Screening and Functional Analyses of Novel Cecropins from Insect Transcriptome

SIMPLE SUMMARY: Antimicrobial peptides (AMPs) are widely present in organisms, exhibiting broad-spectrum antimicrobial activity and rarely developing resistance. Currently, research on AMPs in many species is still limited, and they have great potential for exploration. In this study, a class of AMPs called Cecropin was identified from insect transcriptome using bioinformatics. Novel Cecropin genes were discovered in several insects and three of them were selected for experimental validation. These peptides possess antimicrobial activity against Gram-negative bacteria, exhibit a structure and mechanism of action similar to known Cecropins, and are non-toxic to mammalian cells. This study provides a reliable analytical method in the search for AMPs, which hold promising application prospects in diverse fields such as medicine, food, and beyond. ABSTRACT: Antibiotic resistance is a significant and growing threat to global public health. However, antimicrobial peptides (AMPs) have shown promise as they exhibit a broad spectrum of antibacterial activities with low potential for resistance development. Insects, which inhabit a wide range of environments and are incredibly diverse, remain largely unexplored as a source of novel AMPs. To address this, we conducted a screening of the representative transcriptomes from the 1000 Insect Transcriptome Evolution (1KITE) dataset, focusing on the homologous reference genes of Cecropins, the first identified AMPs in insects known for its high efficiency. Our analysis identified 108 Cecropin genes from 105 insect transcriptomes, covering all major hexapod lineages. We validated the gene sequences and synthesized mature peptides for three identified Cecropin genes. Through minimal inhibition concentration and agar diffusion assays, we confirmed that these peptides exhibited antimicrobial activities against Gram-negative bacteria. Similar to the known Cecropin, the three Cecropins adopted an alpha-helical conformation in membrane-like environments, efficiently disrupting bacterial membranes through permeabilization. Importantly, none of the three Cecropins demonstrated cytotoxicity in erythrocyte hemolysis tests, suggesting their safety in real-world applications. Overall, this newly developed methodology provides a high-throughput bioinformatic pipeline for the discovery of AMP, taking advantage of the expanding genomic resources available for diverse organisms.