Characterization of four prophage endolysins specific for clostridium perfringens
Inventors
Donovan, David M. • Waters, Jerel • Rowley, Dayana T. • Swift, Steven M. • Oakley, Brian B.
Assignees
US Department of Agriculture USDA • Western University of Health Sciences • University of Maryland College Park
Publication Number
US-10544406-B2
Publication Date
2020-01-28
Expiration Date
2038-01-05
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Abstract
Clostridium perfringens can cause food poisoning and is a major agent in necrotic enteritis. As laws banning the use of antibiotics in animal feed become more common, the need for alternatives to antibiotics becomes greater. Peptidoglycan hydrolases that target the cell wall of specific bacteria are one such alternative. Genes for four endolysins, PlyCP10, PlyCP18, PlyCP33, and PlyCP41, were found within clusters of phage associated genes, likely prophages from strains Cp10, Cp18, Cp33, and Cp41. PlyCP18 and PlyCP33 harbor L-alanine amidase catalytic domains, and PlyCP10 and PlyCP41 have glycosyl hydrolase catalytic domains as predicted by BlastP and PFAM searches. All four genes were synthesized with E. coli codon optimization, expressed in E. coli expression vectors with a 6×His tag for nickel column purification, and the recombinant proteins purified. The four endolysins were capable of lysing the 66 C. perfringens strains tested but not the other bacteria tested.
Core Innovation
Clostridium perfringens is a Gram-positive, spore forming, anaerobic bacterium responsible for severe infections such as necrotic enteritis in animals including poultry, newborn cattle, and swine. Necrotic enteritis causes high mortality and economic losses in poultry production. Increasing restrictions and bans on antibiotics in animal feed have created a need for alternative antimicrobials effective against pathogens like C. perfringens.
The invention discloses nucleic acid molecules encoding four bacteriophage endolysins—PlyCP10, PlyCP18, PlyCP33, and PlyCP41—that specifically target and lyse C. perfringens by degrading its peptidoglycan cell wall. PlyCP18 and PlyCP33 harbor L-alanine amidase catalytic domains, whereas PlyCP10 and PlyCP41 have glycosyl hydrolase catalytic domains. These endolysins, expressed recombinantly, exhibit lytic activity against a wide range of C. perfringens strains but not against other tested bacteria.
The invention addresses the problem of antibiotic resistance and the need for alternatives to antibiotics in food animal production by providing highly specific antimicrobial proteins capable of lysing C. perfringens without promoting broad-spectrum resistance. The specificity and mode of action of phage endolysins make them potentially refractory to resistance development. The invention also includes constructs, vectors, and host cells for producing these endolysins, as well as compositions useful as therapeutic agents or feed supplements for controlling diseases caused by C. perfringens.
Claims Coverage
The patent includes multiple claims with two independent claims relating to recombinant DNA encoding endolysins and methods for producing them. There are six main inventive features disclosed.
Recombinant cDNA encoding antimicrobial peptidoglycan hydrolase proteins
The invention provides recombinant cDNA sequences encoding antimicrobial peptidoglycan hydrolase enzymes specifically represented by amino acid sequences SEQ ID NO: 2, 4, 6, 8, and 10, corresponding to PlyCP10, PlyCP18, PlyCP33, and PlyCP41 endolysins.
Nucleic acid sequences encoding endolysins
The cDNA sequences encoding the above antimicrobial proteins are specified as nucleic acid sequences SEQ ID NO: 1, 3, 5, 7, and 9.
Constructs operably linked to heterologous promoters
Nucleic acid constructs comprise the recombinant cDNA operably linked to heterologous promoters to enable expression in host cells.
Vectors comprising the recombinant constructs
Vectors contain the recombinant constructs allowing their introduction and replication in host cells for expression of the endolysin proteins.
Host cells transformed with the recombinant cDNA or constructs
Host cells including bacterial, fungal, plant, or mammalian cells are transformed with the recombinant cDNA or constructs encoding the endolysins to produce the antimicrobial proteins.
Methods for producing recombinant endolysin proteins
Methods comprise introducing the recombinant nucleic acid encoding specific endolysins into host cells, culturing the cells under suitable conditions, and recovering the expressed proteins in purified or partially purified forms.
The claims cover recombinant nucleic acids encoding specific phage endolysin proteins targeting C. perfringens, vectors and constructs for their expression, transformed host cells capable of producing these proteins, and methods for recombinant production of the endolysins in various host organisms.
Stated Advantages
High specificity to C. perfringens, lysing multiple strains without affecting other bacteria.
Potentially refractory to resistance development due to evolutionary co-adaptation of phage endolysins and bacterial hosts.
Viable alternatives to traditional antibiotics in animal feed, addressing public and regulatory concerns about antibiotic resistance.
Can be expressed recombinantly in various host organisms facilitating scalable production.
Documented Applications
Use of purified endolysin proteins as therapeutic agents to treat diseases caused by Clostridium perfringens in animals such as poultry, cattle, and swine.
Incorporation of endolysin compositions into nutritional or feed supplements for livestock to prevent or control C. perfringens infections.
Use in compositions formulated with pharmaceutically acceptable carriers for veterinary applications.
Potential development of kits comprising these endolysins for treating infections caused by C. perfringens.
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