebocin Peptide 1A (LP1A) and Odorranain M, predicted to form β-hairpins but, interestingly, to lack the intramolecular disulfide bonds, cation-π or aromatic interactions that generally stabilize such AMP structures. Other examples include Ponericin Q42, Latarcin 4a, Kassinatuerin 1, Ceratotoxin D, and CPF-B1 peptide, which have α-helical folds, as well as mixed αβ folds of human Histatin 2 peptide and Garvicin A which are, to the best of our knowledge, the first linear αββ fold AMPs lacking intramolecular disulfide bonds. In addition to fold matches to experimentally derived structures, unique folds were also obtained, namely for Microcin M and Ipomicin. These results help in understanding the range of protein scaffolds that naturally bear antimicrobial activity and may facilitate protein design efforts towards better AMPs.

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Progressive Structuring of a Branched Antimicrobial Peptide on the Path to the Inner Membrane Target

Cited by 33 publications

References 81 publications

The Antimicrobial Activity of Sub3 is Dependent on Membrane Binding and Cell‐Penetrating Ability

The Antimicrobial Activity of Sub3 is Dependent on Membrane Binding and Cell‐Penetrating Ability

Structure, Function, and Biosynthetic Origin of Octapeptin Antibiotics Active against Extensively Drug-Resistant Gram-Negative Bacteria

Large scale ab initio modeling of structurally uncharacterized antimicrobial peptides reveals known and novel folds

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