Optimized production of a biologically active Clostridium perfringens glycosyl hydrolase phage endolysin PlyCP41 in plants using virus-based systemic expression

Authors: Hammond, Rosemarie; Swift, Steven; Foster Frey, Juli; KOVALSKAYA, NATALIA - Oak Ridge Institute For Science And Education (ORISE); Donovan, David

Submitted to: BMC Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/6/2019
Publication Date: 12/21/2019
Publication URL: https://handle.nal.usda.gov/10113/6795073
Citation: Hammond, R., Swift, S.M., Foster Frey, J.A., Kovalskaya, N.Y., Donovan, D.M. 2019. Optimized production of a biologically active Clostridium perfringens glycosyl hydrolase phage endolysin PlyCP41 in plants using virus-based systemic expression. BioMed Central (BMC) Biotechnology. 19/101. https://doi.org/10.1186/s12896-019-0594-7.
DOI: https://doi.org/10.1186/s12896-019-0594-7

Interpretive Summary: Clostridium perfringens, a gram-positive, anaerobic, rod-shaped bacterium, is the third leading cause of human foodborne bacterial disease and causes necrotic enteritis, which can lead to significant levels of mortality and lost productivity in poultry. The disease is commonly controlled using antibiotics in drinking water or feed, widespread use of which may lead to development of resistant bacteria. Bacteriophage-encoded endolysins that degrade the bacterial cell wall are potential replacements for the antibiotics. ARS Researchers in Beltsville, Maryland demonstrated the expression in plants of the soluble, functional bacteriophage CP41 endolysin using a plant virus-based expression system. The endolysin retained activity against C. perfringens in crude plant sap. The incorporation of the lysin in plant cells eliminates the need to purify the protein for addition to animal feed as an effective antimicrobial agent against C. perfringens. These results are of value to animal production scientists who are developing alternatives to antibiotics to control disease in poultry and livestock.

Technical Abstract: Clostridium perfringens, a gram-positive, anaerobic, rod-shaped bacterium, is the third leading cause of human foodborne bacterial disease and necrotic enteritis in poultry. It is controlled using antibiotics, widespread use of which may lead to development of resistant bacteria. Bacteriophage-encoded endolysins that degrade peptidoglycans in the bacterial cell wall are potential replacements for antibiotics. Phage endolysins have been identified that exhibit antibacterial activities against several Clostridium strains. An Escherichia coli codon-optimized gene encoding the glycosyl hydrolase endolysin (PlyCP41) engineered for expression in plants (PlyCP41p) and a plant codon-optimized gene (PlyCP41pc), both containing a polyhistidine tag, were expressed in Nicotiana benthamiana plants using a potato virus X-based transient expression vector. PlyCP41p accumulated to ~1% total soluble protein (100ug/gm f. wt leaf tissue) without any obvious toxic effects on plant cells, and both the purified protein and plant sap containing the protein lysed C. perfringens strain Cp39 in a plate lysis assay. Optimal expression of PlyCP41p was achieved at 2 weeks-post-infection. PlyCP41pc did not accumulate to higher levels than PlyCP41p in infected tissue. In conclusion, we demonstrated that a functionally active bacteriophage PlyCP41 endolysin can be produced in plants with the potential to be an effective antimicrobial agent against C. perfringens.