Formation of Pores in <i>Escherichia coli</i> Cell Membranes by a Cecropin Isolated from Hemolymph of <i>Heliothis virescens</i> Larvae

Abstract

The insect humoral defense system produces antibacterial peptides called cecropins. Cecropins were initially isolated from Hyalophora cecropia pupae and have since been isolated and identified in various insects. In this study, we have isolated and identified a cecropin from Heliothis virescens larvae. Rabbit IgG were raised against synthetic cecropin B. Affinity chromatography with the rabbit anti-(cecropin B) IgG was used to isolate a cecropin from hemolymph of H. virescens larvae. Acid gel electrophoresis followed by a bacterial-overlay analysis showed that Heliothis cecropin is a basic peptide of low molecular mass with bactericidal activity against Escherichia coli K12 D31. Heliothis cecropin is therefore analogous to synthetic cecropin B. One unresolved issue concerning cecropins and other antibiotic peptides is the mode of action by which they kill bacteria. By means of electron microscopy and immunocytochemistry with gold-labeled rabbit anti-cecropin IgG, binding of purified and synthetic cecropin to the cell membranes of E. coli K12 D31 cells was observed. Small lesions in the cell membrane were seen that had a diameter of 9.6 nm and internal pore of 4.2 nm. The Heliothis cecropin was found to be a pore-forming molecule that causes lesions in the cell membrane of E. coli K12 D31. The lesions lead to leakage of cytoplasmic contents and death of bacteria.