The Activation Of NLRC4Inflammasome And Its Role In The Defense Against Salmonella Infection

Salmonella is a kind of zoonotic pathogen. After infection, it can survive in the host cells and lead to severe carrier state. Salmonella-carrying food animal is one of the major sources of salmonellosis both in human being and animals. Therefore, the inhibition of bacteria survival in host cells will be benefit to the defense against salmonellosis. Macrophages (Mφs) are one of the key immune cells involved in the innate immune response. However, it is also the major host cell of Salmonella infection. Therefore, the Mcp is a good model to study the responses against Salmonella infection. In Mcps, NLR (NOD-like receptor) can recognize microbial components in cytoplasm related with pathogenicity and induce inflammasome signaling which is benefit to the clearance of intracellular bacteria. Because Salmonella can escape the inflammasome-induced responses in host cells, it is suggested that Salmonella can be cleared through enhancing intracellular inflammasome activity. As reported, the utilization of inflammasome enhancing strategy for the design of attenuated live vaccines of intracellular pathogen could promote their protection efficacy.In the present study, the responses of Mφs against Salmonella infection were firstly analysed. Then the recombinant Salmonella strain was constructed to enhance the activation of inflammasome. Finally, after infection of mice, the effect of enhancing intracellular inflammasome activation on the defence against Salmonella infection was analyzed.1. Responses of macrophages against Salmonella infectionMcps can clear dead Salmonella through the induction of phagosome maturation, while in the live Salmonella-infected Mφs, besides the formation of phagosome, the Salmonella-containing vacuoles are formed due to the regulation function of bacteria to promote their survival in Mφs. It will be benefit to explore the Mφs responses against live Salmonella compared with phagosome mechanism. To explore the responses of Mcps against Salmonella, the early responses of peritoneal macrophages (PMs) after infection with S. Enteritidis C50041(MOI=150) were analyzed, compared with the phagocytosis of dead C50041(C50041-d). Results indicated that the cell membrane of C50041-infected Mφs were seriously damaged compared with C50041-d-treated and non-infected cells. The cytotoxicity induced by live C50041was higher than that for C50041-d (P<0.05) based on the lactate dehydrogenase (LDH) release assay. After stimulation, both live and dead C50041could upregulate the expression of co-stimulatory molecules. For the cell function, similar changing trends of mitochondrial membrane potential, intracellular concentration of calcium ions, reactive oxygen species and nitric oxide were found between them, but the changes induced by live C50041were earlier than those for C50041-d. Four hours post infection (p.i.), the LC3(autophagy marker) expression of Mφs and the ratio of LC3-II/LC3-I induced by C50041were lower than that for C50041-d. C50041significantly inhibited the production of tumor necrosis factor and interleukin (IL)-6(P<0.05). Whereas the intracellular caspase-1activation and IL-1β release induced by C50041were stronger than those for C50041-d (P<0.05). It is suggested that the live Salmonella can induce more higher activation of inflammasome signaling compared with the phagocytosis of C50041-d.2. Construction and identification of recombinant Salmonella enhancing inflammasome activationIn the early stage of infection, Salmonella can activate inflammasome pathway, after survival in the MφS, Salmonella can decrease or escape the inflammasome response by regulating cell function. So, the recombinant bacteria are needed to be constructed to evaluate the effect of intracellular inflammasome activation on the colonization of Salmonella. In the previous studies, the N-terminal oi Salmonella SspH2protein was shown to transport exogenous antigen into the cytoplasm, and the C-terminal of E. coli EscI protein could activate the NLRC4inflammasome in Mcps.In this study, the5’-terminal fragment of sspH2gene (1-453bp) of S. Enteritidis C50041and the3’-terminal fragment (205-426bp) of escl gene of E.coli O:157were ligated by overlap polymerase chain reaction and then the SspH2-EscI gene containing flag-tag gene was cloned into pMD20T vector for sequencing. Gene sequencing results indicated that the content of C+G in SspH2-EscI gene (711bp) was49.79%. There are some different nucleotides found in5’- terminal sequences of sspH2genes of different Salmonella strains and3’-terminal sequences of escl genes of different E.coli strains.The SspH2-EscI gene (720bp) was subcloned into plasmid pYA3334through Nco I and Sal I double digestion. The recombinant plasmid pYA3334-SspH2-EscI was constructed and in turn transformed into S. Typhimurium X3730and X4550. SDS-PAGE and Western blotting results showed that the recombinant X4550(pYA3334-SspH2-EscI) successfully expressed the fusion protein SspH2-EscI (about32kDa).The infection experiments were performed to verify the ability of X4550(pYA3334-SspH2-EscI) to enhance the inflammasome activation in Mφs. As shown that X4550(pYA3334-SspH2-EscI) could significantly promote the murine PMs to secret IL-1β after infection in vitro, compared with X4550(pYA3334) infection (P<0.05). After intravenously infection in vivo, the number of X4550(pYA3334-SspH2-EscI) in mice spleen and liver were decreased significantly than those for X4550(pYA3334)(P<0.05). One week later, the mice infected with X4550(pYA3334-SspH2-EscI) could obviously inhibit the early colonization of X4550(pYA3334)(P<0.05). These data implied that the recombinant bacteria X4550(pYA3334-SspH2-EscI) could enhance the intracellular inflammasome activation.3. Immune protective responses induced by the recombinant bacteria against Salmonella infectionThe immune responses of recombinant bacteria, X4550(pYA3334-SspH2-EscI), in mice against Salmonella infection were analyzed, compared with X4550(pYA3334) infection.In the intravenously infection (1×106cfu/mouse) experiments, results indicated that the spleens of X4550(pYA3334-SspH2-EscI)-infected mice were similar as those of the non-immunized control mice, while those of X4550(pYA3334)-infected mice showed significantly swelled. The necrosis level of the livers and spleens of X4550(pYA3334-SspH2-EscI)-infected mice was lower than those for X4550(pYA3334)-infected mice one week post infection. Two weeks later, the ratio of T lymphocyte subsets (CD4/CD8) of X4550(pYA3334-SspH2-EscI)-infected mice decreased significantly (P<0.05) compared with the non-immunized control mice, while that for X4550(pYA3334)-infected mice slightly increased. Besides, both could significantly activate T lymphocytes of immunized mice compared with negative control group (P<0.05), but the activation of T lymphocytes in X4550(pYA3334-SspH2-EscI)-infected mice was lower than that for X4550(pYA3334)-infected mice. Low levels of cytokines in sera were found in both infection groups. One week post immunization, the immune protection induced by X4550(pYA3334-SspH2-EscI) against challenge of wild-type virulence Salmonella strain D6was significantly higher than that for X4550(pYA3334).After intraperitoneal injection (1×106cfu/mouse), results indicated that the colonization of X4550(pYA3334-SspH2-EscI) was decreased in mice spleens compared with X4550(pYA3334) immunization group, while no obvious promotion in its immune protection against challenge of Salmonella strain D6. Furthermore, the mice immunized with X4550(pYA3334-Ssph2-EscI) showed decreased health status than that for X4550(pYA3334).These data suggested that the different immunization routes could influence the safety and immunity of Salmonella enhancing inflammasome activity. From the above analysis, it would be benefit for the study of inflammasome mechanism and its application in the defense against intracellular bacteria infection.