| Background and aims:Adherent-invasive Escherichia coli(AIEC)is a gram-negative bacterium of the genus Enterobacteriaceae and is believed to be associated with human inflammatory bowel disease.AIEC can survive and reproduce in macrophages and stimulate macrophages to secrete TNF-α,IL-6 and other pro-inflammatory factors.The sly A gene in Salmonella typhi regulates the bacterial survival in macrophages.The aer gene sequence of LF82 strain had 82.27%homology with that of Salmonella typhimurium,S.Typhimurium LT2 strain sly A.In this study,we investigated the effect of aer gene on AIEC survival in macrophages and its mechanism.Aer gene regulates the effect of AIEC on host tissue proinflammatory response.Methods:1.Discovery of aer gene in AIEC and construction of deletion strain1)Blast analysis and comparison were performed on the genomic sequences of sly A gene of LT2 strain of S.Typhimurium and LF82 strain of AIEC strain.X fragment of sly A gene with high homology sequence was amplified by PCR and sent to Sipukin for sequencing,and X fragment was analyzed by software.2)The upstream fragment F1 and the downstream fragment F2 of aer gene were amplified by PCR,and then the fragment F1F2 was ligated with the suicide plasmid p DS132.The recombinant plasmid pDS132-F1F2 was screened on the antibiotic plate,and the recombinant plasmid was transformed into SM10λ pir by calcium chloride method,and then conjugated with LF82.Aer deletion strains were screened by LB plate containing 15% sucrose,and the colonies that did not grow on the double antibody plate but only grew on the monoclonal antibody plate were selected and identified by PCR,and sent to the company for sequencing.The aer gene fragment was amplified by PCR and inserted into the plasmid p ACYC184,which was transformed into △aer by calcium chloride method,and the recombinant plasmid clones of p ACYC 184-aer were screened on antibiotic plate.2.Aer gene regulates the effect of AIEC on the intracellular survival of macrophages and its mechanism1)The growth curves of Wild Type(WT),△aer,C△aer and DH5α strains of LF82 in LB medium were compared by turbidimetric method.The difference of intracellular survival in RAW264.7 macrophages was compared by colony plate counting and light microscopy.2)Flow cytometry was used to compare the average fluorescence intensity of reactive oxygen species produced by WT,△aer and C△aer infected macrophages.The diameters of inhibitory rings at different concentrations of WT,△aer and C△aer were compared.The survival rates of WT,△aer and C△aer in different concentrations of H2O2,acid or hypertonic LB medium were compared.q RT-PCR was used to compare the transcription levels of IL-6,TNF-α and INF-γ in RAW264.7 macrophages infected with WT and △aer for 12 h.CCK-8 cytotoxicity assay was used to compare the toxicity of WT,△aer and C△aer to macrophages.3)q RT-PCR was used to compare the transcriptional levels of fim H and ibe A in WT,△aer and C△aer.The promoter region of kat G gene pkat G and the promoter region of fim H gene pfim H were amplified and ligated with fluorescent plasmid pet28a-e GFP to form recombinant plasmid.The fluorescent plasmids were transfected into WT and △aer respectively by calcium chloride method.The fluorescence intensity of the recombinant plasmid in WT and△aer was compared under fluorescence microscope,and the transcription level of e GFP gene in WT and △aer was compared by q RT-PCR.3.Aer gene regulates the effect of AIEC on host tissue proinflammatory response1)LF82 strains WT,△aer and DH5α were injected into the abdominal cavity of mice respectively,and the viability of LF82 strains in liver,spleen and kidney of mice was compared by plate counting method.The liver,spleen and kidney tissues of mice were taken and photographed for pathological sections and HE staining.2)The mice were divided into 8 groups :PBS group,LF82 group,DH5α group,△aer group,DSS group,DH5α+DSS group,LF82(WT)+DSS group and △aer +DSS group.Ulcerative enteritis was induced by 2.0% DSS for 5 days in mice.Except PBS group,the bacteria were intragastric for 8 days.Mental state and body weight were observed,DAI score was performed,colon length was measured,pathological sections were prepared and HE staining was performed.q RT-PCR was used to detect the gene transcription of inflammatory factors IL-6,TNF-α and INF-γ in mouse colon tissue.Results:1.Discovery of aer gene in AIEC and construction of deletion strain1)The X sequence analyzed by software is composed of 435 bp open reading framework,with promoter region,and named as AIEC regulating gene(aer).2)The deleted strains were screened by PCR and sent to the company for sequencing.The sequencing results showed that the homology of this section of base was 100% with F1F2,and the bacteria obtained was named △aer.3)PCR was used to screen the transforters of p ACYC 184-aer recombinant plasmid,and the product was sent to the company for sequencing.The sequencing results showed that the homology with aer fragment was 100%,and the bacteria obtained was named C△aer.2.Aer gene regulates the effect of AIEC on macrophage intracellular survival and its mechanism1)There was no difference in the growth curves of LF82 and DH5α in LB medium;LF82could survive and multiply in macrophages,but DH5α could not.The growth curves of WT,△aer and C△aer of LF82 strains showed no difference in LB medium,and the number of LF82 strains survived in macrophages was higher than △aer.2)Compared with LF82 strain WT and C△aer,flow cytometry showed that the fluorescence intensity of reactive oxygen species produced by △aer infected cells was significantly increased.△aer bacteriostatic ring diameter increased significantly.The survival rate decreased significantly in different concentrations of H2O2,acid or hypertonic LB medium.The transcription levels of inflammatory cytokines IL-6,TNF-α and INF-γ were significantly decreased after △aer was infected with macrophages for 12 h.The cytotoxicity of △aer was significantly reduced.3)Compared with WT and C△aer,transcription levels of fim H and ibe A genes in △aer were significantly reduced.The fluorescence intensity of recombinant plasmid in WT was higher than that of △aer,and the gene transcription level of e GFP in WT was higher than that of △aer.3.Aer gene regulates the effect of AIEC on host tissue proinflammatory response1)The viability of DH5α in liver,spleen and kidney was significantly lower than that of LF82 after intraperitoneal injection of mouse bacterial solution.The size of liver,spleen and kidney in LF82 group was larger than that in DH5α group.LF82 had toxicity to liver,spleen and kidney of mice,but DH5α had no toxicity to these organs.The survival ability of △aer in liver,spleen and kidney was significantly lower than WT.WT has certain toxic effects on liver,spleen and kidney of mice,while △aer has no toxic effects on the above-mentioned organs.2)Compared with PBS group,there were no significant differences in mental status,body weight,DAI score,colon length and inflammatory factor levels in colon tissue between LF82 group and DH5α group;Compared with DSS group,LF82+DSS group reduced these indexes,while DH5α+DSS group had no significant difference in these indexes.There were no significant differences in mental status,body weight,DAI score,colon length and inflammatory factors in colon tissue between WT and △aer groups.Compared with DSS group,these indexes in WT+DSS group decreased,but there was no significant difference in△aer+DSS group..Conclusion:1.The aer gene deletion strain and the complementary strain of AIEC were constructed.2.aer gene affects the bactericidal mechanism of AIEC bacteria against the oxidation and acidity of macrophages,thus facilitating the survival of bacteria in macrophages.3.aer gene positively regulates the expression of AIEC target genes fim H,ibe A,htr A,gad B,sod C,kat G,dsb A and vat A at the transcription level.4.aer gene affects the cell toxicity of AIEC bacteria and induces the release of TNF-α from macrophages.5.aer gene affects the survival ability of AIEC bacteria in mice and affects the effect of AIEC bacteria on DSS mice. |
PDF Full Text Request