Atomic Force Microscopy Study of the Effect of Antimicrobial Peptides on the Cell Envelope of Escherichia coli

Abstract

The influences of the antibacterial magainin 2 and PGLa from the African clawed frog (Xenopus laevis) and the hemolytic bee venom melittin on Escherichia coli as the target cell were studied by atomic force microscopy (AFM). Nanometer-scale images of the effects of the peptides on this gram-negative bacterium's cell envelope were obtained in situ without the use of fixing agents. These high-resolution AFM images of the surviving and intact target cells before and after peptide treatment showed distinct changes in cell envelope morphology as a consequence of peptide action. Although all three peptides are lytic to E. coli, it is clear from this AFM study that each peptide causes distinct morphological changes in the outer membrane and in some cases the inner membrane, probably as a consequence of different mechanisms of action.Bacterial resistance to conventional antibiotics has become a major problem worldwide, and certain strains of bacteria are already resistant to all available drugs (3, 29). The development of a new family of antibiotics is therefore an important research topic. Antimicrobial peptides are considered good candidates for the next group of antibiotics because their proposed mode of action is different from those of conventional antibiotics (12,25). To advance antimicrobial peptides to the status of a new group of antibiotics, it is important to understand the mechanisms of action of these agents and the reason for their selectivity against microbes. In this study we compared the actions of three different peptides, namely, melittin, the toxic and hemolytic 26-residue peptide from the European honeybee Apis mellifera (6, 11), and two antibacterial peptides from the African clawed frog Xenopus laevis, an amide analogue of the 23-residue magainin 2 (32, 33) and the 21-residue PGLa (28), on the membrane of Escherichia coli. All three peptides are cationic ␣-helical antimicrobial peptides with markedly different sequential distributions of polar and nonpolar amino acids. However, all three peptides are amphipathic and highly membranolytic, with PGLa and magainin 2 having a better selectivity, via electrostatic discrimination, toward prokaryotic cells (13, 22) than the hemolytic melittin (which is a nonselective lytic peptide).Model membrane studies have provided a good understanding of antimicrobial peptides and their mechanisms of action. Different models for the interaction of a peptide with the bacterial membrane have been proposed: the barrel stave model (2), the toroidal pore or wormhole model (5, 31), the carpet model (23), and the peptide aggregate model (19,30). Most antimicrobial peptides are thought to follow the toroidal pore model and/or carpet model and not the barrel stave model, in which the peptides in the pore need to span the membrane without deformation of the lipid bilayer. Peptides that form toroidal pores remain associated with the phospholipid head groups and insert perpendicularly to the membrane plane by causing the lipid bilayer to fold back on itself. It has been proposed ...

Escherichia coli Cell Surface Perturbation and Disruption Induced by Antimicrobial Peptides BP100 and pepR

“…The average measured length, width, and height of the untreated cells (n ϭ 23) were 3.42 Ϯ 0.78 m, 1.18 Ϯ 0.18 m and 0.22 Ϯ 0.05 m, respectively. The dimensions of the air-dried E. coli cells reported here compare well with those found in the literature (20,21). E. coli cells were then treated with the minimum concentration of peptide required to inhibit bacterial growth by 50% for a period of 0.5, 2, and 5 h, and imaged.…”

“…Several studies investigating the interaction of cationic AMPs with model bacterial membranes have shown that an overcompensation in zeta potential at high peptide concentrations is associated with membrane insertion via hydrophobic interactions (22)(23)(24). A similar trend was also reported for the interaction of the cationic peptide rBPI 21 with LPS aggregates (24). The overcompensation in E. coli zeta potential at high pepR concentrations (ՆMICs) may indicate: 1) that hydrophobic interactions contribute to membrane interaction in addition to electrostatic attraction, or 2) not all positive charges of pepR contribute to electroneutralization.…”

Investigation into the mechanism of action of the antimicrobial peptides Os and Os-C derived from a tick defensin

“…Meincken et al previously found an increase in the membrane roughness of E. coli cells exposed to Mel [22]. The authors attributed this membrane roughness to an increase in the surface area of the membrane, after Mel incorporation into the outer membrane of the bacterial cell [22]. The ruffled appearance observed in the present study may similarly be caused by peptide integration into the membrane.…”

“…Therefore, it is likely that the cells exposed to Mel were under enough stress to cause DNA damage. Meincken et al previously found an increase in the membrane roughness of E. coli cells exposed to Mel [22]. The authors attributed this membrane roughness to an increase in the surface area of the membrane, after Mel incorporation into the outer membrane of the bacterial cell [22].…”

High-resolution imaging of pelagic bacteria by Atomic Force Microscopy and implications for carbon cycling

“…Such an increase in roughness has also been reported for E. coli 057 and the lytic phage A 157 phage-host system (Dubrovin et al, 2008). Further, AFM studies have shown that mechanical and chemical damage of cell surface results in increased roughness-including damage caused by EDTA (Amro et al, 2000), detergents (Camesano et al, 2000), proteases (F Malfatti, unpublished) (Camesano et al, 2000) and antibiotics (Braga and Ricci, 1998;Meincken et al, 2005). Marine phages lyse 10-50% of bacteria production in the upper ocean, and it would be interesting to observe if phage-infected bacteria and archaea could be identified through a surface 'signature'.…”