Designing potent antimicrobial peptides by disulphide linked dimerization and N-terminal lipidation to increase antimicrobial activity and membrane perturbation: Structural insights into lipopolysaccharide binding

• Published in: Journal of colloid and interface science Vol. 461; pp. 335 - 345
• Main Authors: Datta, Aritreyee, Kundu, Pallob, Bhunia, Anirban
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.01.2016
• Subjects: Anti-Bacterial Agents - chemical synthesis; Anti-Bacterial Agents - chemistry; Anti-Bacterial Agents - pharmacology; Antibacterial activity; Antimicrobial Cationic Peptides - chemical synthesis; Antimicrobial Cationic Peptides - chemistry; Antimicrobial Cationic Peptides - pharmacology; Antimicrobial peptide; Binding Sites; Cell Membrane - drug effects; Disulfides - chemistry; Dose-Response Relationship, Drug; Escherichia coli - drug effects; Hemolytic activity; Lipids - chemistry; Lipopolysaccharides - chemistry; LPS binding; LPS bound conformation; LPS neutralization; Membrane permeabilization; Nuclear Magnetic Resonance, Biomolecular; Peptide modifications; Protein Multimerization; Pseudomonas aeruginosa - drug effects; Spectrometry, Fluorescence; Structure-Activity Relationship; trNOESY(Transferred Nuclear Overhauser Effect Spectroscopy); Xanthomonas campestris - drug effects; AMP; MIC; trNOESY; Peptide modifications; LPS; LPS binding; Membrane permeabilization; LPS neutralization; trNOESY(Transferred Nuclear Overhauser Effect Spectroscopy); Antibacterial activity; MDR; Hemolytic activity; LPS bound conformation; Antimicrobial peptide
• ISSN: 0021-9797; 1095-7103
• DOI: 10.1016/j.jcis.2015.09.036

[Display omitted] The remarkable rise in multi-drug resistant Gram-negative bacterial pathogens is a major concern to the well being of humans as well as susceptible plants. In recent years, diseases associated with inflammation and septicemia have already become a global health issue. Therefore, there is a rising demand for the development of novel “super” antibiotics. In this context, antimicrobial peptides offer an attractive, alternate therapeutic solution to conventional antibiotics. Microbroth dilution assay was performed to investigate the antimicrobial activities of the two designed peptides against Gram negative bacterial pathogens. Fluorescence studies including NPN dye uptake assay, Calcein entrapped vesicle leakage assay, quenching and anisotropy in presence of lipopolysaccharide (LPS) were performed to elucidate binding interactions and enhanced membrane permeabilisation. Hemolytic assay and endotoxin/LPS neutralisation assay were performed to study the hemolytic effects and LPS scavenging abilities of the peptides. High resolution NMR studies were performed to obtain insights into LPS-peptide interaction at the molecular level. Here, we report more potent analogues of previously designed peptide VG16KRKP, designed through dimerization via Cys–Cys disulphide linkage and N-terminal lipidation. Similar to the parent peptide, VG16KRKP, the modified analogue peptides are non hemolytic in nature, but possessed, 2–10-fold increase in antibacterial activities against E. coli, human pathogen Pseudomonas aeruginosa and the devastating plant pathogen, Xanthomonas campestris pv. campestris as well as membrane permeabilization, and endotoxin neutralization. LPS bound solution structure of both analogues, as determined by NMR spectroscopy, reveal that the conserved hydrophobic triad motif, formed by Trp5, Leu11 and Phe12 is compactly organized and stabilized either by the acyl chain or disulphide bond. This structural constraint accounts for the separation of polar face from the hydrophobic face of the peptides. Our novel peptides designed through Cys–Cys dimerization and N-terminal lipidation, will serve as a template to develop more potent antimicrobials in future, to control plant and human diseases.