Type 1 Fimbriae Of Salmonella Typhimurium Mediate Bacterial Virulence And Invasion To Antigen Presenting Cells In Mice

Salmonella enteria serovar Typhimurium(S.typhimurium) is gram-negative facultative intracellular bacterium,who is a member of the genus Salmonella belonging to Enterobacteriaceae,causing infections in a wide range of animal species including mice, poultry,pigs,sheep,cattle,horses,and humans.S.typhimurium plays an important role in people and animals infecting Salmonella(Salmonellosis),and it is also important to public health.Type 1 fimbriae of S.typhimurium are proteinaceous filaments produced by many members of the family Enterobacteriaceae.It is responsible for the ability of bacterial adherence and colonization.Furthermore,it is the only fimbriae which can express in stationary culture.The previous studies have proved that type 1 fimbriae can induce the bacterial adherence and colonization to host cells in vitro.The present study was focused on research in vivo and aimed to demonstrate type 1 fimbriae of S.typhimurium will influence the bacterial virulence and invasion.The results were summarized as follows:1.In order to test the activity of bacteria,the AJB3 strain who is derived from a wild-type S.typhimurium SR-11,a triple-deletion mutant strain AJB3fim^-lpf^-invA^-(marked as 117#),the recombinant strain 117#-pAZ44-h which can express type 1 fimbriae,and the recombinant strain 117#-pAZ33-h which can not express type 1 fimbriae,were adopted to study bacterial adhesion to HEp-2 cells and myeloid dendritic cells(MDC) tissue culture cells.We confirmed that the mutant strain 117# is fewly able to adhere to HEp-2 cells and MDC,so the results indicated that triple-deletion mutant was successfully constructed.While we also found that the S.typhimurium strain 117#-pAZ44-h can adhere more to HEp-2 cells and myeloid dendritic cells(MDC) than the recombinant strain 117#-pAZ33-h,and a few more than the AJB3 strain.So,the results showed that type 1 fimbriae could specially enhance fimbrate 117#-pAZ44-h strain adherence,which can express type 1 fimbriae.These findings laid a good foundation for further study of S.typhimurium type 1 fimbriae.2.Mice were immunized at intragastrical route with the equal mixture of the fimbriate and afimbriate bacteria Three mice were killed at various time-points of 12h, 24h,36h,48h,72h,96h,120h,144h,168h,and Peyer' patches,mesentery lymph nodes, spleen were collected.The tissue was homogenized,then 100μL of each supernatant was plated on MacConkey agar which containing different antibiotics.The numbers of the bacteria growing on the plates were counted.The result showed that afimbriate bacteria invaded the body more effectively than fimbriate strain.We have performed acute toxicity test for the two strains in Balb/c mice,and the results showed that the virulence of the fimbriate bacteria expressing type 1 fimbriae was decreased shown by LD₅₀ increase of 42-fold,compared to the other one.Then the same tests were conducted by intravenous route,and three mice were also killed at various time-points of 4h,12h,24h,36h,48h, 72h,96h,120h,and bacteria were recovered as described previously in intragastrical route.However,the results indicated that the bacteria that expressed type 1 fimbriae invaded the body more effectively than ones not expressing type 1 fimbriae and the virulence of the bacteria expressing type 1 fimbrial was increased shown by LD₅₀ decrease of 19-fold,compared to the other one.However,at both intragastrical and intravenous routes,type 1 fimbriae expressing bacteria associated with DC and B cells significantly higher those afimbriated bacteria.In contrast,there was no difference in the bacterial association with macrophage.3.We have analyzed gene expression profiling through 32K genome arry oligo chip (contenting 32256 genes) at 1 hour after the two S.typhimurium strains stimulated myeloid dendritic cells.The data of microarray suggests that the bacteria who can express type 1 fimbriae mainly down-regulated gene expression and the afimbriate strain up-regulated a few gene expression.From the analysis of the data,the differential genes focused on several kind genes,such as genes relating to transcription and translation about 21.6%and signal transduction about 13.8%and protein metabolism about 24.1%, which are differenfilly expressed when bacteria stimulating MDC.Altogether,These results might play a significant role in elucidate the pathogenesis of Salmonella typhimurium and design novel gene engineered vaccine to both Salmonella and other pathogens by using Salmonella as the live vector.