Function Exploration Of Flagella Surface Display In The Plasmid-Free Clone Strain Of Escherichia Coli Nissle 1917

Escherichia coli Nissle 1917 (EcN) was firstly isolated from the feces of a soldier by german doctor Alfred Nissle, the soldier did not develop infectious diarrhea when staying in the region which was heavily contaminated with enteropathogens at that time. Since then EcN as the active component of the human medicine Mutaflor(?) plays an important role in treatment of various intestinal diseases in Germany and some other European countries. In recent years, more and more study is involved in EcN as a diagnosis and treatment carrier for foreign gene expression. Plasmid pMUT2 in EcN, Type I secretion system and Type V secretion system were used to display heterologous antigen on the EcN surface. Flagellin display is based on fusion of foreign peptides into the variable domain of flagellin FliC which subsequently assembled into flagellum filaments to display heterologous antigens. Flagellin display technology was reported in some Salmonella while not applied in EcN. We construct plasmid-free clone strain of EcN (EcN cured of its two cryptic plasmids pMUT1 and pMUT2; EcNc) and then explore its flagellin display.EcN own two cryptic plasmids called pMUT1 and pMUT2 which are stably maintained in subculture. Plasmid pMUT1 carries a replication system of ColEl-type and plasmid pMUT2 contains a ColE2-like replication system, whereas there are few informations about them. Some reaserch reported that pathogenicity of Escherichia coli were partly controlled by the plasmid, and the resistance gene of some Escherichia coli strains are relevant to the cryptic plasmid. In order to reduce the influence on recombinant plasmid transformation and genome DNA operation, as well as the human and animal, suicide vector and cryptic plasmids were respectively digested with the restriction enzyme Sph I, subsequently ligated to construct recombinant plasmids pMUTl-pRE112 and pMUT2-pRE112. Through the competitive inhibition the recombinant suicide plasmid and cryptic plasmid, curing of two intrinsic cryptic plasmids in EcN was achieved, by passaging on LB medium containing 10% sucrose, the recombinant suicide plasmids could be eliminated by themseleves. The plasmid-free clone strain of EcN (Escherichia coli Nissle 1917 cured of its two cryptic plasmids pMUT1 and pMUT2, EcNc) was acquired in our study. Both EcN and EcNc did not exhibit different changes on growth, biochemical, serological, morphological and the ability of adherence in vitro. The EcNc will be conducive to plasmid transformation and related biological operation.Baced on the fliC gene sequence encoding flagellin of EcN, we deleted fliC gene of EcNc using λ phage Red homologous recombination. First, the PCR products containing homology arms of fliC gene and chloramphenicol resistance gene(cat) expression cassette was purifiedand transformed into EcNc strainsalready carrying pKD46 plasmid, under the action of Red homologous recombination enzymes Gam, Exo, Beta, through the homologous recombination between PCR fragment flanking by two homologous sequence wings and the target gene sequence on the chromosome, we obtained theprimary recombinant strain EcNc △fliC::cat by screening on the chloramphenicol resistance plate. Then pCP20 plasmid was transformed into EcNc AfliC::cat strain by electroporation to eliminate chloramphenicol resistance gene between FRT sites on the PCR fragment through expression of Flp recombinase at 42℃ After comfirned by PCR and sequencing, the secondary recombinant fliC deletion strain, EcNc △fliC, was successfully constructed. The complemented strain EcNc △fliC/pBR322-fliC was constructed by transform recombinant plasmid pBR322-fliC into mutant strain EcNc △fliC At the same time, usingconstructed recombinant plasmid pUC18-fliC as a DNA template, the fragments fliC1,fliC2 lacking 859th-888thaa,829th-858thaa of flagellin were constructed by inverse PCR, and ligated to the suicide vector pRE112 to construct the recombinant suicide vector pRE112-fliC1, pRE112-fliC2 after double digestion. The pRE112-fliC1, pRE112-fliC2 were introduced into EcNc strain sequently obtaining mutant strains EcNc fliCl, EcNc fliC2 with partial delection in hypervariable region of flagellin gene through primary recombination, secondary recombination and sequencing. The successful constructions of EcNc, EcNc△fliC, EcNc AfliC/pBR322-fliC, EcNc fliCl, EcNc fliC2 will contribute to further study the impact of sequence changes of flagellin hypervariable region on traits of EcNc.The growth performance, biochemical characteristics, movability, antigenicity of flagella, the formation of flagella, and the ability of adherence in vitro and the colonization in intestine in vivo of EcNc、EcNc △fliC, EcNc △fliC/pBR322-fliC, EcNc fliCl、EcNc fliC2 were detected. The results showed that the biochemical characteristics of each strain of bacteria was the same and growth performance showed little difference; EcNc AfliC showed no movability on the semisolid medium, and the expression of flagellin was not detected using Western blot and the morphology of flagella was not observedunder the electron microscope, meanwhile, compared to EcNc, EcNc fliCl, EcNc fliC2, the adherence ability of EcNc AfliC were declined by 64%(p=0.002),52%(p=0.018),50%(p=0.01) respectively. The complemented strain EcNc AfliC/pBR322-fliC have the same characteristics as EcNc and its ability of adherence to IPEC-J2 recovered to the same level as wild type strain EcNc; it could be detected for the expression of flagellin of EcNc fliC1、EcNc fliC2 using West blot and their filament could also be found under electron microscope, but they lost mobility on the semisolid medium; Compared to EcNc, the ability of adherence of EcNc fliC1、EcNc fliC2 were declined by 26%(p=0.124), 29%(p=0.586) respectively, but no significant difference; but the the adherence ability of EcNc fliCX and EcNc fliC2 were increased by 52%(p=0.018) and 50%(p=0.01) respectively, compared to EcNc AfliC. In the in vivo intestine colonizationexperiment, we respectively collected samples on day 1, day 3 and day 7 after inoculating, the abilities of intestine colonization of each group shown no remarkable difference; but it could be found that the number bacteria colonizing on cecum was more than that on jejunum and colon, especially on day 1, and the colonization number of EcNc/pBR322、EcNc AfliOpBR322-fliC、EcNc fliCl/pBR322、EcNc fliC2/pBR322 on on cecum were significant higher than that on jejunum.. some adherence mediated factor F1A、F1C and curli on EcNc jointly mediate and promote bacteria colonize in vivo leading to EcNc △fliC/pBR322 couldcolonize efficiently in the mice’s intestine. The deletion of 10 amino acid on the sites 287th-296th or 277th-286th on the hypervariable region of flagellin does not affect the expression of the flagellinand formation of the filament while this doesn’t influence the ability of colonization in the mice’s intestine, which means that it is tolerant for the deletion of partial amino acids on hypervariable region of EcNc.Using recombinant plasmid pUC18-fliC as a template, we designed two pair of inverse PCR primers containing hexahistidine peptide (6His) gene. The inverse PCR product was cyclized using Dpn Ⅰ, ExnaseTMⅡ, obtaining two recombinant plasmid pUC18-fliC01and pUC18-fliC02 containing chimeric flagellar gene fragments (fliC01、 fliC02). The hexahistidine peptide (6His) gene was inserted into the site of 774th-775th and 792th-811th in fliC hypervariable region in these two chimeric flagellar gene fragments (fliC01、fliC02). These fliC01、fliC02 fragments were respectively inserted into suicide vector pRE112 to construct recombinant plasmid pRE112-fliC01、pRE112-fliC02, subsequently transformed into EcNc separately. Chimeric flagellar genes were integrated into EcNc genome through primary recombination and secondary recombination, generating the recombinant strains EcNc fliC01 and EcNcy fliC02 without resistance gene cassette. We analyzed two recombinant strains through the following experiments such as growth characteristic, formation of flagella and in vitro cell adhesion assay. The results showed that the growth of EcNc fliC1 and EcNcy fliC02 were not affected, the chimeric flagellin could be recognized by H1 polyclonal antibody expression or His-Tag monoclonal antibody and could assembled to form the flagellar filament. Compared to EcNc, no significant difference was found in the in vitro adherence ability and in vivo intestinal colonization ability. The flagellin of EcNc containing 6His gene in fliC hypervariable region could be displayed on the cell surface.Using recombinant plasmid pUC18-fliC as a template, we designed two pair of inverse PCR primers containing B cell and T cell epitopes gene from E2 protein of BVDV (Bovine viral diarrhea virus) NADL. The inverse PCR product was cyclized using Dpn I, ExnaseTM II, obtaining recombinant plasmid pUC18-fliCB and pUC 18-fliCT containing chimeric flagellar gene fragments (fliCB and fliCT). The B cell and T cell epitope genes were inserted into the site of 792th-811th in fliC hypervariable region in these two chimeric flagellar gene fragments (fliCB and fliCT).These two gene fragments were respectively inserted into suicide vector pRE112 to construct recombinant plasmid pRE112-fliCB and pRE112-fliCT, subsequently transformed into EcNc separately. Chimeric flagellar genes were integrated into EcNc genome through primary recombination and secondary recombination, generating the recombinant strains EcNc fliCB and EcNc fliCT without resistance gene cassette. Compared to EcNc, no significant difference was found in growth characteristic, formation of flagella and in vitro cell adhesion assay of two recombinant strains. In the semisolid medium, EcNc fliCB is nonmotile while EcNc fliCT is motile. SPF BALB/c mice were grouped and immunized by gavage of EcNc, EcNc fliCB and EcNc fliCT separately.Sample of blood and intestine were collected to test anti-B and anti-T cell epitope specific antibody IgA and IgG generated from the immunized mice. The result shows that EcNcfliCB could stimulate the production of anti-B cell epitope IgG (0.552±0.027). Meanwhile, EcNc fliCT could stimulate the production of anti-T cell epitope IgG (0.552±0.027). IgG of corresponding envelope protein was not detected in control group (immunized by EcN). Anti-B cell epitopes IgA antibody (0.367±0.030) could be detected in the intestinal of mouse immunized with EcNc fliCB, which showed significant difference (0.040±0.017;P=0.040) compared to the control group; Anti-T cell epitopes IgA antibody (0.371±0.034) could be detected in the intestinal of mouse immunized with EcNc fliCT, which showed significant difference (0.024±0.010; p=0.025) compared to the control group; EcNc fliCB, EcNc fliCT could induce humoral and mucosal immune responses that B cell and T cell epitopes antigen-specific.In a word, above results showed foreign antigens can be carried in the flagellin hypervariable region of EcNc and displayed on the cell surface, which laid the foundation for the study of potential application of high-quality probiotic EcN.