Characterization of antimicrobial peptides deriving from insects and their application in the biomedical field

Antibiotics are the current drugs used to treat pathogenic bacteria, but their prolonged use contributes to the development and spread of drug-resistant microorganisms. The antibiotic resistance issue led to the need to find new alternative molecules, which should be less prone to bacterial resistance. Antimicrobial peptides (AMPs) aroused great interest as potential next-generation antibiotics. AMPs are involved in several defence-related processes such as the binding and neutralization of endotoxins, the modulation of the immune responses to infection and the killing of pathogens. Antimicrobial peptides are small molecules with an amino acid composition ranging from 10 to 100 residues and are biosynthesized by all living organisms but it is known that the class of insects represents the largest source of these molecules. This aspect is related to insect’s biodiversity and their ability to live in hostile environments rich of pathogens. Most insect AMPs are cationic molecules due to the presence of basic residues and according to their amino acid sequences and structures, they can be classified in four different groups: cysteine-rich peptides (e.g., defensins), the α-helical peptides (e.g., cecropins), glycine-rich proteins (e.g., attacins) and proline-rich peptides (e.g., drosocins). Insect AMPs have demonstrated to be useful in several applications concerning the pharmaceutical as well as the agricultural fields. Moreover, insect AMPs aroused great interest for their biomedical application thanks to the increasing number of peptides that can inhibit human pathogens. For this reason, this Ph.D. project aimed to the identification of antimicrobial peptides deriving from insects, particularly from the Black Soldier Fly Hermetia illucens (L.) (Diptera: Stratiomyidae). Through a combination of transcriptomics and bioinformatics analysis, 57 antimicrobial peptides have been identified from H. illucens insect. Through an in silico analysis, the biological activity have been predicted and the physio-chemical properties have been calculated for all the identified peptides. Based on the bioinformatics results, the in vitro production of the most promising sequences has been performed through molecular cloning strategies in order to evaluate the antibacterial activity in vitro. Particularly, some of the identified peptides (C16571, C46948, C16634, and C7985) showed the ability to inhibit E. coli growth at a concentration value of 3 μM. For the C15867 peptide, recombinantly produced and expressed, a MIC (Minimum Inhibitory Concentration) value of 18 μM has been determined. Moreover, an in vivo approach was carried out for the identification of antimicrobial peptides by extracting the hemolymph from the H. illucens larvae, recovering then the peptides fraction from the larvae’s plasma and its antibacterial activity has been evaluated against both Gram-positive and Gram-negative bacteria. The performed analysis showed that a small amount (7.5/15 μL) of the peptide fraction recovered from the larvae’s plasma was able to inhibit the cell growth of different bacterial strains.