Construction Of Integration Recombinant Strains Based On Probiotic Escherichia Coli Nissle1917 Plasmid-free Clone Strain And Assessment Of Its Immunologic Function As Live Vaccine Candidates

E.coli Nissle 1917(EcN)was the kind of probiotic strain firstly isolated by the German doctor Alfred Nissle from the feces of an anti-diarrhea soldier in the epidemic area of diarrhoea in 1917.Due to"Strong Efficacy and Antagonistic Activity" from this strain present in the intestinal tract,the soldier is free of diarrhea in the endemic areas.As we know,E.coli Nissle1917 is a non-pathogenic probiotic strain and has not yet been found to have known harmful effects on the host and can confer health benefits to various host.In Germany and other European countries,EcN plays an important role in the treatment of various intestinal diseases as the active ingredient in the human medicament Mutaflor(?).In recent years,it has been often reported that the probiotic Nissle1917 strain is applied as a disease diagnosis and treatment carrier in clinic.The probiotic has been used for the development and research for vaccines,oncology treatments,health products and diagnostic preparations.The EcNc(EcN cured of its two cryptic plasmids pMUT1 and pMUT2;EcNc)is capable to carry more(larger)homologous or heterologous genes than the parent strain.This strain has been prepared previously in our laboratory.In order to achieve overexpression of homologous or heterologous proteins in microorganisms,plasmid is often used for overexpression.This method is widely used due to easy manipulation,but this method is usually genetically unstable.The metabolic burden of plasmid replication,overexpression of antibiotic resistance genes and other heterologous genes could cause depletion of host cell,loss of yield,and even loss of host cell initial function.In addition,in order to maintain the presence of plasmids in bacterial cells,antibiotics or other selective agents need to be used,thereby increasing the cost of the entire bioprocessing,and at the same time increasing the probability of spreading of antibiotic resistant genes.In recent years,chromosomal expression of homologous or heterologous genes has the advantages of strong genetic stability and low metabolic burden on host cells.Bacterial chromosome expression is favored in the field of synthetic biology and biomedicine.How to integrate the target gene into chromosomal target site and stably genetically express a heterologous gene under the normal physiological conditions of the host bacteria?It does need to be exploited.To integrate homologous or heterologous DNA gene molecules in the bacterial genome,the commonly used molecular biological methods includes the use of transposons,bacteriophage,endonucleases and recombinase systems,CRIM(conditional-replication,integration,and modular)plasmid-host system is one of the classic molecular techniques and it is a phage-derived molecular integration technique,which can integrate the target gene into a chromosome targeting site(phage attachment site attB)and can stably express foreign genes under the normal physiological conditions of the host bacteria.In our study,the CRIM system was used to construct the recombinant E.coli probiotic Nissle1917 stably carrying green fluorescent protein(GFP)as a screening tag.PCR primers for GFP amplification were designed based on the sequence of the plasmid pGFPmut3.1.The green fluorescent protein coding sequence was amplified from the plasmid pGFPmut3.1 and cloned into CRIM plasmid pCAH63,and then transformed it into the competent cell,an engineered strain containing pir-encoded protein(pir+),to construct the recombinant plasmid pCAH63GFP.The pCAH63GFP recombinant plasmid was then electrotransformed into competent cell Nisslel917/pINT-ts containing the helper plasmid pINT-ts,and the pCAH63GFP plasmid was integrated into the genome of E.coli Nissle 1917 by the X phage-specific integrase with the helper plasmid pINT-ts.After that,the pINT-ts helper plasmid was eliminated by incubation at 42?.Through Western blotting assay,the expression of GFP protein on the recombinant plasmid pCAH63GFP was confirmed.The GFP fluorescence signal from the Nissle1917::p63GFP integrant strain could be detected under the fluorescence microscope.This study establishes a recombinant model probiotic Nissle1917 that can stably carry a stable screened tagged GFP fluorescent protein,whichprovided the tool for the in vivo study of Nissle1917.Post weaning diarrhea(PWD)and edema disease(ED)are common clinical diseases in the swine industry.The dominating pathogens causing PWD and ED are F4 and F18 positive enterotoxigenic Escherichia coli(ETEC).The pathogen colonizes in the susceptible piglets mainly through fimbriae adhesion and colonization,F4 and F18 ETEC infections cause diarrhea and high mortality in piglets,weight loss,slow growth,and expensive medicine cost.Currently,the prevention and treatment for this disease in the clinic mainly depends on antimicrobial agents,but with the emergence of drug-resistant bacteria in the breeding industry,alternative prevention and control measures are urgently needed.To explore to integrate fimbriae adhesin cluster genes into the genome of non-pathogenic E.coli strain and E.coli probiotics,to enhancing the adhesion and colonization capacity of E.coli strain for developing a new oral live vaccine strain,we tried to manipulate the chromosome of E.coli SE5000 and E.coli probiotic EcNc by integrating F18ab and K88ac fimbrial antigen cluster genes into E.coli SE5000 and E.coli probiotic EcNc and expressed on the surfaces of the recombinants.To achieve this goal,we firstly integrate the K88ac fimbriae operon gene and the F18ab fimbriaeoperon gene into the chromosomes of E.coli SE5000 strain and EcNc strain respectively by site-specific recombination of the CRIM plasmid-host system.Whole cell lysates of recombinant SE5000::p63K88 were analyzed for K88 expression using SDS-PAGE and Western blot and a 27 kDa FaeG protein band was detected and confirmed.Using F18ab rabbit antiserum The agglutination test showed the F18ab rabbit antiserum agglutinated EcNc::p63F 18.In vitro cell adhesion assays showed that the integrated recombinant strains could improve their adhesion to pig intestinal epithelial cells,especially SE5000::p63K88 with more better binding capability.In addition,the anti-K88ac rabbit polyclonal serum was able to inhibit the adhesion of SE5000::p63K88 to IPEC-J2 cells.These data demonstrated that recombinant could express K88ac fimbriae in engeenieered E.coli SE5000 strain and E.coli probiotic EcNc,and thus they can be effective probiotic vaccines candidates.Conventional integration techniques or molecular biology methods as described above have certain advantages in bacterial chromosome,but they also have their own limitations,such as the difficulty in integration of long-length genes,low efficiency,and high off-target rates.In recent years,CRISPR/Cas(Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated(Cas)genes)systems are being developed into revolutionary biotechnologies in the field of life sciences and medical biology.The CRISPR/cas system is derived from the ancient bacteria's immune system and is now widely used in the genetic editing of various organisms.In this study,the two plasmids based CRISPR/cas9 system was used to integrate heterologous DNA fragment into the chromosome of the probiotic Nissle1917 plasmid-free clone strain(F4 fimbriae operon gene is about 8Kb-long,F18 fimbriae operon gene is about 5.6Kb-long).This experiment was based on the clear background of the bacterial genomic sequence of the Nissle1917 plasmid-free clone EcNc prepared previously in our lab.The non-essential genes X(yjcS,pcadA,lacZ,yieN/trkD,maeB,nth/tppB)on the genome were selected as an insertion site.Using the two plasmids based CRISPR/cas9 system,we constructed the CRISPR/cas9 recombinant plasmid pTargetT-X::PtetF18/F4 containing E.coli F18 or F4 fimbriae operon genes on both sides flanking with the homology arms of insertion site.The recombinant plasmid pTargetT-X::PtetF18/F4 was further transformed into a recombinant Nissle 1917/pCas host strain,and the cas9 protein was expressed on the pCas plasmid,the sgRNA transcripted from the recombinant plasmid pTargetT-X::PtetF18/F4 direct the cas9 protein to specifically cleave target sites.Homologous recombinase(Gam,Exo,Bet)led to homologous recombination using the introduced homologous fragment from pTargetT-X::PtetF18/F4 as a template,and completed the integration of the F18 or F4 fimbriae operon gene into the corresponding site of the genome of EcNc.Then IPTG was used to induce the initiation of the IPTG-inducible promoter(Ptrc)on the pCas plasmid,thereby activating the Ptrc promoter to transcript the sgRNA(sgRNApMB1)on the downstream of the Ptrc promoter which targets to the pMB1 replicon on the pTargetT-X::PtetF18/K88 plasmid for efficient cleavage.The replication initiation site(pMB1)of the pTargetT-X::PtetF18/K88 plasmid would be cleaved to eliminates the pTargetT-X::PtetF18/K88 plasmid of previous round,and eliminating the spectinomycin antibiotic resistance screening marker at the same time.After completing six rounds of integration recombination reactions,cultured the recombinant strain at 42? for eliminating the temperature-sensitive pCas plasmid.Finally,the integration mutant recombinant E.coli Nissle 1917 plasmid-free clone strain that did not contain any antibiotic resistance gene and expressed F18 and F4 fimbriae was obtained.We selected two integration mutant recombinant strains,EcNcAnth/tppB::PtetF4 andEcNc ?yjcS::PtetF 18?pcadA::PtetF 18?lacZ::PtetF 1 8?yieN/trkD::PtetF18?maeB::PtetF4Anth/tppB::PtetF 4,for the following experiments.This integration mutant recombinant strains were cultured for several generations(at least 30 passages)in a non-resistant culture medium.The stable expression of the F18 and F4 fimbriae in these two recombinant strains was confirmed through Western blotting,and the growth cycle curve of integration mutant recombinant strains and the parental strain are identical,and this integration method does not affect the biological characteristics of the host cell,such as growth rate.Through in vitro adhesion assay,compared to the EcNc,the recombinant strains can effectively improve their adhesion ability to the intestinal cell line(IPEC-J2 and IPEC-I),and animal experiments were performed through two rounds of oral immunization in 8-week-old BALB/c mice.14 days after second time immunization,the immunized mouse serum had IgG titers against the F18 fimbriae and the major subunit FaeG protein of F4 fimbriae,and its serum could significantly reduce the pathogenic F18+ and F4+ ETEC strains(eg,farm field isolate 8516 and virulent strain 3030-2)adhere to pigs intestinal epithelial cell line IPEC-J2.These integration recombinant strains are expected to be vaccine candidates for live vaccines against post weaning diarrhea and edema disease.The method we prepare the integration recombinant strain constructed in this paper is expected to become a method for constructing live vaccine candidates for post-weaning diarrhea and edema disease caused by F4+ and F18+ ETEC strains.It provides inspired ideas and strategies to prevent and control post-weaning diarrhea and edema disease.In addition,the biological molecular methods(CRIM plasmid-host system and CRISPR/cas9 system)we used in constructing the integration recombinant strains in thisstudy could be used as a platform for the genetic modification in the chromosomes of other bacterial strains.