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Engineered Recombinant Escherichia coli Probiotic Strains Integrated with F4 and F18 Fimbriae Cluster Genes in the Chromosome and Their Assessment of Immunogenic Efficacy in Vivo.

Abstract

F4 (K88) and F18 fimbriaed enterotoxigenic Escherichia coli (ETEC) are the predominant causes of porcine postweaning diarrhea (PWD), and vaccines are considered the most effective preventive approach against PWD. Since heterologous DNA integrated into bacterial chromosomes could be effectively expressed with stable inheritance, we chose probiotic EcNc (E. coli Nissle 1917 prototype cured of cryptic plasmids) as a delivery vector to express the heterologous F4 or both F4 and F18 fimbriae and sequentially assessed their immune efficacy of anti-F4 and F18 fimbriae in both murine and piglet models. Employing the CRISPR-cas9 technology, yjcS, pcadA, lacZ, yieN/trkD, maeB, and nth/tppB sites in the chromosome of an EcNc strain were targeted as integration sites to integrate F4 or F18 fimbriae cluster genes under the Ptet promotor to construct two recombinant integration probiotic strains (RIPSs), i.e., nth integration strain (EcNcΔnth/tppB::PtetF4) and multiple integration strain (EcNc::PtetF18x4::PtetF4x2). Expression of F4, both F4 and F18 fimbriae on the surfaces of two RIPSs, was verified with combined methods of agglutination assay, Western blot, and immunofluorescence microscopy. The recombinant strains have improved adherence to porcine intestinal epithelial cell lines. Mice and piglets immunized with the nth integration strain and multiple integration strain through gavage developed anti-F4 and both anti-F4 and anti-F18 IgG immune responses. Moreover, the serum antibodies from the immunized mice and piglets significantly inhibited the adherence of F4+ or both F4+ and F18+ ETEC wild-type strains to porcine intestinal cell lines in vitro, indicating the potential of RIPSs as promising probiotic strains plus vaccine candidates against F4+/F18+ ETEC infection.

Authors+Show Affiliations

College of Veterinary Medicine , Yangzhou University , Yangzhou 225009 , China. College of Life Science , Zhaoqing University , Zhaoqing 526061 , China. Jiangsu Co-Innovation Center for Important Animal Infectious Diseases and Zoonoses , Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China , Yangzhou 225009 , China. Diagnostic Medicine/Pathobiology , Kansas State University College of Veterinary Medicine , Manhattan , Kansas 66506 , United States.College of Veterinary Medicine , Yangzhou University , Yangzhou 225009 , China. Jiangsu Co-Innovation Center for Important Animal Infectious Diseases and Zoonoses , Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China , Yangzhou 225009 , China.College of Veterinary Medicine , Yangzhou University , Yangzhou 225009 , China. Jiangsu Co-Innovation Center for Important Animal Infectious Diseases and Zoonoses , Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China , Yangzhou 225009 , China.College of Veterinary Medicine , Yangzhou University , Yangzhou 225009 , China. Jiangsu Co-Innovation Center for Important Animal Infectious Diseases and Zoonoses , Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China , Yangzhou 225009 , China. College of Animal Science , Guizhou University , Guiyang 550025 , China.College of Life Science , Zhaoqing University , Zhaoqing 526061 , China.College of Veterinary Medicine , Yangzhou University , Yangzhou 225009 , China. Jiangsu Co-Innovation Center for Important Animal Infectious Diseases and Zoonoses , Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China , Yangzhou 225009 , China.College of Life Science , Zhaoqing University , Zhaoqing 526061 , China.College of Veterinary Medicine , Yangzhou University , Yangzhou 225009 , China. Jiangsu Co-Innovation Center for Important Animal Infectious Diseases and Zoonoses , Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China , Yangzhou 225009 , China.College of Veterinary Medicine , Yangzhou University , Yangzhou 225009 , China. Jiangsu Co-Innovation Center for Important Animal Infectious Diseases and Zoonoses , Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China , Yangzhou 225009 , China.College of Veterinary Medicine , Yangzhou University , Yangzhou 225009 , China. Jiangsu Co-Innovation Center for Important Animal Infectious Diseases and Zoonoses , Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China , Yangzhou 225009 , China.College of Veterinary Medicine , Yangzhou University , Yangzhou 225009 , China. Jiangsu Co-Innovation Center for Important Animal Infectious Diseases and Zoonoses , Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China , Yangzhou 225009 , China.Diagnostic Medicine/Pathobiology , Kansas State University College of Veterinary Medicine , Manhattan , Kansas 66506 , United States.Diagnostic Medicine/Pathobiology , Kansas State University College of Veterinary Medicine , Manhattan , Kansas 66506 , United States.Diagnostic Medicine/Pathobiology , Kansas State University College of Veterinary Medicine , Manhattan , Kansas 66506 , United States. Department of Pathobiology , University of Illinois at Urbana-Champaign , Urbana , Illinois 61802 , United States.Diagnostic Medicine/Pathobiology , Kansas State University College of Veterinary Medicine , Manhattan , Kansas 66506 , United States.College of Veterinary Medicine , Yangzhou University , Yangzhou 225009 , China. Jiangsu Co-Innovation Center for Important Animal Infectious Diseases and Zoonoses , Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China , Yangzhou 225009 , China. Diagnostic Medicine/Pathobiology , Kansas State University College of Veterinary Medicine , Manhattan , Kansas 66506 , United States.College of Life Science , Zhaoqing University , Zhaoqing 526061 , China.Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology , Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences , Shanghai 200032 , China.Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology , Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences , Shanghai 200032 , China.Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology , Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences , Shanghai 200032 , China.Biology and Microbiology Department , South Dakota State University , Brookings , South Dakota 57007 , United States.College of Life Science , Zhaoqing University , Zhaoqing 526061 , China.Diagnostic Medicine/Pathobiology , Kansas State University College of Veterinary Medicine , Manhattan , Kansas 66506 , United States. Department of Pathobiology , University of Illinois at Urbana-Champaign , Urbana , Illinois 61802 , United States.College of Veterinary Medicine , Yangzhou University , Yangzhou 225009 , China. Jiangsu Co-Innovation Center for Important Animal Infectious Diseases and Zoonoses , Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China , Yangzhou 225009 , China.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

31944664

Citation

Ou, Bingming, et al. "Engineered Recombinant Escherichia Coli Probiotic Strains Integrated With F4 and F18 Fimbriae Cluster Genes in the Chromosome and Their Assessment of Immunogenic Efficacy in Vivo." ACS Synthetic Biology, 2020.
Ou B, Jiang B, Jin D, et al. Engineered Recombinant Escherichia coli Probiotic Strains Integrated with F4 and F18 Fimbriae Cluster Genes in the Chromosome and Their Assessment of Immunogenic Efficacy in Vivo. ACS Synth Biol. 2020.
Ou, B., Jiang, B., Jin, D., Yang, Y., Zhang, M., Zhang, D., ... Zhu, G. (2020). Engineered Recombinant Escherichia coli Probiotic Strains Integrated with F4 and F18 Fimbriae Cluster Genes in the Chromosome and Their Assessment of Immunogenic Efficacy in Vivo. ACS Synthetic Biology, doi:10.1021/acssynbio.9b00430.
Ou B, et al. Engineered Recombinant Escherichia Coli Probiotic Strains Integrated With F4 and F18 Fimbriae Cluster Genes in the Chromosome and Their Assessment of Immunogenic Efficacy in Vivo. ACS Synth Biol. 2020 Jan 29; PubMed PMID: 31944664.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Engineered Recombinant Escherichia coli Probiotic Strains Integrated with F4 and F18 Fimbriae Cluster Genes in the Chromosome and Their Assessment of Immunogenic Efficacy in Vivo. AU - Ou,Bingming, AU - Jiang,Boyu, AU - Jin,Duo, AU - Yang,Ying, AU - Zhang,Minyu, AU - Zhang,Dong, AU - Zhao,Haizhou, AU - Xu,Mengxian, AU - Song,Haoliang, AU - Wu,Wenwen, AU - Chen,Mingliang, AU - Lu,Ti, AU - Huang,Jiachen, AU - Seo,Hyesuk, AU - Garcia,Carolina, AU - Zheng,Wanglong, AU - Guo,Weiyi, AU - Lu,Yinhua, AU - Jiang,Yu, AU - Yang,Sheng, AU - Kaushik,Radhey S, AU - Li,Xinchang, AU - Zhang,Weiping, AU - Zhu,Guoqiang, Y1 - 2020/01/29/ PY - 2020/1/17/pubmed PY - 2020/1/17/medline PY - 2020/1/17/entrez KW - CRISPR/cas9 KW - E. coli Nissle 1917 KW - Engineered recombinant probiotic strains KW - F18 fimbriae KW - F4(K88) fimbriae KW - Genomic integration JF - ACS synthetic biology JO - ACS Synth Biol N2 - F4 (K88) and F18 fimbriaed enterotoxigenic Escherichia coli (ETEC) are the predominant causes of porcine postweaning diarrhea (PWD), and vaccines are considered the most effective preventive approach against PWD. Since heterologous DNA integrated into bacterial chromosomes could be effectively expressed with stable inheritance, we chose probiotic EcNc (E. coli Nissle 1917 prototype cured of cryptic plasmids) as a delivery vector to express the heterologous F4 or both F4 and F18 fimbriae and sequentially assessed their immune efficacy of anti-F4 and F18 fimbriae in both murine and piglet models. Employing the CRISPR-cas9 technology, yjcS, pcadA, lacZ, yieN/trkD, maeB, and nth/tppB sites in the chromosome of an EcNc strain were targeted as integration sites to integrate F4 or F18 fimbriae cluster genes under the Ptet promotor to construct two recombinant integration probiotic strains (RIPSs), i.e., nth integration strain (EcNcΔnth/tppB::PtetF4) and multiple integration strain (EcNc::PtetF18x4::PtetF4x2). Expression of F4, both F4 and F18 fimbriae on the surfaces of two RIPSs, was verified with combined methods of agglutination assay, Western blot, and immunofluorescence microscopy. The recombinant strains have improved adherence to porcine intestinal epithelial cell lines. Mice and piglets immunized with the nth integration strain and multiple integration strain through gavage developed anti-F4 and both anti-F4 and anti-F18 IgG immune responses. Moreover, the serum antibodies from the immunized mice and piglets significantly inhibited the adherence of F4+ or both F4+ and F18+ ETEC wild-type strains to porcine intestinal cell lines in vitro, indicating the potential of RIPSs as promising probiotic strains plus vaccine candidates against F4+/F18+ ETEC infection. SN - 2161-5063 UR - https://www.unboundmedicine.com/medline/citation/31944664/Engineered_recombinant_Escherichia_coli_probiotic_strains_integrated_with_F4_and_F18_fimbriae_cluster_genes_in_the_chromosome_and_their_assessment_of_immunogenic_efficacy_in_vivo L2 - https://dx.doi.org/10.1021/acssynbio.9b00430 DB - PRIME DP - Unbound Medicine ER -