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Published on: December 30, 2016
Purification and characterization of a novel antibacterial peptide against Clostridium perfringens
Alex Novodvorski1,2, Avalene Kong1,2, Hai Yu1
1Guelph Research and Development Centre, Agriculture and Agri-Food Canada, Guelph, Ontario, N1G 5C9, Canada.
Abstract:
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 IPHG88, 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 IPHG88 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 IPHG88 was also predicted using AlphaFold 2.0.
Insights
A novel antibacterial peptide, IPHG88, was identified from Bacillus velezensis HG88. This peptide effectively inhibits Clostridium perfringens, a key cause of necrotic enteritis in chickens.
Area of Science:
- Microbiology
- Bacteriology
- Poultry Science
Background:
- Necrotic enteritis, caused by Clostridium perfringens, poses a significant threat to chicken health and the poultry industry.
- Antibiotic use in poultry production is increasingly restricted, necessitating the development of alternative disease control strategies.
Purpose of the Study:
- To isolate and characterize novel antimicrobial compounds from antibiotic-free raised chickens.
- To identify and validate a specific inhibitor of Clostridium perfringens growth.
Main Methods:
- Isolation of Bacillus velezensis HG88 from chicken ileal mucosa.
- Analysis of cell-free supernatant (CFS) for antimicrobial activity against Clostridium perfringens.
- Protein identification via peptide mass fingerprinting and genomic analysis.
- Gene cloning, recombinant peptide purification, and in vitro antimicrobial assays.
- 3D structure prediction using AlphaFold 2.0.
Main Results:
- Bacillus velezensis HG88 CFS demonstrated specific inhibitory activity against Clostridium perfringens.
- A novel antibacterial peptide, IPHG88, was identified and characterized.
- Purified IPHG88 inhibited Clostridium perfringens growth with minimum bactericidal concentrations of 57.0 µg/mL (with His-tag) and 39.1 µg/mL (without His-tag).
- IPHG88 shares sequence similarity with bacterial SH3 domains, suggesting a peptidoglycan-binding mechanism.
Conclusions:
- Bacillus velezensis HG88 produces a potent antibacterial peptide, IPHG88, effective against Clostridium perfringens.
- IPHG88 represents a promising candidate for developing novel therapeutic strategies against necrotic enteritis in poultry.
- This discovery supports the use of beneficial bacteria as a sustainable alternative to antibiotics in animal agriculture.
