Identification and characterization of new insect-derived antimicrobial peptides: an innovative approach against drug resistance

This thesis investigates insect-derived antimicrobial peptides (AMPs) as an innovative and potentially scalable approach to tackling antimicrobial resistance (AMR). This work integrates discovery, functional validation, and rational optimization into a single translational framework. The research begins by placing AMPs in the broader context of the AMR crisis and framing insects as an extremely rich source of innate immunity effector molecules, while outlining proteomics-based strategies useful for “deconvoluting” complex biological matrices and prioritizing peptide candidates. Building on this foundation, the experimental work establishes H. illucens hemolymph as a platform for inducing, recovering, and profiling bioactive peptide fractions. Immune stimulation followed by hemolymph recovery yields peptide-enriched extracts with broad antibacterial and antifungal activity, including efficacy against clinically relevant drug-resistant pathogens. In addition, this work demonstrates that hemolymph-derived peptide extracts from immunologically stimulated larvae can enhance the antibacterial activity of conventional antibiotics, such as ampicillin and kanamycin, against both Gram-negative and Gram-positive bacterial targets, supporting their possible use as adjuvants in combination therapies. Beyond antimicrobial potential, the thesis explores the activity of insect peptide fractions in oncology-relevant models. Hemolymph-derived fractions suppress cancer cell lines growth in a dose- and time-dependent manner and may enhance standard therapeutic efficacy. This thesis also develops an experimental and proteomic workflow to identify infection-induced AMPs directly from larval hemolymph. In particular, the larvae are subjected to an immune challenge with Gram-negative and Gram-positive bacteria, then the peptides derived from the hemolymph are extracted and fractionated using reverse-phase high-performance liquid chromatography (RP-HPLC). The fractions obtained are tested in vitro for antibacterial activity against a panel of pathogens and then further characterized by LC-MS/MS to identify candidate AMPs, accompanied by shotgun proteomics on unfractionated extracts for quantitative comparisons between experimental conditions. To complete the process, in silico analysis with predictive tools highlights physicochemical profiles and machine learning-based scores consistent with an antimicrobial function, supporting the prioritization of candidates. To move from complex mixtures to defined therapeutic leads, the thesis evaluates complementary methods for selecting and improving candidates. A first approach considers a selected set of AMPs from H. illucens, prioritized in silico and produced by chemical synthesis, to be subjected to experimental screening; a second approach implements an integrated computational-experimental pipeline that fragments insect-derived AMPs into short “lead” sequences and refines them through sequence rational modifications. Overall, the thesis proposes a roadmap from discovery to optimization of insect-derived AMPs. It demonstrates that immunologically stimulated H. illucens hemolymph contains peptide repertoires with both antimicrobial and potentially antitumoral relevant activities, as well as the ability to potentiate the activity of conventional antibiotics in vitro, and provides practical design principles for transforming insect-derived AMPs into optimized short peptides suitable for rigorous preclinical evaluation.