Antimicrobial potential of a ponericin-like peptide isolated from Bombyx mori L. hemolymph in response to Pseudomonas aeruginosa infection

The main effectors in the innate immune system of Bombyx mori L. are antimicrobial peptides (AMPs). Here, we infected B. mori with varied inoculum sizes of Pseudomonas aeruginosa ATCC 25668 cells to investigate changes in morpho-anatomical responses, physiological processes and AMP production. Ultraviolet–visible spectra revealed a sharp change in λ(max) from 278 to 285 nm (bathochromic shift) in the hemolymph of infected B. mori incubated for 24 h. Further, Fourier Transform InfraRed studies on the hemolymph extracted from the infected B. mori showed a peak at 1550 cm(−1), indicating the presence of α-helical peptides. The peptide fraction was obtained through methanol, acetic acid and water mixture (90:1:9) extraction, followed by peptide purification using Reverse Phase High Performance Liquid Chromatography. The fraction exhibiting antibacterial properties was collected and characterized by Matrix-Assisted Laser Desorption/Ionization-Time of Flight. A linear α-helical peptide with flexible termini (LLKELWTKMKGAGKAVLGKIKGLL) was found, corresponding to a previously described peptide from ant venom and here denominated as Bm-ponericin-L1. The antibacterial activity of Bm-ponericin-L1 was determined against ESKAPE pathogens. Scanning electron microscopy confirmed the membrane disruption potential of Bm-ponericin-L1. Moreover, this peptide also showed promising antibiofilm activity. Finally, cell viability and hemolytic assays revealed that Bm-ponericin-L1 is non-toxic toward primary fibroblasts cell lines and red blood cells, respectively. This study opens up new perspectives toward an alternative approach to overcoming multiple-antibiotic-resistance by means of AMPs through invertebrates’ infection with human pathogenic bacteria.