Purification and characterization of a novel antibacterial peptide against Clostridium perfringens.

Bacillus velezensis HG88 was isolated from ileal mucosa samples of egg layer hens that were raised without the use of antibiotics. Its cell-free supernatant (CFS) was found to inhibit the growth of Clostridium perfringens, the causative agent of necrotic enteritis in chickens. The inhibitory compound was determined to be proteinaceous due to its susceptibility to protease digestion. The antimicrobial activity was specific towards C. perfringens, as the CFS did not inhibit the growth of Gram-positive or Gram-negative bacteria across nine different species and two yeast fungi. Separation of proteins from the CFS followed by peptide mass fingerprinting and genomic analyses of the strain enabled the identification of a putative antibacterial peptide with an export signal for secretion from the cell. The peptide from B. velezensis HG88, named IP_HG88, has sequence similarity to bacterial SH3 domains that are known to bind to the peptide portion of peptidoglycan. The gene encoding this peptide was cloned, and the peptide was purified from recombinant Escherichia coli as an N-terminal His-tagged peptide. The IP_HG88 with or without the His-tag inhibited the growth of C. perfringens with a minimum bactericidal concentration of ~57.0 or 39.1 µg ml^-1, respectively. The 3D structure of IP_HG88 was also predicted using AlphaFold 2.0.