Preliminary Study On The Molecular Mechanism Of OmpR Regulating The Virulence Of Salmonella Enterica Serover Typhi

Objective: OmpR is closely related to the virulence and viability in various stress microenvironments during S.Typhi infection.This study selected virulence-related genes under specific stress microenvironment as the research object,aiming to elucidate the transcriptional regulation mechanism of OmpR on it under the stress microenvironment,and to understand how OmpR can control virulence and survivability of S.Typhi under specific stress microenvironment through the expression of virulence-related genes,and to provide a theoretical basis for further understanding the pathogenic mechanism of S.Typhi.Methods: 1.Rescue of genes in the deletion mutant of S.Typhi: The recombinant vector containing the OmpR target fragment was constructed using the p BAD33 plasmid.The recombinant vector was electronically transferred into(35)OmpR,then the OmpR gene deletion supplementary strain could be obtained(denoted as C-(35)OmpR).At the same time,empty vector p BAD33 was transferred into WT and(35)OmpR as the corresponding control strains(denoted as WT-p BAD33 and(35)OmpR-p BAD33).2.Measurement of growth curve: OD600 values were taken as Y-axis,the time as X-axis,and the OD600 measured hourly.The growth of WT-p BAD33,(35)OmpR-p BAD33 and C-(35)OmpR at logarithmic metaphase(OD600=0.8)under acid stress,and the growth of WT,(35)OmpR and(35)hfq under low osmotic conditions were observed.3.q RT-PCR detects gene transcription levels: The total RNAs of the corresponding strains were extracted and reverse transcribed into c DNA for q RT-PCR assay to detect the m RNA level of the relevant genes.4.Lac Z gene fusion experiment: The promoter region of the corresponding gene was cloned between two restriction sites upstream of the p HRP309 plasmid β-galactosidase gene.The recombinant plasmid was electroporated into WT and ΔOmpR,and the activity of both β-galactosidase was compared.5.Expression and purification of OmpR protein: The expression of OmpR protein in the OmpR-p ET28a-BL21 strain was induced by adding IPTG.The bacteria cultures was centrifuged after ultrasonic decomposition and the His-OmpR protein was separated and purified by Ni-column affinity chromatography and preserve it at-80 °C.6.Electrophoretic mobility shift assay: The phosphorylated His-OmpR protein was co-incubated with the corresponding DNA promoter region fragment,then electrophoretic mobility shift assay was carried out using 6% non denatured polyacrylamide gel.Finally,the binding of His-OmpR and DNA fragments was analyzed by gel imager.7.The He La cells invasion assay: WT-p BAD33,(35)OmpR-p BAD33 and C-(35)OmpR were cultured to the early logarithm phase,co-incubated with the He La cells for 90 minutes,and the culture broth was discarded.The 0.5%(v/v)Triton X-100 was added to half of the wells,plated and cultured,the number of colonies was counted as T0.After gentamicin was added to half of the cell wells for 90 minutes,cells were lysed by adding 0.5%(v/v)Triton X-100,plated and cultured,the number of colonies was counted as T90,then T90/T0 indicates the invasiveness of S.Typhi on He La epithelial cells.8.The THP-1 cells intracellular survival assay: WT-p BAD33,(35)OmpR-p BAD33 and C-(35)OmpR were cultured to log-phase(OD600=0.4)and added to 24-well plate cultured with THP-1 cells.After one hour,gentamicin was added,and after one hour,the 1/3-well cells were ruptured,and the cells were collected and plated overnight.The number of colonies(T0)indicates the phagocytosis level of the basal bacteria.The other 2/3-well cells were cultured for 12 h and 24 h,and plated overnight after rupture.The number of colonies(T12,T24)represents intracellular bacterial proliferation,then T12/T0 and T24/T0 indicate the survivability of S.Typhi in THP-1 cells.Results:1.The OmpR gene defect supplementary strain,the wild control strain and the defective control strain were successfully constructed.2.The results of growth curve showed that the growth rate of(35)OmpR-p BAD33 was significantly faster than that of WT-p BAD33 under acid stress,while the growth of WT-p BAD33 was consistent with that of C-(35)OmpR.Under low osmotic conditions,the growth rates of both ΔOmpR and Δhfq were much lower than that of the WT.3.q RT-PCR results showed that acid stress can increase the m RNA levels of cad B and cad C,and OmpR can down-regulate the m RNA levels of cad B and cad C.OmpR promotes the transcription of hfq and Vi antigen-related genes,whereas Hfq feedback represses OmpR and Vi antigen-related genes,both OmpR and Hfq auto-positively regulate their own genes transcription.OmpR positively regulates transcription of invasion-related genes iag A,inv F,prg H and survivability-related genes ssr A,ssr B,spi C.4.The results of Lac Z gene fusion assay showed that OmpR can activate the expression of cad B and cad C under acid stress conditions.OmpR can activate the expression of ssr A,ssr B and spi C.5.Electrophoretic mobility shift assay showed that OmpR can directly bind to the promoter region of the following genes,including cad B,cad C,tvi A,OmpR,invasion-related gene prg H,survivability-related genes ssr A,ssr B and spi C.6.Compared with the WT-p BAD33,the invasive ability of(35)OmpR-p BAD33 on He La cells decreased,and the invasive ability of C-(35)OmpR partially recovered.It indicates that OmpR can enhance the invasive ability of S.Typhi on epithelial cells.7.Compared with WT-p BAD33,the viability of(35)OmpR-p BAD33 in THP-1 macrophage cells decreased,and the intracellular survivability of C-(35)OmpR partially recovered.It indicated that OmpR can enhance the survivability of S.Typhi in THP-1 macrophage cells.Conclusins:Under acid stress conditions,OmpR inhibits their transcription by direct binding to cad B and cad C,thereby inhibiting the growth of S.Typhi under acid stress conditions.(35)OmpR and(35)hfq attenuate the growth of S.Typhi in low osmotic medium.Under hyperosmotic stress conditions,OmpR can promote the transcription of hfq and Vi antigen-related genes,while Hfq feedback inhibits OmpR and Vi antigen-related genes,and OmpR and Hfq can automatically positive regulation of its own gene transcription.OmpR up-regulates the expression of invasion-related genes by directly regulating prg H,indirectly regulating iag A and inv F,and promotes the invasion of S.Typhi.OmpR up-regulates the expression of survivability-related genes by directly regulating ssr A,ssr B and spi C,and enhance the survivability of S.Typhi in macrophages.