Thermophile peptidoglycan hydrolase fusion proteins and uses thereof
Inventors
Donovan, David M. • Swift, Steven M.
Assignees
University of Maryland Baltimore • US Department of Agriculture USDA • University of Maryland College Park
Publication Number
US-11236315-B2
Publication Date
2022-02-01
Expiration Date
2039-04-04
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Abstract
The disclosure relates to chimeric recombinant lysins comprising at least one thermophile endolysin catalytic domain and at least one cell wall binding domain. Also disclosed are polynucleotides encoding the chimeric recombinant lysins, host cells expressing the chimeric recombinant lysins, and use of such chimeric recombinant lysins.
Core Innovation
The invention relates to chimeric recombinant lysins comprising at least one thermophile endolysin catalytic domain selected from the N-terminal catalytic domains of PlyGspY412, PlyGspY4, and PlyGVE2, fused with at least one cell wall binding domain derived from Clostridium perfringens endolysins PlyCP10, PlyCP18, PlyCP33, PlyCP41, or PlyCP26. These chimeric lysins optionally include linkers between the catalytic and cell wall binding domains and may contain a polyhistidine tag. The chimeric recombinant lysins are capable of killing several strains of C. perfringens but do not affect other Gram-positive bacteria.
The problem addressed by this invention arises from the pathogenicity of Clostridium perfringens, which causes severe infections in animals and humans, including necrotic enteritis in poultry, food poisoning, and gas gangrene. Current control of necrotic enteritis relies heavily on antimicrobial drugs used as growth promoters in feed or water. However, there is an increasing global consensus and regulatory actions to ban or limit the use of antibiotics in animal feeds due to concerns over antibiotic resistance development and public health implications. Such restrictions exacerbate the risk of disease outbreaks in the livestock industry. Therefore, there is a critical need for effective alternatives to traditional antibiotics that effectively prevent and treat diseases caused by C. perfringens, especially in poultry.
The invention exploits the modular domain structure of bacteriophage endolysins, utilizing thermophile-derived catalytic domains fused to C. perfringens-specific cell wall binding domains to produce chimeric lysins with enhanced thermostability and targeted activity. The thermophile-derived catalytic domains offer improved resistance to heat, which is beneficial for industrial applications like incorporation into animal feed that undergoes heat treatments. The cell wall binding domains provide specificity for C. perfringens, limiting off-target effects on beneficial or other bacterial species. This specificity, combined with the modular design and thermostability, addresses the need for alternative antimicrobials against C. perfringens infections that are refractory to resistance development.
Claims Coverage
The patent contains several independent claims focusing on polynucleotides, chimeric lysin polypeptides, host cells, compositions, and methods related to chimeric recombinant lysins comprising thermophile endolysin catalytic domains and C. perfringens cell wall binding domains. Main inventive features are extracted below.
Polynucleotide encoding chimeric recombinant lysin with thermophile catalytic and C. perfringens cell wall binding domains
A polynucleotide encoding a chimeric recombinant lysin comprising: - at least one thermophile endolysin catalytic domain selected from PlyGspY412 (SEQ ID NO: 4), PlyGspY4 (SEQ ID NO: 16), or PlyGve2 (SEQ ID NO: 23), or variants thereof having at least 90% sequence identity; - at least one cell wall binding domain from C. perfringens endolysins PlyCP10 (SEQ ID NO: 5), PlyCP18 (SEQ ID NO: 6), PlyCP33 (SEQ ID NO: 7), PlyCP41 (SEQ ID NO: 8), or PlyCP26 (SEQ ID NO: 9), or variants thereof having at least 90% sequence identity; - optionally a linker nucleic acid molecule between the catalytic and cell wall binding domains; - optionally a nucleic acid molecule encoding a polyhistidine tag; - wherein the chimeric recombinant lysin is not a fusion of PlyGve2 and PlyCP26 with amino acid sequence of SEQ ID NO: 28.
Chimeric recombinant lysin polypeptide comprising thermophile catalytic and C. perfringens cell wall binding domains
A chimeric recombinant lysin polypeptide comprising: - at least one thermophile endolysin catalytic domain from PlyGspY412 (SEQ ID NO: 4), PlyGspY4 (SEQ ID NO: 16), or PlyGve2 (SEQ ID NO: 23), or variants thereof; - at least one cell wall binding domain from C. perfringens endolysins PlyCP10, PlyCP18, PlyCP33, PlyCP41, or PlyCP26, or variants thereof; - optionally a linker between catalytic and cell wall binding domains; - optionally a polyhistidine tag; - wherein the polypeptide does not have the amino acid sequence of SEQ ID NO: 28 when the catalytic domain is from PlyGve2 and binding domain from PlyCP26.
Host cells comprising polynucleotides encoding the chimeric recombinant lysins
Host cells genetically engineered to comprise and express polynucleotides encoding the described chimeric recombinant lysins, including bacterial, fungal, plant, or mammalian cells.
Vectors and nucleic acid constructs comprising the polynucleotides
Nucleic acid constructs where the polynucleotide encoding the chimeric recombinant lysin is operably linked to a promoter, and vectors comprising such constructs for expression in host cells.
Compositions comprising chimeric recombinant lysins
Pharmaceutical or animal feed compositions comprising the chimeric recombinant lysin polypeptides and optionally pharmaceutically acceptable carriers, suitable for oral administration.
Methods of treatment and preparation
Methods of treating infections and diseases caused by C. perfringens in animals including chickens, pigs, and newborn calves, by administering an effective dose of the chimeric recombinant lysin composition or the host cell expressing the lysin. Methods of preparing the chimeric recombinant lysin polypeptides by cultivating host cells under suitable conditions and optionally recovering the polypeptides.
The claims cover polynucleotides encoding chimeric recombinant lysins comprising thermophile endolysin catalytic domains fused to C. perfringens cell wall binding domains, the corresponding polypeptides, genetically engineered host cells, expression vectors, compositions including pharmaceutical and animal feed formulations, and methods for treating C. perfringens infections by administering these lysins or lysin-expressing cells.
Stated Advantages
The chimeric recombinant lysins have improved thermostability compared to mesophile-derived lysins, enabling better tolerance to heat treatments used in animal feed production.
They exhibit highly specific activity against several strains of Clostridium perfringens without affecting other Gram-positive bacteria, reducing off-target effects.
The modular design using thermophile catalytic domains fused with C. perfringens cell wall binding domains makes these lysins refractory to resistance development, addressing antibiotic resistance concerns.
Documented Applications
Treatment and prevention of infections and diseases caused by Clostridium perfringens, including necrotic enteritis, gas gangrene, and other C. perfringens-related diseases in animals such as chickens, pigs, and newborn calves.
Incorporation into oral compositions, including animal feed formulations, for controlling C. perfringens infections in food-producing animals.
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