Thermophile lytic enzyme fusion proteins that target Clostridium perfringens

Authors: SWIFT, STEVEN - Retired ARS Employee; REID, KEVIN - Retired Non ARS Employee; DONOVAN, DAVID - Retired ARS Employee; Ramsay, Timothy

Submitted to: Antibiotics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/4/2019
Publication Date: 11/8/2019
Citation: Swift, S.M., Reid, K.P., Donovan, D.M., Ramsay, T.G. 2019. Thermophile lytic enzyme fusion proteins that target Clostridium perfringens. Antibiotics. https://doi.org/10.3390/antibiotics8040214.
DOI: https://doi.org/10.3390/antibiotics8040214

Interpretive Summary: Clostridium perfringens is an infectious bacterium that causes debilitating gut inflammation in swine and poultry, and is a source of food poisoning in humans. As the swine and poultry industry eliminate antibiotic use in animal feed, alternatives to antibiotics are needed for promoting growth and for treating antibiotic resistant bacteria. The most cost-effective way to treat swine and poultry is through the diet. However, most animal feeds are pelleted which requires heating the diet to very high temperatures. Therefore, heat tolerant anti-microbials are needed for implementation into swine and poultry feed. Enzymes exist that can kill bacteria and thus function as anti-microbials, but unfortunately are not tolerant of high temperatures. A series of thermostable enzymes were made by fusing components of enzymes known to tolerate high temperatures to parts of other enzymes that can kill bacteria. These fusion enzymes were then tested for their ability and efficacy to kill the bacteria Clostridium perfringens in a controlled laboratory environment. The resulting fifteen fusion proteins were more thermostable than the native C. perfringens endolysins, and killed several disease-associated strains of C. perfringens with varying success. Several of these fusion proteins have the potential for further refinement and development for use in pelleted diets as alternatives to antibiotics for promoting growth, health and well-being.

Technical Abstract: Clostridium perfringens is a bacterial pathogen that causes necrotic enteritis in swine and poultry, and is a source of food poisoning and gas gangrene in humans. As agriculture eliminates the use of antibiotics in animal feed, alternatives to antibiotics will be needed. Bacteriophage endolysins are enzymes used by the virus to burst their bacterial host to release bacteriophage particles. This type of enzyme represents a potential replacement for antibiotics for controlling C. perfringens. As animal feed is often heat-treated during production of feed pellets, thermostable enzymes would be preferred for feed. To make thermostable endolysins that target C. perfringens, a set of chimeric lysins were created by fusing thermophile endolysin catalytic domains with cell wall binding domains from different C. perfringens prophage endolysins. Thermostable catalytic domains were derived from prophage endolysins from Geobacillus species Y412MC61 and Y4.1MC1 and the endolysin from the deep-sea thermophilic bacteriophage Geobacillus virus E2 (GVE2) (L-alanine-amidase, glucosaminidase, and L-alanine-amidase, respectively). These domains degrade the peptidoglycan of the cell wall of bacteria. These catalytic domains were fused to the cell wall binding domains from C. perfringens prophage endolysins: PlyCP18, PlyCP10, PlyCP33, PlyCP41, and PlyCP26F. The resulting fifteen chimeric proteins were more thermostable than the native C. perfringens endolysins, and killed several disease-associated strains of C. perfringens.