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Biophysical Journal Jan 2018The permeabilization of model lipid bilayers by cationic peptides has been studied extensively over decades, with the bee-sting toxin melittin perhaps serving as the...
The permeabilization of model lipid bilayers by cationic peptides has been studied extensively over decades, with the bee-sting toxin melittin perhaps serving as the canonical example. However, the relevance of these studies to the permeabilization of real bacterial membranes by antimicrobial peptides remains uncertain. Here, we employ single-cell fluorescence microscopy in a detailed study of the interactions of melittin with the outer membrane (OM) and the cytoplasmic membrane (CM) of live Escherichia coli. Using periplasmic green fluorescent protein (GFP) as a probe, we find that melittin at twice the minimum inhibitory concentration first induces abrupt cell shrinkage and permeabilization of the OM to GFP. Within ∼4 s of OM permeabilization, the CM invaginates to form inward facing "periplasmic bubbles." Seconds later the bubbles begin to leak periplasmic GFP into the cytoplasm. Permeabilization is localized, consistent with possible formation of toroidal pores. Within ∼20 s, first the OM and then the CM re-seals to GFP. Some 2-20 min later, both CM and OM are re-permeabilized to GFP. We invoke a mechanism based on curvature stress concepts derived from model bilayer studies. The permeabilization and re-sealing events involve sequential, time-dependent build-up of melittin density within the outer and inner leaflets of each bilayer. We also propose a mechanical explanation for the early cell shrinkage event induced by melittin and a variety of other cationic peptides. As peptides gain access to the periplasm, they bind to the anionic peptido-crosslinks of the lipopolysaccharide layer, increasing its longitudinal elastic modulus. The cell wall shrinks because it can withstand the same turgor pressure with smaller overall extension. Shrinkage in turn induces invagination of the CM, preserving its surface area. We conclude by comparing the behavior of different peptides.
Topics: Amino Acid Sequence; Cell Membrane; Escherichia coli; Kinetics; Melitten; Permeability
PubMed: 29401434
DOI: 10.1016/j.bpj.2017.10.046 -
Preparative Biochemistry & Biotechnology 2023The antimicrobial peptide was considered an important target for developing novel antibacterial drugs. However, the unstable biological activity and the low...
The antimicrobial peptide was considered an important target for developing novel antibacterial drugs. However, the unstable biological activity and the low antibacterial activity are challenges for the application of recombinant proteins. In this study, the fusion peptide of Melittin-Thanatin (MT) was designed and produced, and its derivative sequence (MT-W) was obtained by replacing three glycines (Gly, G) with tryptophan (Trp, W). The MT-W peptide were synthesized in WB700 by EDDIE self-cleavage protein fusion. Compared with MT, MT-W exhibited 2-4 times higher antibacterial rate against K88. In addition, MT-W showed lower cytotoxicity (IC50 > 300 mg·L) to the red blood cell, and more stable biological activities under the conditions of different temperatures (20, 30, 40, 50, 60, 70, 80, and 90 °C), pH values (2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0) and different proteases. Especially, MT-W showed a broader antibacterial effect on three drug-resistant strains than florfenicol and oxytetracycline calcium. In conclusion, compared with MT, the MT-W showed increased antibacterial activity, stability, lower cytotoxicity, and broader antimicrobial effect. Therefore, it would become a promising alternative to conventional antibiotics.
Topics: Melitten; Tryptophan; Glycine; Recombinant Proteins; Anti-Bacterial Agents; Mutation; Microbial Sensitivity Tests
PubMed: 36508334
DOI: 10.1080/10826068.2022.2151016 -
Analytical Chemistry Jul 2019Antimicrobial peptides (AMPs) are generally cationic and amphipathic peptides that show potential applications to combat the growing threat of antibiotic resistant...
Antimicrobial peptides (AMPs) are generally cationic and amphipathic peptides that show potential applications to combat the growing threat of antibiotic resistant infections. AMPs are known to interact with bacterial membranes, but their mechanisms of toxicity and selectivity are poorly understood, in part because it is challenging to characterize AMP oligomeric complexes within lipid bilayers. Here, we used native mass spectrometry to measure the stoichiometry of AMPs inserted into lipoprotein nanodiscs with different lipid components. Titrations of increasing peptide concentration and collisional activation experiments reveal that AMPs can exhibit a range of behaviors from nonspecific incorporation into the nanodisc to formation of specific complexes. This new approach to characterizing formation of AMP complexes within lipid membranes will provide unique insights into AMP mechanisms.
Topics: Dimyristoylphosphatidylcholine; Gramicidin; Lipid Bilayers; Mass Spectrometry; Melitten; Nanostructures; Phosphatidylglycerols
PubMed: 31251560
DOI: 10.1021/acs.analchem.9b02261 -
Advanced Materials (Deerfield Beach,... Oct 2021Poisoning is a leading cause of admission to medical emergency departments and intensive care units. Supramolecular detoxification, which involves injecting...
Poisoning is a leading cause of admission to medical emergency departments and intensive care units. Supramolecular detoxification, which involves injecting supramolecular receptors that bind with toxins to suppress their biological activity, is an emerging strategy for poisoning treatment; it has few requirements and a broad application scope. However, it is still a formidable challenge to design supramolecular therapeutic materials as an antidote to macromolecular toxins, because the large size, flexible conformation, and presence of multiple and diverse binding sites of biomacromolecules hinder their recognition. Herein, a supramolecular antidote to macromolecular toxins is developed through the coassembly of macrocyclic amphiphiles, relying on heteromultivalent recognition between the coassembled components and toxic macromolecules. The coassembly of amphiphilic cyclodextrin and calixarene strongly and selectively captures melittin, a toxin studied herein; this imparts various therapeutic effects such as inhibiting the interactions of melittin with cell membranes, alleviating melittin cytotoxicity and hemolytic toxicity, reducing the mortality rate of melittin-poisoned mice, and mitigating damage to major organs. The use of the proposed antidote overcomes the limitation of supramolecular detoxification applicability to only small-molecular toxins. The antidote can also detoxify other macromolecular toxins as long as selective and strong binding is achieved because of the coassembling tunability.
Topics: Animals; Antidotes; Antimicrobial Cationic Peptides; Cell Membrane; Cell Survival; Cyclodextrins; HEK293 Cells; Hemolysis; Humans; Liver; Macromolecular Substances; Melitten; Mice; Spider Venoms
PubMed: 34418189
DOI: 10.1002/adma.202104310 -
The Journal of Biological Chemistry Dec 2008The mechanism of pore formation of lytic peptides, such as melittin from bee venom, is thought to involve binding to the membrane surface, followed by insertion at...
The mechanism of pore formation of lytic peptides, such as melittin from bee venom, is thought to involve binding to the membrane surface, followed by insertion at threshold levels of bound peptide. We show that in membranes composed of zwitterionic lipids, i.e. phosphatidylcholine, melittin not only forms pores but also inhibits pore formation. We propose that these two modes of action are the result of two competing reactions: direct insertion into the membrane and binding parallel to the membrane surface. The direct insertion of melittin leads to pore formation, whereas the parallel conformation is inactive and prevents other melittin molecules from inserting, hence preventing pore formation.
Topics: Animals; Bee Venoms; Bees; Cell Membrane; Circular Dichroism; Dose-Response Relationship, Drug; Fluoresceins; Lipids; Liposomes; Melitten; Molecular Conformation; Phosphatidylcholines; Protein Structure, Tertiary; Surface Properties
PubMed: 18819911
DOI: 10.1074/jbc.M805171200 -
International Journal of Molecular... Jan 2024Melittin, a natural antimicrobial peptide, has broad-spectrum antimicrobial activity. This has resulted in it gaining increasing attention as a potential antibiotic...
Melittin, a natural antimicrobial peptide, has broad-spectrum antimicrobial activity. This has resulted in it gaining increasing attention as a potential antibiotic alternative; however, its practical use has been limited by its weak antimicrobial activity, high hemolytic activity, and low proteolytic stability. In this study, N-terminal fatty acid conjugation was used to develop new melittin-derived lipopeptides (MDLs) to improve the characteristics of melittin. Our results showed that compared with native melittin, the antimicrobial activity of MDLs was increased by 2 to 16 times, and the stability of these MDLs against trypsin and pepsin degradation was increased by 50 to 80%. However, the hemolytic activity of the MDLs decreased when the length of the carbon chain of fatty acids exceeded 10. Among the MDLs, the newly designed analog Mel-C8 showed optimal antimicrobial activity and protease stability. The antimicrobial mechanism studied revealed that the MDLs showed a rapid bactericidal effect by interacting with lipopolysaccharide (LPS) or lipoteichoic acid (LTA) and penetrating the bacterial cell membrane. In conclusion, we designed and synthesized a new class of MDLs with potent antimicrobial activity, high proteolytic stability, and low hemolytic activity through N-terminal fatty acid conjugation.
Topics: Melitten; Endopeptidases; Peptide Hydrolases; Anti-Bacterial Agents; Fatty Acids; Lipopeptides
PubMed: 38255940
DOI: 10.3390/ijms25020867 -
Journal of Colloid and Interface Science May 2023It is widely regarded that antimicrobial peptides (AMPs) kill bacteria by physically disrupting microbial membranes and causing cytoplasmic leakage, but it remains...
HYPOTHESIS
It is widely regarded that antimicrobial peptides (AMPs) kill bacteria by physically disrupting microbial membranes and causing cytoplasmic leakage, but it remains unclear how AMPs disrupt the outer membrane (OM) of Gram-negative bacteria (GNB) and then compromise the inner membrane. We hypothesise that different AMPs impose different structural disruptions, with direct implications to their antimicrobial efficacies.
EXPERIMENTS
The antimicrobial activities of three typical AMPs, including the designed short AMP, G, and two natural AMPs, melittin and LL37, against E. coli and their haemolytic activities were studied. Lipopolysaccharide (LPS) and anionic di-palmitoyl phosphatidyl glycerol (DPPG) monolayer models were constructed to mimic the outer membrane and inner membrane leaflets of Gram-negative bacteria. The binding and penetration of AMPs to the model lipid monolayers were systematically studied by neutron reflection via multiple H/D contrast variations.
FINDING
G has relatively high antimicrobial activity, low cytotoxicity, and high proteolytic stability, whilst melittin has significant haemolysis and LL37 has weaker antimicrobial activity. G could rapidly lyse LPS and DPPG monolayers within 10-20 min. In contrast, melittin was highly active against the LPS membrane, but the dynamic process lasted up to 80 min, with excessive stacking in the OM. LL37 caused rather weak destruction to LPS and DPPG monolayers, leading to massive adsorption on the membrane surface without penetrating the lipid tail region. These findings demonstrate that the rationally designed AMP G was well optimised to impose most effective destruction to bacterial membranes, consistent with its highest bactericidal activity. These different interfacial structural features associated with AMP binding shed light on the future development of active and biocompatible AMPs for infection and wound treatments.
Topics: Lipopolysaccharides; Antimicrobial Peptides; Melitten; Escherichia coli; Anti-Infective Agents; Gram-Negative Bacteria; Bacteria; Cell Membrane; Anti-Bacterial Agents
PubMed: 36701864
DOI: 10.1016/j.jcis.2023.01.051 -
Animal Biotechnology Dec 2023This study intended to assess the properties of in-ovo administration of Melittin (MLT) on hatchability, chick yield, hematology, immunological indices and relative...
This study intended to assess the properties of in-ovo administration of Melittin (MLT) on hatchability, chick yield, hematology, immunological indices and relative organs weight of Alexandria chickens at hatch. A total of 600 eggs with an average weight of (45.12 g), were gathered and split into five groups: a non-injected group or negative control (NC), a saline injection group or positive control (PC), and three concentrations of MLT (5, 10 and 15 µg of MLT per egg, respectively). On day 18 of incubation, eggs from the injection groups were injected into the amniotic fluid from the large end with the in-ovo injection solutions (0.2 ml per egg). Results indicated that 10 µg MLT/egg positively affected the weight and yield of chicks. In addition, our findings indicated that the in-ovo administration with 10 or 15 µg MLT/egg was superior in most of the immunological indicators (spleen and bursa relative weights, immunoglobulins IgG and IgM, T cells and B cells). In conclusion, in order to improve the immune efficiency (early immune acceleration) of Alexandria chicks, which may contribute to offering a significant boost to their future performance, this study suggests injecting eggs with 5 or 10 µg MLT/egg.
Topics: Animals; Chickens; Melitten; Injections; Ovum
PubMed: 37688385
DOI: 10.1080/10495398.2023.2255063 -
Toxins Jun 2022Melittin is a membrane-active peptide with strong anticancer activity against various cancers. Despite decades of research, the role of the singular Trp in the...
Melittin is a membrane-active peptide with strong anticancer activity against various cancers. Despite decades of research, the role of the singular Trp in the anticancer activity and selectivity of melittin remains poorly understood. Here, we propose a theranostic solution based on the substitution of Trp19 with a noncanonical fluorescent amino acid (Dap). The introduction of Dap residue in melittin stabilized the helical structure of the peptide, as evaluated by circular dichroism spectra and molecular dynamics simulations. In vitro hemolytic and anticancer activity assays revealed that introducing Dap residue in melittin changed its mode of action with the cell membrane, resulting in reduced hemolytic toxicity and an improved the selectivity index (SI), with up to a five-fold increase compared to melittin. In vitro fluorescence imaging of Dap-labeled melittin (MEL) in cancer cells demonstrated high membrane-penetrating activity, with strong nuclear and nucleolar localization ability. These findings provide implications for novel anticancer therapies based on Trp-substituted designs and nuclear/nucleolar targeted therapy.
Topics: Amino Acids; Circular Dichroism; Melitten; Peptides; Tranexamic Acid; Tryptophan
PubMed: 35878166
DOI: 10.3390/toxins14070428 -
Endeavour 1988
Review
Topics: Amines; Animals; Bee Venoms; Bees; Hyaluronoglucosaminidase; Melitten; Peptides; Pheromones; Phospholipases
PubMed: 2458907
DOI: 10.1016/0160-9327(88)90082-8