Effects Of SpvB Gene In Salmonella Typhimurium On Autophagy Of Host Cells And Its Molecular Mechanism

Objective: spv gene Salmonella typhimurium is an important pathogenic factor in Salmonella infection.spv contains three essential genes--the positive transcriptional regulatory gene spvR and the two structural genes named spvB and spvC.In this study,a model of Salmonella typhimurium infection with human intestinal mucosal epithelial cells was established to observe the influence of downregulation of spvB gene expression on autophagy and autophagy related proteins by knocking spvB out.In addition,we explored the effect of p38 MAPK signaling pathway on the regulation of autophagy in intestinal epithelial cells by the spvB gene of Salmonella typhimurium.All of these provide new research ideas and targets for the clinical treatment of Salmonella infection.It lays the foundation for further study of the difference in the effect of spvB on autophagy and its molecular mechanism in drug-resistant and non drug resistant salmonk strains.Methods:1.the effect of spvB gene on autophagy in intestinal mucosal epithelial cells.S.typhimurium strains STM-211 which contains the spvB gene,STM-?spv B which the spvB was knocked out and the spvB complemented strain STM-c-spvB were used to co-culture with Henle-407 cells in intestinal mucosa epithelial cells in the logarithmic phase of growth period.Afterwards transmission electron microscope(TEM)was used to observe the ultrastructure of the cells;MDC staining was observed under the microscope;counting the intracellular bacteria after dilution;Western Blot(WB)was used to detect the expression of autophagy related protein--LC3 and p62,and the level of autophagy was compared between the infection group and the control group..2.The role of spvB gene in the process of autophagy in Henle-407 cells is mediated by p38 MAPK signaling pathway.P38MAPK pathway inhibitor SB203580 was added to the original infection model for intervention control.The cells were collected at 1h and 3h after co-culture.The phosphorylation level of p38 and the expression of autophagic marker protein LC3 and p62 were detected by WB,and the expression and distribution of autophagic protein LC3 were observed by immunofluorescence staining(IF).Resulst:1.the effect of spvB gene on autophagy in intestinal mucosal epithelial cells.(1)The ultrastructure of Henle-407 cells after infection of S.typhimurium was observed by transmission electron microscopy.The normal cell structure was seen in the 1h after infection,and no obvious autophagy was found in the control group.In the wild strain STM-211 infection group and STM-?spvB infection group,the structure of autophagosome and autophagosome,as well as a large number of Salmonella-containing vacuoles were found.(2)The MDC staining showed that the green dots fluorescence of cells in each group increased discriminatively at 1h after infection.In the control group,there was only a little fluorescence distribution,and the number of fluorescent cells was also significantly weaker than that of the infected group.The intracellular fluorescence intensity and the number of fluorescent cells in STM-?spvB infection group were significantly higher than those in STM-211 infection group.(3)Counting of surviving bacteria in cells after dilutionThe total amount of bacteria in the culture medium of each infection group was not significantly different at 1h after the bacterial infection.From 2h,the bacterial survival of the STM-?spvB infection group was significantly lower than that of the STM-211 and STM-c-spvB infection group(P<0.05).While the number of bacteria increased at 4h,STM-?spvB group was still much more than that in STM-211 and STM-c-spvB group obviously.(4)WB detect the autophagy level in wild,mutant and complemented strains infection groups.Compared with the control group,the expression level of LC3 II at 1h after infection in STM-?spvB group was significantly higher than that in the STM-211 group and the STM-c-spv B group(P<0.05).The level of the substrate p62 protein was lower than that in the STM-211 and STM-c-spvB infection group(P<0.05).2.The role of spvB gene in the process of autophagy in Henle-407 cells ismediated by p38 MAPK signaling pathway.(1)Expression level of p38 MAPK pathway in different infection groupsIn different groups of Salmonella at different time after co-culture,the p38 phosphorylation level of each infection group was higher than that of the control group.The phosphorylation level of p38 at 1h of STM-?spvB group after infection was significantly higher than that of STM-211 and STM-c-spvB infection groups.This trend continued to 2h and 4h.Moreover,the levels of p38 phosphorylation in STM-211,STM-?spvB or STM-c-spvB infected groups increased with time.(2)WB detected autophagy level in each group after inhibition of P38 pathway.After SB203580 treatment,the level of LC3 II in the control group,STM-211 and STM-c-spv B infection group had no significant difference(P>0.05),but the STM-?spvB infection group decreased significantly(P<0.05),and the p62 protein level in STM-?spvB infection group was significantly higher than that before the SB203580 treatment,while there was no significant difference between the other groups before treatment.At 3h after infection,the expression LC3 II and p62 protein kept the trend at 1h after infection.After the pretreatment of SB203580,the expression of LC3 II in STM-? spvB group increased and the p62 expression decreased significantly.However,LC3 II and protein levels in STM-211 and STM-c-spvB group had no significant changes.(3)The autophagy level of each group was detected by immunofluorescence after inhibition of p38 pathway.The results showed that after 2h of the co-culture,the LC3 point structure of the cell nucleus of each infection group increased significantly compared with the control group,and the STM-?spvB group was significantly more than the STM-211 group.After treatment with SB203580,the number of fluorescent particles in STM-? spvB group decreased significantly,while the STM-211 group did not change obviously compared with that before the SB203580 treatment.Conclusions: 1.SpvB gene inhibits autophagy in Salmonella infected intestinal epithelial cells Henle-407,which is beneficial to the survival of bacteria.2.The p38 pathway is activated in Salmonella infection,and the inhibition of on p38 pathway by spvB is one of the key mechanisms to autophagic inhibition.