| As one important member of the gram-negative probiotics,Escherichia coli Nissle 1917(EcN)has been widely studied and applied in many fields.Despite its advantages,the probiotics still possesses problems like the unstable effects and difference among individuals.To solve these problems,one method is to explore functional components in the probiotics and their mechanisms of action.According to previous experiments and existing studies,EcN could modulate some physiological reactions of the host via secreting some bioactive components.Among which,outer membrane protein takes an important part.As an essential component of EcN,it is worth of further studying on the role that outer membrane protein plays in the beneficial functions of EcN.Furthermore,it has been reported that Escherichia coli(E.coli)outer membrane protein A(OmpA)and outer membrane protein T(OmpT)could modulate interactions between bacteria and the host,especially on epithelial barrier.Since one of EcN's beneficial funtions is to enhance the gut barrier,we selected OmpAEcN and OmpTEcN as targets in the initiating stage of this study to figure out the functions of outer membrane protein in enhancing host gut barrier.Firstly,with the ?-Red recombination technology,we constructed EcN?ompA deletion stain and EcN?ompT deletion stain.At the same time,we also construct complementary strains of EcN?ompA/pBR322ompA and EcN?ompT/pBR322ompT.Bacterial growth tests demonstrated that deletion of ompAEcN and ompTEcN gene did not influence the normal growth of EcN.After that,with human colorectal adenocarcinoma cells(Caco-2)as an in vitro model of gut barrier,we compared changes of tight junction protein Claudin-1 and ZO-1 in terms of their mRNA and protein expression levels among the Caco-2 cells co-cultured with EcNWT,EcN?ompA,EcN?ompT,EcN?ompA/pBR322ompA,EcN?ompT/pBR322ompT and E.coliDH5? strain respectively,as well as the control group.The results showed that Caco-2 cell treated with had significantly lower mRNA(decrease by 61%,P<0.01)and protein(decrease by 52%,P<0.01)level of Claudin-1 than that of the EcNWT treated group,which was similiar to that in the control group.Moreover,the deletion of ompAEcN gene had no impact on the expression of ZO-1.In comparison,deletion of ompTEcN had no impacts on expressing level of Claudin-1 and ZO-1.These results demonstrated that only OmpAEcN protein could influence gut barrier via changing the expression of Claudin-1.Further studying the how OmpAEcN protein influences the expression of Claudin-1,we detected changes of the OmpAEcN receptor gp96 in terms of its mRNA and protein level,as well as the variations in the mRNA level of tight junction associated cytokine IL-22,IL-6 and TNF?.And results showed that compared with EcNWT group,Caco-2 cell treated with EcN?ompA strain had a significantly lower mRNA(decreased by 63%,P<0.01)level and protein expressing level(decreased by 48%,P<0.01)of gp96.Similarly,the transcriptional level of IL-22 in the EcN?ompA treated group was significantly lower than that in the EcNWT treated group(decreased by 49%,P<0.01).Besides,the deletion of ompAEcN gene exerted no impact on the mRNA level of IL-6 and TNF?.These results demonstrated that OmpAEcN could activate the gut gp96 protein,which could promote the expression of Claudin-1 and IL-22,and finally enhance the gut barrier.To further confirm the function of OmpAEcN,we attempted to transfer the plasmid pBR322,which contains the whole ompAEcN gene,into a commensal bacterial strain named E.coliG58-1,constructing the recombinant strain E.coliG58-1/pBR322ompA.Then,through qRT-PCR and Western blot experiments,we detected the variations in terms of the mRNA and protein expressing levels of Claudin-1 and gp96,as well as mRNA levels of IL-22,IL-6 and TNFa among different treatment groups(including Control group,EcNWT group,E.coliG58-1 group and E.coliG58-1/pBR322ompA group).Results showed that compared with E.coliG58-1 treated group,E.coliG58-1/pBR322ompA recombinant strain obtained the ability to significantly increase the expression level of Claudin-1,gp96 and IL-22,which were similiar to that of EcNWT treated group.And there was no difference in mRNA level of IL-6 and TNFa between treatment groups of E.coliG58-1 and E.coliG58-1/pBR322ompA.These results further confirmed the function of OmpAEcN in modulating Claudin-1 expression of Caco-2 cell.Finally,we confirmed the function of OmpAEcN in the complex in vivo mice model.By intragastric administration,we managed to inoculate different groups of mice,which are pretreated with antibiotics,with EcNWT,E.coliDH5?,EcN?ompA,EcN?ompA/pBR322ompA,E.coliG58-1 and E.coliG58-1/pBR322ompA recombinant strains,respectively.Then we compared their difference in the mRNA and protein level of Claudin-1 and gp96,as well as difference in mRNA levels of IL-22,IL-6 and TNF?.Results showed that compared with EcNWT,mice treated with EcN?ompA strain had significant lower mRNA(decreased by 53%,P<0.01)and protein(decreased by 57%,P<0.01)levels of Claudin-1;as well as a decreased mRNA level(decreased by 61%,P<0.01)and protein level(decreased by52%,P<0.01)of gp96;meanwhile mice intragastrically administrated with E.coliG58-1/pBR322ompA strain showed higher mRNA(increased by 88%and 81%respectively,P<0.01)and protein(increased by 106%and102%respectively,P<0.01)levels of Claudin-1 and gp96 than that of the E.coliG58-1 treatment group,which are similar to that of EcNWT treated group.Moreover,after treatment with EcN?ompA strain,IL-22 in the mice had a lower mRNA level than that of EcNWT treated group,which decreased by 50%(P<0.01),while IL-22 in E.coliG58-1/pBR322ompA strain treated mice has an increased mRNA level(increased by 82%,P<0.01)compared that of E.coliG58-1 treated group.Both IL-6 and TNFa expression are free from being affected by OmpAEcN.These results are in accordance with results of in vitro experiments.In conclusion,results from both in vitro and in vivo demonstrated that OmpAEcN could promote the expression of gut Claudin-1 and IL-22 through activating gp96 receptor in the gut,which eventually improved the gut barrier. |
PDF Full Text Request