title: A Thermophilic Phage Endolysin Fusion to a Clostridium perfringens-Specific Cell Wall Binding Domain Creates an Anti-Clostridium Antimicrobial with Improved Thermostability.
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 ɸGVE2 to the cell-wall binding domain (CWB) from the endolysin of the C. perfringens-specific bacteriophage ɸCP26F. The resulting protein, PlyGVE2CpCWB, lysed C. perfringens in liquid and solid cultures. PlyGVE2CpCWB was most active at pH 8, 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 thermostable chimeric endolysin that could potentially be utilized as a feed additive to control the bacterium during poultry production.
authors.affiliation: Animal Biosciences and Biotechnology Laboratory, Beltsville Agricultural Research Center, Agricultural Research Service, U.S. Department of Agriculture, 10300 Baltimore Avenue, Bldg. 230, BARC-East, Beltsville, MD 20705, USA. steven.swift@ars.usda.gov.
authors.forename: Steven M
authors.initials: SM
authors.lastname: Swift
authors.affiliation: Poultry Microbiological Safety and Processing Research Unit, Richard B. Russell Agricultural Research Center, Agricultural Research Service, U.S. Department of Agriculture, 950 College Station Road, Athens, GA 30605, USA. sealb@onid.oregonstate.edu.
authors.forename: Bruce S
authors.initials: BS
authors.lastname: Seal
authors.affiliation: Poultry Microbiological Safety and Processing Research Unit, Richard B. Russell Agricultural Research Center, Agricultural Research Service, U.S. Department of Agriculture, 950 College Station Road, Athens, GA 30605, USA. johnna.garrish@ars.usda.gov.
authors.forename: Johnna K
authors.initials: JK
authors.lastname: Garrish
authors.affiliation: Poultry Microbiological Safety and Processing Research Unit, Richard B. Russell Agricultural Research Center, Agricultural Research Service, U.S. Department of Agriculture, 950 College Station Road, Athens, GA 30605, USA. boakley@westernu.edu.
authors.forename: Brian B
authors.initials: BB
authors.lastname: Oakley
authors.affiliation: Poultry Microbiological Safety and Processing Research Unit, Richard B. Russell Agricultural Research Center, Agricultural Research Service, U.S. Department of Agriculture, 950 College Station Road, Athens, GA 30605, USA. keli.hiett@ars.usda.gov.
authors.forename: Kelli
authors.initials: K
authors.lastname: Hiett
authors.affiliation: Poultry Microbiological Safety and Processing Research Unit, Richard B. Russell Agricultural Research Center, Agricultural Research Service, U.S. Department of Agriculture, 950 College Station Road, Athens, GA 30605, USA. hungyueh.yeh@ars.usda.gov.
authors.forename: Hung-Yueh
authors.initials: HY
authors.lastname: Yeh
authors.affiliation: Nevada Proteomics Center, University of Nevada School of Medicine, Manville Medical Building, 1664 N. Virginia Street, Reno, NV 89557-MS0330, USA. rebekahw@unr.edu.
authors.forename: Rebekah
authors.initials: R
authors.lastname: Woolsey
authors.affiliation: Nevada Proteomics Center, University of Nevada School of Medicine, Manville Medical Building, 1664 N. Virginia Street, Reno, NV 89557-MS0330, USA. schegg@unr.edu.
authors.forename: Kathleen M
authors.initials: KM
authors.lastname: Schegg
authors.affiliation: Poultry Microbiological Safety and Processing Research Unit, Richard B. Russell Agricultural Research Center, Agricultural Research Service, U.S. Department of Agriculture, 950 College Station Road, Athens, GA 30605, USA. eric.line@ars.usda.gov.
authors.forename: John Eric
authors.initials: JE
authors.lastname: Line
authors.affiliation: Animal Biosciences and Biotechnology Laboratory, Beltsville Agricultural Research Center, Agricultural Research Service, U.S. Department of Agriculture, 10300 Baltimore Avenue, Bldg. 230, BARC-East, Beltsville, MD 20705, USA. David.Donovan@ars.usda.gov.
authors.forename: David M
authors.initials: DM
authors.lastname: Donovan
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000378:metabolism*; D001433:Bacteriolysis; D001435:Bacteriophages / Q000201:enzymology / Q000235:genetics; D002473:Cell Wall / Q000187:drug effects*; D002645:Chickens; D003016:Clostridium perfringens / Q000187:drug effects*; D010450:Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020536:Enzyme Activators / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D019649:Food Industry / Q000379:methods; D059022:Food Safety; D015870:Gene Expression; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011485:Protein Binding; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012965:Sodium Chloride / Q000378:metabolism; D013696:Temperature
chemical_list: D000900:Anti-Bacterial Agents; D020536:Enzyme Activators; D011993:Recombinant Fusion Proteins; D012965:Sodium Chloride; D010450:Endopeptidases; C001143:endolysin
keywords: alternative antimicrobial; bacteriophage; endolysin; feed additive; food safety; peptidoglycan hydrolase; thermostabile; thermostable