Characteristics Of Salmonella Typhimurium Effector SrfA And Its Function For Regulating Host Inflammatory Responses During Bacterial Infection

Salmonella enterica serovar typhimurium (S. typhimurium) strains are Gram-negative intracellular pathogens and considered as the major Salmonella serotype that causes the infection in humans. As one of the most common food-borne diseases, Salmonellosis usually leads to varied manifestations of self-limited diseases from gastroenteritis to enteric fever in adults. However, Salmonellosis infection causes life-threatening diseases including bacteremia and lethal sepsis in susceptible population like children and immunocompromised individuals, which mortality is 10%-15% without suitable treatment. With the emergence of new multidrug resistant Salmonella strains and lack of suitable effective Salmonella vaccine, understanding the pathogenesis of invasive Salmonella is very important for infectious disease prevention and control.A series of effectors and structures such as type III secretion system (T3SS), virulence effectors and regulatory system encoded by genes of Salmonella pathogenicity islands (SPIs) are the molecular basis of the pathogenesis of Salmonella-induced diseases. Innate immune responses provide the first line of defense against invasive Salmonella infection. During Salmonella infection, effectors encoded by SPIs play a key role in promoting bacterial intracellular dissemination and pathogenesis by regulating NF-kB signaling activation.In this study, we focused on the S. typhimurium effector protein SrfA (SsrA-SsrB regulated factor) to investigate its role for pathogenesis of invasive Salmonella. The results demonstrated that SrfA is not associated with S. typhimurium growth. SrfA was secreted into the cytoplasm and promoted NF-?B activation in S. typhimurium infected macrophages.A yeast two hybrid (YTH) assay was used to investigate the SrfA-interacting host proteins. The full-length SrfA was used as bait to screen a human fetal brain-derived cDNA library, and SrfA-interacting cytoplastic protein, Tollip (Toll interacting protein), was identified. The interaction of SrfA and Tollip was also confirmed by GST-pull down, Co-IP and sub-cellular co-localization assays. The SrfA-Tollip interaction repressed the combination of Tollip to IRAK-1 and led to IRAK-1 phosphorylation and downstream NF-?B pathway.The CUE aa219-272 domain of Tollip and the MDaa207-226 and CTaa257-337 domains of SrfA mediated the interaction between SrfA and Tollip, based on the interaction assay between a series of truncated Tollip mutants and SrfA, respectively. tollip-RNA interference and srfA-delc4m4 mutant further confirmed that S. typhimurium effector SrfA activated NF-?B signaling by interaction of SrfA with Tollip.We also investigated the effect of srfA on Salmonella pathogenesis in a mouse model. Our results showed that mice infected with wt S. typhimurium all died within 9 days p.i., whereas 70% mice infected with ?srfA S. typhimurium survived. The concentration of cytokines including IFN-y and TNF-a in the sera of mice infected with AsrfA S. typhimurium was remarkable low level compared with that of mice infected with wt S. typhimurium. Histopathological analysis showed that microabscesses and necrotic areas existed in the spleen and liver of mice infected with wt bacteria, whereas histological lesions were not observed in the spleen or liver of mice infected with the AsrfA mutant. The Colony-Forming Units (CFU) counts of organ lysates showed that AsrfA mutant exhibited significantly higher bacterial loads than wt Salmonella at 5 days p.i., however, there was no significant difference of the CFU number of between wt and AsrfA Salmonella in the spleens or livers at 9 days p.i., suggesting that SrfA mediated bacterial dissemination at early stage of S. typhimurium infection.In summary, our results demonstrated that SrfA is a secretory virulence effector and regulates inflammatory responses to S. typhimurium infection. Our findings provide new theoretical foundations to understanding Salmonella pathogenesis.