Purification Of Antimicrobial Peptides From Housefly Larvae And Their Effects On Bacterial Membrane And DNA

Insect antimicrobial peptides are the important components of insect immune systems with the advantages of small molecule weight, heat-stability and broad antibacterial activity. Antimicrobial peptides (AMPs) from edible insects may serve as a potentially significant group of food preservatives. Study on the antimicrobial peptides from the Chinese traditional edible housefly (Musca domestica) larvae is of therefore both theoretical and practical importance.Based on the membrane binding property of AMPs, an innovative method was developed to isolate AMPs from housefly larvae. Combining with gel filtration chromatography followed by reverse phase high-performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry analysis, two novel antimicrobial peptides MDpep5 and MDpep9 were successfully isolated. These two peptides were active against both Gram-negative (Escherichia coli and Salmonella typhimurium) and Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis), with MIC values ranged from 9 to 36μg/mL, but no antimicrobial activity for fungi (Penicillium expansum and Aspergillus niger) and yeast (Saccharomyces cerevisiae) was detected up to the concentration of 576μg/ml.Changes of the A630nm, membrane permeability and the morphological of bacteria showed that the cytoplasmic cell membrane is the target for peptides which can exert its antimicrobial action by disrupting and disintegrating bacterial cell membranes, leading ultimately to loss of cytoplasmic membrane integrity. Moreover, sublethal stage existed in the bactericidal process through flow cytometry determination.Measuring the change of A260nm showed that the peptides interacted with the bacterial ultraviolet-absorbing materials such as DNA, RNA and nucleotides, and make them released. The electrophoresis, ultraviolet spectra and fluorescence spectra, and circular dichroism results indicated that the antimicrobial peptides could binding to bacterial genomic DNA, change its conformation, loosen the double helix of DNA, facilitating the interaction of EcoRⅠand DNA, finally accelerate DNA degradation.All the results demonstrated that antimicronial peptides MDpep5 and MDpep9 have dual mechanisms of bactericidal activity: disrupting bacterial cell membranes and binding to bacterial genomic DNA to accelerate DNA degradation by facilitating the interaction of EcoRⅠand DNA, ultimately leading to cell death. The results of bactericidal mode may be contributive in designing new and promising antimicrobial peptides for food preservatives, and exploitation and utility of the antibacterial peptide from the housefly.