The Effect And Mechanism Of Cellulose And Curli Fimbriae On Salmonella Typhimurium Escaping From The Intestinal Mucosal Immune Response

Salmonella typhimurium is an important foodborne pathogen,which seriously threatens the global public health.The host innate immune response plays a significant role in resisting infection.Intestinal mucosal dendritic cells(DCs)are specialized antigen presenting cells,which play an important role in the intestinal mucosal immune system.Submucosal DCs as“sentries”patrol under the epithelium use their transepithelial dendrites(TEDs)to monitor luminal“antigens”.The antigen-loaded DCs quickly migrated into draining lymph nodes for initiating the adaptive immune response,which is important for the clearance of pathogen.Therefore,whether submucosal DCs can be mobilized,which is a pivotal step for initiating the downstream innate and adaptive immune response.The DCs recruitment and TEDs formation mainly depend on the activation ability of pattern recognition receptors(PRRs)of mucosal epithelial cells,and Toll-like receptors(TLRs)belong to important PRRs.Salmonella usually exists with the biofilm state in the environment,and cellulose and curli fimbriae are the main components of biofilm,which are encoded by hcsA and csgA genes,respectively.The formation of biofilm by Salmonella typhimurium significantly improved the pathogenicity of bacteria to mice.It is not clear whether cellulose and curli fimbriae affect the pathogenicity of Salmonella typhimurium.In this study,Salmonella typhimurium wild strain(S025)and gene mutations(S025△bcsA,S025△csgA,and S025△bcsA/csgA)were used.Firstly,the pathogenicity of bacteria to mice was determined;Secondly,the DCs recruitment and TEDs formation were evaluated;Finally,the innate immunity levels of DCs were analyzed.The study provided a new research approach for Salmonella typhimurium escaping from the host immune response.1.Pathogenicity of Salmonella typhimurium△bcsA,△csgA and △bcsA/csgA in miceFirstly,the biological characteristics and pathogenicity of bacteria in mice were evaluated.The results showed that △bcsA strain had reduced biofilm forming ability,the △csgA and △bcsA/csgA strains did not form biofilm.Then,components of biofilm were analyzed.The results showed that the expression of cellulose and curli fimbriae significantly decreased in △bcsA and △csgA strains,respectively.Murine pathogenicity assay in vivo showed that,survival rate of mice and secretion of proinflammatory cytokine were significantly increased,the bacteria-load in liver and spleen,the expression of anti-inflammatory cytokines were obviously decreased when infection of the △csgA and △bcsA/csgA strains compared to the wild strain with biofilm state.However,the bacteria-load in liver and spleen were significantly declined after the infection of the △ bcsA strain.In addition,the adhesion and invasion abilities of △bcsA,△csgA,and △bcsA/csgA strains to intestinal epithelial cells were markedly declined compared to the wild strain with biofilm state.The above results indicated that △bcsA,△csgA and △bcsA/csgA strains improved the pathogenicity of Salmonella typhimurium to mice.2.Effects of Salmonella typhimurium △bcsA,△csgA,and △bcsA/csgA on DCs recruitment and TEDs formationIn vivo experiment,mice were inoculated with Salmonella typhimurium into ligated intestines.The results showed that,the △csgA and △bcsA/csgA strains recruited more DCs into the intestinal epithelium region to take up bacteria by more TEDs,the expression levels of CCL20,TLR4 and TLR5 in intestinal epithelial cells were up-regulated,while the expression of TLR2 significantly declined.However,there was no significant change in △bcsA strain.These data demonstrated that the △csgA and △bcsA/csgA strains improved the DCs to take up bacteria by TEDs in intestinal epithelial cells.3.Effects of Salmonella typhimurium △bcsA,△csgA,and △bcsA/csgA on the immunity of murine DCsDCs were incubated with Salmonella typhimurium.Compared by the wild strain with biofilm state,the △csgA and △bcsA/csgA strains remarkably up-regulated the expression of MHC□,CD40,CD80,and CD86.Similarly,the △csgA and △bcsA/csgA strains significantly increased the release of cytokines(IL-12p70,IL-6,TNF-α,IL-17A,and IL-1β),inhibited the secretion of anti-inflammatory cytokines(IL-10 and TGF-β1).The DCs from the △csgA and △bcsA/csgA strains groups strongly enhanced the proliferation of allogeneic T cells compared to that from the control group.Furthermore,the expression levels of TLR4 and TLR5 were up-regulated,while the expression of TLR2 significantly declined.The △bcsA strain significantly up-regulated the expression of MHC□,CD40,CD80,and CD86.Therefore,the △csgA and △bcsA/csgA strains improved the immunity of DCs.