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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Animal Biosciences & Biotechnology Laboratory » Research » Publications at this Location » Publication #316953

Research Project: DEVELOPING GENETIC BIOTECHNOLOGIES FOR INCREASED FOOD ANIMAL PRODUCTION, INCLUDING NOVEL ANTIMICROBIALS FOR IMPROVED HEALTH & PRODUCT SAFETY

Location: Animal Biosciences & Biotechnology Laboratory

Title: A thermophilic phage endolysin fusion to a Clostridium perfringens-specific cell wall binding domain creates an anti-clostridium antimicrobial with improved thermostability

Author
item Swift, Steven
item Seal, Bruce
item Garrish, Johnna
item Oakley, Brian
item Yeh, Hung-yueh
item Woolsey, Rebekah - University Of Nevada School Of Medicine
item Schegg, Kathleen - University Of Nevada School Of Medicine
item Line, John - Eric
item Donovan, David
item Hiett, Kelli

Submitted to: Viruses and Bacteriophages
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/12/2015
Publication Date: 6/12/2015
Citation: Swift, S., Seal, B.S., Garrish, J.K., Oakley, B., Yeh, H., Woolsey, R., Schegg, K.M., Line, J.E., Donovan, D.M., Hiett, K.L. 2015. A thermophilic phage endolysin fusion to a Clostridium perfringens-specific cell wall binding domain creates an anti-clostridium antimicrobial with improved thermostability. Viruses and Bacteriophages. 7(6):3019-3034.

Interpretive Summary: The bacteria Clostridium (C.) perfringens is the third leading cause of human foodborne bacterial disease and the cause of a serious disease of poultry (UC Necrotic enteritis). Treatment of poultry with antibiotics is becoming less acceptable so alternatives to antibiotics to control the bacteria are needed. Bacteriophages are viruses that infect and kill bacteria. One of the molecular tools that bacteriophage use are endolysins. Endolysins are proteins that can degrade the bacterial cell wall and cause the cells to rupture. Endolysins are thus potential replacements for antibiotics. With the goal to add these endolysins to chicken feed to control C. perfringens, the proteins must be able to withstand high temperatures (be thermostable) due to the high temperatures seen during animal feed processing. To produce a thermostable endolysin for feeding to poultry, a gene was synthesized that fused an endolysin from a thermophilic bacteriophage to a endolysin of a C. perfringens-specific bacteriophage. The result was the creation of a new fusion endolysin with increased thermostability, that could potentially be utilized as a feed additive to control the bacterium during poultry production. This would be a huge benefit to the poultry industry if successful.

Technical Abstract: Clostridium perfringens is the third leading cause of human foodborne bacterial disease and is the presumptive etiologic agent of Necrotic enteritis among chickens. Treatment of poultry with antibiotics is becoming less acceptable. Endolysin enzymes are potential replacements for antibiotics. Many enzymes are added to animal feed during production and are subjected to high-heat stress during feed processing. To produce a thermostabile endolysin for treating poultry, an E. coli codon-optimized gene was synthesized that fused the N-acetylmuramoyl-L-alanine amidase domain from the endolysin of the thermophilic bacteriophage to the cell wall binding domain from the endolysin of a C. perfringens-specific bacteriophage. The resulting protein lysed C. perfringens in liquid and solid cultures, was most active at pH8, had peak activity at 10 mM NaCl, 40% activity at 150 mM NaCl and was still 16% active at 600 mM NaCl. The protein was able to withstand temperatures up to 50°C and still lyse C. perfringens. Herein we report the construction and characterization of a thermostabile chimeric endolysin that could potentially be utilized as a feed additive to control the bacterium during poultry production.