Abstract
Cecropins constitute an important family of insect antimicrobial peptides involved in humoral innate immune response. In comparison with the highly basic cecropins A and B, cecropins D are less cationic and more hydrophobic. Interestingly, cecropins D were described only in lepidopteran insects, e.g., the greater wax moth Galleria mellonella. In the present study, interactions of neutral cecropin D (pI 6.47) purified from hemolymph of G. mellonella with living Escherichia coli cells were investigated. Fluorescence lifetime imaging microscopy using fluorescein isothiocyanate-labeled cecropin D revealed very fast binding of the peptide to E. coli cells. Fourier transform infrared spectroscopy analyses showed that G. mellonella cecropin D interacted especially with E. coli LPS and probably other lipid components of the bacterial cell envelope and exhibited an ordering effect with regard to lipid chains. This effect is consistent with the peptide binding mechanism based upon its incorporation into the lipid phase of the cell membrane. The interaction resulted in permeabilization of the bacterial cell membrane. Upon cecropin D binding, the cells lost characteristic surface topography, which was accompanied by altered nanomechanical properties, as revealed by atomic force microscopy. The interaction of the peptide with the bacterial cells also led to intracellular damage, i.e., loss of the cell envelope multilayer structure, formation of membrane vesicles, and enlargement of periplasmic space, which eventually caused death of the bacteria. In summary, it can be concluded that amphipathic character of α-helices, exposure of small positively charged patches on their polar surfaces and hydrophobic interactions are important physicochemical characteristics related to effective binding to E. coli cells and antibacterial activity of neutral G. mellonella cecropin D.
Highlights
Antimicrobial peptides (AMPs) are small, amphipathic, and mainly cationic molecules that upon interaction with microbial membranes lead to disruption of the membrane1 3 Vol.:(0123456789)A
The exposure of E. coli JM83 cells to G. mellonella cecropin D at the concentration range of 2.5–20 μM led to permeabilization of the bacterial cell membrane with similar kinetics of the peptide action during the 60-min incubation time, regardless of the concentration (Fig. 1)
As revealed by the cell membrane permeabilization assay, cecropin D used at the 2.5 μM concentration effectively permeabilized E. coli JM83 cell membranes
Summary
Antimicrobial peptides (AMPs) are small, amphipathic, and mainly cationic molecules that upon interaction with microbial membranes lead to disruption of the membrane. Cecropins constitute a family of invertebrate AMPs, found especially in insects. They are amphipathic and mostly highly basic linear peptides composed of 31–39 amino acids, which can form two flexible α-helices bound with a hinge region in a hydrophobic environment. The distribution of hydrophilic and hydrophobic groups is conserved among all cecropins. They possess a hydrophilic N-terminal part containing many charged residues, a very hydrophobic stretch between residues 22–30, and a more hydrophilic C-terminal end. The N-terminal part of a cecropin molecule is strongly basic, whereas the C-terminal part is hydrophobic. Cecropins D were described only in lepidopteran insects, suggesting that this subfamily appeared relatively late in the evolution of insects (Gudmundsson et al 1991)
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