Characterization of two glycosyl hydrolases, putative prophage endolysins, that target Clostridium perfringens

Authors: Swift, Steven; DONOVAN, DAVID - Retired ARS Employee; OAKLEY, BRIAN - Western University Of Health Sciences; WATERS, JEREL - US Department Of Agriculture (USDA); ROWLEY, DAYANA - Retired ARS Employee; Ramsay, Timothy

Submitted to: FEMS Microbiology Letters
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/11/2018
Publication Date: 7/13/2018
Citation: Swift, S.M., Donovan, D., Oakley, B.B., Waters, J.J., Rowley, D.T., Ramsay, T.G. 2018. Characterization of two glycosyl hydrolases, putative prophage endolysins, that target Clostridium perfringens. FEMS Microbiology Letters. https://doi.org/10.1093/femsle/fny179.
DOI: https://doi.org/10.1093/femsle/fny179

Interpretive Summary: The bacteria, Clostridium perfringens, causes disease in chickens, pigs, and cows that leads to weight loss and death, which results in loss of income for farmers. The use of antibiotics in animal feed may contribute to development of bacteria that are resistant to antibiotics, which has become a health and safety concern for people. Replacements for antibiotics are needed for treating or preventing infections of farm animals by the Clostridium perfringens bacteria. We discovered two proteins, PlyCP10 and PlyCP41, that can kill these bacteria by damaging the cell wall of the bacteria. We also identified conditions that were favorable for these proteins to kill the bacteria. These proteins killed Clostridium perfringens bacteria from different infected farm animal populations, proving their versatility in combatting infection. These proteins may be able to cure or prevent infections of farm animals by the Clostridium perfringens bacteria, thereby increasing farm productivity, improving animal welfare and averting loss of income for farmers and commercial animal producers.

Technical Abstract: Clostridium perfringens, a spore-forming anaerobic bacteria, causes food poisoning and gas gangrene in humans, and is an agent of necrotizing enteritis in poultry, swine and cattle. Endolysins are peptidoglycan hydrolases from bacteriophage that degrade the bacterial host cell wall causing lysis and thus harbor antimicrobial therapy potential. The genes for the PlyCP10 and PlyCP41 endolysins were found in prophage regions of the genomes from Clostridium perfringens strains Cp10 and Cp41, respectively. The gene for PlyCP10 encodes a protein of 351 amino acids, while the gene for PlyCP41 encodes a protein of 335 amino acids. Both proteins harbor predicted glycosyl hydrolase domains. Recombinant PlyCP10 and PlyCP41 were expressed in E. coli with C-terminal His-tags, purified by nickel chromatography, and characterized in vitro. PlyCP10 activity was greatest at pH 6.0, and between 50-100 mM NaCl. PlyCP41 activity was greatest between pH 6.5 and 7.0, and at 50 mM NaCl, with retention of activity as high as 600 mM NaCl. PlyCP10 lost most of its activity above 42°C, whereas PlyCP41 survived at 55°C for 30 minutes and still retained >80% activity. Both enzymes had lytic activity against 75 C. perfringens strains (isolates from poultry, swine and cattle) suggesting therapeutic potential.