Bioactivity and the First Transmission Electron Microscopy Immunogold Studies of Short De Novo -Designed Antimicrobial Peptides

Abstract

In light of the era of microbial drug resistance, the current study aimed to better understand the relationships between sequence, higher-order structure, and mechanism of action for five designed peptides against multidrug-resistant (MDR) pathogens. All peptides studied were 15 residues long, were polycationic, adopted alpha-helical structures within hydrophobic environments (excluding the D-amino acid-substituted peptide MA-d), and contained N-terminal glycine residues, a novel antimicrobial peptide (AMP) design principle. Increasing hydrophobicity enhanced MICs (<500 g/ml to <7.4 g/ml) without significantly increasing hemolytic activity (18% maximum hemolysis at 3,400 g/ml). To the best of our knowledge, this is the first study to have successfully adapted and used a transmission electron microscopy (TEM) immunogold method to investigate the mechanism of action of short (ϳ15 residues long) AMPs within bacteria. We propose a "floodgate" mechanism to possibly explain membrane deformation and the relative absence of membraneassociated peptides 10 h into incubation.

Imaging the Antistaphylococcal Activity of CATH-2: Mechanism of Attack and Regulation of Inflammatory Response

“…The observation of morphological changes in S. aureus cells correlated with the observed intracellular localization and membrane binding of CATH-2 at sub-MBCs. Comparable findings with small (~15-amino-acid) alpha-helical peptides (MAw-1) were obtained by Azad and colleagues ( 34 ). These peptides were shown to cause deformation of the membrane, the peptidoglycan layer, and the outer acidic surfaces.…”

“…These peptides were shown to cause deformation of the membrane, the peptidoglycan layer, and the outer acidic surfaces. Based on immunolabeling of the TEM sections, it was observed that the peptides are localized next to the induced membrane folds, suggesting that these membrane folds are aggregates of toroid-like pores produced earlier ( 34 ). This might also occur in the presence of CATH-2, as with lower peptide concentrations, similar binding of peptides near folds was detected.…”

A Peptide Derived from Phage Display Library Exhibits Antibacterial Activity against E. coli and Pseudomonas aeruginosa

“…The test microorganisms mentioned earlier in the experimental procedures were used to evaluate the bactericidal activity of EC5 peptide. The minimum inhibitory concentrations (MICs) of the peptide was determined by standard dilution assay as recommended by Clinical and Laboratory Standard Institutes (CLSI) guidelines [28]. Overnight cultures of bacterial cells were diluted in cation-supplemented Mueller-Hinton broth (MHB) (Becton Dickinson, Sparks, MD) and grown to mid-logarithmic phase on an orbital shaker (37°C) and diluted to a 0.5 McFarland standard to a final volume of 1 ml.…”

Cell envelope disruption of E. coli Exposed to ϵ‐polylysine by FESEM and TEM technology

“…FESEM is very well suited to provide a quick overview of sample and can capture the surface details of specimens, but the information on the interior of cell can be visualized only by TEM after sectioning cell and staining (Milillo et al 2011). The key factor of the desired and lossless image is specimen processing for electron microscopy, conventional specimen preparation methods involve more steps for example, chemical fixation, staining and dehydration by replacement (Diestra et al 2005;Azad et al 2011), and undergo more centrifugations (Anderson et al 2004;Bouhdid et al 2009), which seriously hinders the observation of flexible and fine ultrastructure and even leads to artifacts (Liu et al 2012).…”