Deciphering Novel Antimicrobial Peptides from the Transcriptome of Papilio xuthus

SIMPLE SUMMARY: Insects live in a pathogenic microorganism rich environment. Thus, insects develop a stronger defense in terms of innate immunity. Antimicrobial peptides (AMPs) are one of the key tools in the insect’s innate immune system, which kills the invading pathogens. In this study, we used Papilio xuthus, the Asian swallowtail butterfly, to identify the AMPs from their genomic product. We utilized next generation sequencing technology and in silico analysis tools to decipher the possible novel AMPs. The obtained novel AMPs were then tested for the antibacterial and antifungal activities. Seven novel peptides were identified as the antimicrobial agent, and these can be used as a lead candidate in the process of antibacterial therapy development against various infectious diseases. ABSTRACT: An insect’s innate immune system is the front line of defense against many invading microorganisms. One of the important components of this defense system is antimicrobial peptides (AMPs). Papiliocin is a well-studied antimicrobial peptide (AMP) isolated from the swallowtail butterfly, Papilio xuthus, and it was previously reported to be effective against Gram-positive bacteria, Gram-negative bacteria, and fungi, particularly in drug resistant Gram-negative bacteria. Hence, we aimed to identify novel AMPs from Papilio xuthus using its transcriptome. We immunized the swallowtail butterfly with Escherichia coli, Staphylococcus aureus, Candida albicans, and the total RNA was isolated. De novo transcriptome assembly and functional annotations were conducted, and AMPs were predicted using an in-silico pipeline. The obtained 344,804,442 raw reads were then pre-processed to retrieve 312,509,806 (90.6%) total clean reads. A total of 38,272 unigenes were assembled with the average length of 1010 bp. Differential gene expression analysis identified 584 and 1409 upregulated and downregulated genes, respectively. The physicochemical, aggregation, and allergen propensity were used as filtration criteria. A total of 248 peptides were predicted using our in-house pipeline and the known AMPs were removed, resulting in 193 novel peptides. Finally, seven peptides were tested in vitro and three peptides (Px 5, 6, and 7) showed stronger antimicrobial activity against Gram-negative bacteria and yeast. All the tested peptides were non-allergens. The identified novel AMPs may serve as potential candidates for future antimicrobial studies.