FORMATION OF PORES IN ESCHERICHIA-COLI CELL-MEMBRANES BY A CECROPIN ISOLATED FROM HEMOLYMPH OF HELIOTHIS-VIRESCENS LARVAE

The insect humoral defense system produces antibacterial peptides call ed cecropins. Cecropins were initially isolated from Hyalophora cecrop ia pupae and have since been isolated and identified in various insect s. In this study, we have isolated and identified a cecropin from Heli othis virescens larvae. Rabbit IgG were raised against synthetic cecro pin B. Affinity chromatography with the rabbit anti-(cecropin B) IgG w as used to isolate a cecropin from hemolymph of H. virescens larvae. A cid gel electrophoresis followed by a bacterial-overlay analysis showe d that Heliothis cecropin is a basic peptide of low molecular mass wit h bactericidal activity against Escherichia coli K12 D31. Heliothis ce cropin is therefore analogous to synthetic cecropin B. One unresolved issue concerning cecropins and other antibiotic peptides is the mode o f action by which they kill bacteria. By means of electron microscopy and immunocytochemistry with gold-labeled rabbit anti-cecropin IgG, bi nding of purified and synthetic cecropin to the cell membranes of E. c oli K12 D31 cells was observed. Small lesions in the cell membrane wer e 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.