In this study, for the first time, the mechanism of protection provided by immunization with the gidA mutant STM strain was characterized. GidA was originally thought to be involved in cell division due to the filamentous morphology observed when the cells were grown in rich medium supplemented with glucose . More recent studies done in E. coli have shown GidA modulates several bacterial factors by a post-transcriptional mechanism to modify tRNA by the addition of a cmnm group at the 5 position of the wobble uridine (U34) of tRNAs [15–19, 30, 31]. It has been proposed that tRNA modification can serve as a regulatory mechanism to modulate gene expression. Furthermore, it has been suggested that secreted proteins are particularly vulnerable to U34 hypomodification, and many codons in bacteria require proper U34 modification for efficient decoding . Studies will need to be conducted in Salmonella to see if GidA modifies tRNA in the same fashion as in E. coli. Such studies are currently underway in this laboratory.
Immunization of mice with the gidA STM mutant strain provided full protection from a lethal dose challenge of WT STM. All of the immunized mice survived a lethal dose challenge, while all the naïve mice died within 4 days of challenge. Furthermore, none of the immunized mice displayed any visual signs of illness or septic shock associated with Salmonella infection. We chose to challenge the immunized mice with a WT STM dose of 1 x 105 CFU which is highly lethal. In our initial GidA study, this dose was approximately 1000 times higher than the LD50 of the WT STM strain . We chose such a high challenge dose because we feel it is more reflective of the amount of Salmonella animals are exposed to in the environment.
Antibody responses are known to contribute to Salmonella immunity [34–36]. It has been proposed that antibodies made by IgM memory B cells are the first-line defense mechanism against all infections and these antibodies are the only defense against T cell-independent antigens . Studies in B cell deficient mice have shown that B cells are required for efficient protection from both primary and secondary Salmonella infection . Our data indicates a strong humoral response to immunization with the gidA mutant STM strain. The Th2 marker, IgG1, showed a marked increase in sera of mice immunized with the gidA mutant STM strain. Naïve mice receiving sera from immunized mice were more protected than naïve mice receiving a passive transfer of cells from immunized mice. Further, the level of the Th2 cytokine IL-10 showed a significant increase in induction when splenocytes from immunized mice were treated with STM cell lysate. The strong Th2 response, however, was not accompanied by an increase in IL-4 induction. IL-4, along with IL-10, induces differentiation of uncommitted T cells toward a Th2 phenotype [38, 39]. One possible explanation for this could be reasoned from the study by Okahashi et al. In their study, IL-4 knockout mice which were unable to generate classical Th2-type responses were still capable of producing significant antibody responses to inoculation with Salmonella.
Since Salmonella is a facultative intracellular pathogen, cellular immune responses are considered to be a crucial component of protective immunity. Protective cellular mediated immunity is mediated by CD4+ cells which results in the activation of macrophages and delayed-type hypersensitivity responses. Numerous gene target studies have shown the importance of CD4+ activation in resistance to Salmonella infection [41, 42]. Our data indicates a cellular immune response in mice immunized with the gidA mutant STM strain. Although the flow cytometric analysis showed no induction of memory T cells, or difference in CD8+ cells, it shows an increase in CD4+ population in the immunized mice at both day 7 and 42 post-immunization. It has been shown that CD4+ cells are more important than CD8+ in resistance to Salmonella infection [43, 44]. The passive transfer of cells to naïve mice from immunized mice did not confer full protection, and was not as significant as the serum passive transfer, but there was enough cell mediated immunity activated to protect a portion of the mice from a lethal dose challenge. Furthermore, splenocytes from immunized mice proliferated at a much higher rate than splenocytes from control mice when treated with STM cell lysate. The IgG1 induction was significantly more prominent than the induction of IgG2a, but the level of IgG2a was still significantly higher in the immunized mice than in that of the sera of the control mice. Furthermore, the induction of the Th1 cytokines, IL-2 and IFN-γ, shows a strong indication of cell mediated immunity induced by immunization. In particular, IFN-γ showed a marked increase in cell culture supernatant when splenocytes from immunized mice were treated with STM cell lysate.
The general consensus is that the ideal Salmonella vaccine should generate both humoral and cell mediated immunity. This is due to protective immunity to Salmonella in mice being attributed to a balance between humoral and cell mediated immunity with an emphasis on development of the Th1 and Th2 subsets [45, 46]. In this study, the gidA mutant vaccine strain generated both Th1 and Th2 immunity with the Th2 immune response being the more prominent of the two. This was somewhat surprising since Salmonella is a facultative intracellular pathogen. One possible explanation for this could be found in our initial GidA study comparing the gidA mutant to the WT STM strain. The gidA mutant showed an approximate 1000-fold reduction in the ability to invade T84 intestinal epithelial cells, as well as a marked reduction in ability to cause systemic infection in mice. Additionally, transcriptional and proteomic profiling identified a significant down-regulation in numerous genes and proteins responsible for invasion. Overall, the gidA mutant vaccine strain provides full protection to mice when challenged with a highly lethal dose of WT STM. The passive transfer experiments show the importance of both humoral and cell mediated immunity in this protective mechanism. This is an initial study in which a proof of principle of protective immunity has been established suggesting a gidA mutant STM strain could be a good candidate for use in a live-attenuated Salmonella vaccine. Future studies will be conducted using oral immunization in order to establish the optimal immunization route. Once the immunization route is established, further studies will be conducted in a target host animal to determine efficacy and long-term protection. Based on our initial data, we believe a gidA mutant STM strain used in a live-attenuated vaccine could provide superior protection against highly lethal levels of Salmonella by stimulating humoral, cellular immunity and potentially mucosal immunity.