The Effect And Mechanism Of Biofilm On Salmonella Typhimurium Escaping From The Immune Surveillance Of Dendritic Cells

Salmonella typhimurium is a common zoonotic pathogen,which mainly infects the host through the digestive tract and can cause a variety of foodborne diseases and food poisoning.The main clinical symptoms are diarrhea,gastroenteritis,and sepsis.After invasion of Salmonella typhimurium from the intestinal mucosa,intestinal mucosal dendritic cells(DCs)can capture bacteria via transepithelial dendrites,and then DCs are activated and matured,migrated to mesenteric lymph nodes,and early innate immunity are established.Finally,DCs presented the antigen and initiated the adaptive immune response.Therefore,DC is a bridge connecting innate immune response and adaptive immune response.Salmonella usually exists in the biofilm state in the environment,which enhances the resistance to the adverse growth environment and antibiotics.Pathogen-associated molecular patterns in the intestinal cavity can be specifically identified by toll-like receptor(TLR)of the intestinal mucosa,which can induce DCs to form transepithelial dendrites for uptake of bacteria.It is not clear whether biofilm affects the immune surveillance function of intestinal mucosal DCs.In this study,Salmonella typhimurium wild strain(S025)and absence of gene mutations(S025?csgD?S025?rpoN)were used.Firstly,the biofilm on the influence of Salmonella typhimurium infection in mice was determined;Secondly,the ability of transepithelial dendrite formation of DCs for capturing Salmonella typhimurium in vivo was evaluated;Finally,the effect of biofilm of Salmonella typhimurium on DC maturation in vitro was determined.This study analyzed the relationship between Salmonella typhimurium biofilm and intestinal mucosal DCs,which laid a foundation for further elucidating the pathogenic mechanism of Salmonella.The content is mainly divided into the following two parts:1.The effect of biofilm on infection ability of Salmonella typhimurium in miceIn this study,the biological characteristics of the Salmonella typhimurium wild strain(S025)and its gene deletion mutant strains(S025 ?csgD,S025?rpoN)were studied,including growth characteristics,biofilm formation ability,pathogenicity to mice and bacterial load in viscera,and adhesion invasion ability to intestinal epithelium.The results showed that,compared with wild strain,the growth rate of bacteria did not change after the deletion of csgD or rpoN gene.Notably,?csgD strain lost biofilm formation ability,while this ability of ?rpoN strain was enhanced.The LD50 of wild strain with plankton state,wild strain with biofilm state,?csgD,or ?rpoN strain in mice was 3.3×106,5.3×105,3.9×106,or 8.2×105,respectively.The survival rate of mice after 15 d in the group of the wild strain with biofilm state and ?rpoN strain was only 10%,the survival rate of mice in the group of the wild strain with planktonic state was 50%,and the survival rate of mice in the group of?csgD strain was 40%.In comparison of the wild strain with biofilm state,the survival rate was significantly improved after infection of wild strain with planktonic state and ?csgD strain.And the adhesion and invasion ability of these two strains to colon cancer epithelial cells(CMT-93)of mice were decreased in vitro.?rpoN strain had no significant effect on the survival rate of mice and the adhesion and invasion ability to CMT-93.The bacteria-load in feces,liver,spleen,kidney,and blood of mice infected with the wild strain with biofilm state was significantly higher than that with planktonic state.The results suggested that the formation of a biofilm by Salmonella typhimurium significantly improved the pathogenicity of bacteria to mice.2.The effects of biofilm on the transepithelial uptake and maturation of dendritic cells induced by Salmonella typhimuriumIntestinal mucosal DCs of has the ability of transepithelial uptake of bacteria.In this experiment,mice were inoculated with Salmonella typhimurium into ligated intestines,after 30 min,intestinal tissues were collected for confocal observation and flow cytometry.The results showed that,compared with the wild strain with biofilm state,the number of transepithelial dendrites of DCs in the group of wild strain with planktonic state and ?csgD strain was significantly increased,the number of DCs in the interepithelial cells and lamina propria was significantly increased,the secretion of CCL20 from the epithelial cells was significantly increased,the amount of bacteria-loaded DCs migrated into the mesenteric lymph nodes was also significantly increased.These data suggested that biofilm can suppress the transepithelial uptake of DCs induced by Salmonella typhimurium.In order to investigate the effect of biofilm on the level of innate immune response of DCs induced by Salmonella typhimurium in mice,this primary murine DCs were infected with Salmonella typhimurium for 24 h,cells and their supernatant were collected for detecting the phenotypic markers,cytokine secretion,and the proliferation of CD4+ T cells.Firstly,the results showed that the phagocytosis ability of wild strain with planktonic state and ?csgD strain but not ?rpoN strain was significantly increased compared with wild strain with biofilm state.Secondly,compared with wild strain with biofilm state,?csgD strain significantly promoted the expression of phenotypic marker(CD80,CD86,CD40,and MHC ?),early activation marker(CD69),migration marker(CCR7),the proinflammatory cytokines(IL-1?,IL-6,and TNF-?)of DCs.In the mixed lymphoid response assay,?csgD strain significantly promoted the proliferation of CD4+T cells.Collectedly,these data suggested that biofilm can suppress the activation and maturation of DCs induced by Salmonella typhimurium.