The Construction Of Pic Gene Deletion Mutant Of Escherichia Coli Probiotics Nissle 1917 And Relevant Function Analysis

In 1917,German doctor Alfred Nissle isolated a non-pathogenic Escherichia coli strain from the feces of a soldier with enteritis infected with Shigella.and the isolate was named Nissle 1917(EcN).EcN can survive and colonize in intestinal tract well.Also,EcN does not carry any virulence factors and has obvious probiotic characteristics.So it has become one of the most studied probiotics except some lactobacillus as probiotics.Research showed that EcN has certain efficacy on human intestinal diseases.The commercial drug Mutaflor with the main component EcN has been applied in the treatment of inflammatory bowel disease(IBD)in many European and American states.In recent years,Nissle 1917,as a vector delivering exogenous proteins to the target,has played a more and more important role in the treatment of human intestinal diseases.In the genome of EcN,We found the pic gene coding the serine protease of Enterobacteriaceae(SPATE).Based on the gene sequence found in NCBI,we designed ?-Red homologous recombination system-based the primers for the construct of pic gene deletion mutant.The 5'end of primer is homologous to the upstream and downstream of pic gene and the 3'end of primer is homologous to cat gene with chloramphenicol resistance in plasmid pKD3.We knock out the pic gene by ?-Red homologous recombination system technology.First,we used plasmid pKD3 as template DNA to PCR amplify cat gene carrying the homologous arm of pic gene.Then we transformed the purified PCR products into EcN carrying plasmid pKD46 by electroporation.The pic gene was replaced by cat gene with the involvement of recombination enzymes.We selected successful recombinant bacteria with pic gene deletion by combination of chloramphenicol LB plate screen and PCR detection.Next,we transformed plasmid pCP20 containing FLP recombinase into the first run of recombinant bacteria.The expression of FLP recombinase can activate in the second recombination and knock out the cat gene.The successful construction of the pic deletion for EcN?pic mutant strains were confirmed by PCR and DNA sequencing.Finally,we constructed complemental strains EcN?pic/c/ppic on the base of EcNA?pic with the involvement of plasmid pBR322 expressing pic.The result of bacterial growth curve determination indicated that there was no significant difference among wild type EcN,EcN?pic and EcN?pic/ppic;Bio film formation assay indicated that biofilm formation ability was significantly decreased in EcN?pic;In vitro,The adhesion assay to IPEC-J2 indicates that adhesion ability of EcN?pic was decreased by 41%compared to wild type EcN(p<0.05);In the adhesion competition assay between EcN and Adherent-invasive Escherichia coli LF-82,the adhesion ability of LF82 to Caco-2 cells in co-incubation with EcN?pic was 52%higher than that in co-incubation with wild type EcN(p<0.05);The invasion competition assay between EcN and LF-82 indicates that the invasion ability of LF82 to Caco-2 cells has no significant difference in group EcN and group EcN?pic.It is probably that the Pic protein secreted by EcN just promote EcN to colonize the intestinal cells.But it does not have inhibit activity on LF82;The result of Real-time quatitative PCR showed that transcription level of pic in 30? and 42? was significantly decreased by 40%and 54%compared to 37?(p<0.05);The CD 14 blocking assay on macrophage MV411 indicated that the transcription level of inflammatory cytokine TNF-??IL-1??IL-6?IL-8?IL-10 in CD14 blocking group was significantly decreased by 84.4%(p<0.01),84.7%(p<0.01),63.0%(p<0.05),71.6%(p<0.01)and 45.5%(p<0.05)compared to control group after been incubated with Pic protein.It is probably that the the immune stimulation of LPS to macrophages MV411 was enhanced after Pic cleaved CD 14.Then it promoted the release of inflammatory mediators;When Caco-2 cells were incubated with EcN?pic,the transcription level of TNF-?,IL-1?,IL-6,IL-8 and IL-10 was decreased by 69.5%(p<0.01),79.1%(p<0.01),77%(p<0.01),55.0%(p<0.05)and 43.6%(p<0.05)compared to incubate with wild type EcN.Maybe it was associated with the expression of CD 14 on the surface of cells;Mouse enteritis model was induced by Citrobacter rodentium Then,different types of EcN was given to mouse by gavage.However,the result of transcription level of inflammatory cytokine in mice intestines indicated that there is no significant difference between different groups.It is probably that the amount of EcN and concentration of Pic was at a lower level compared to vitro test.This study focused on the role of pic gene in colonization and immune regulation.In vitro,Pic protein secreted by EcN promoted colonization of bacteria to intestinal cells.It can also increase the immune response of cells to some extent.But deep function in vivo still needs to further studied.