Typhimurium with a nonfunctional SPI-2 is considered as an avirulent and a potential vaccine strain [37
]. In this study we have experimentally proved that S
. Typhimurium diarrhea vaccine strain with nonfunctional SPI-2 system can be further attenuated without impeding the immunogenicity in immunocompromised hosts. We additionally mutated mig-14
. Typhimurium strain. The ssaV
double mutant was found to be highly attenuated in wild-type C57BL/6 mice and in immunocompromised mice like Nos2−/−, Il-10−/−
. These transgenic immunocompromised mice were selected for this study because of their high susceptibility to different infections [33
]. One of the characteristic features of Salmonella
infections in humans is that few infected individuals can become chronic carriers. Such individuals comprise about 1–6% of the total infected population [19
] acting as reservoirs, and restricting the pathogen within the human populations. Previous studies have established that the successive progression of host-adapted Salmonella
species has led to an increased virulence because of their association with the host along with increased invasiveness and long-term persistence [51
]. The virulence factors essential for long-term persistence of the pathogen in their respective hosts are therefore likely to be important for its evolutionary success.
Mig-14 is an important factor for Salmonella
resistance to IFN-γ-mediated host responses and to different anti-microbial peptide during the establishment of infection as well as survival in the macrophages [16
]. It has also been reported that mig-14
mutant can establish an infection but cannot persist for longer periods in the host system [53
]. These reports support the contribution of Mig-14 in Salmonella
long-term virulence. Although the mechanism of Mig-14 action is not completely established, the binding of Mig-14 deficient Salmonella
to cathelin-related antimicrobial peptide (CRAMP) proves its active involvement in Salmonella
antimicrobial peptide resistance [40
]. Mechanistically, Mig-14 protein is a periplasmic protein which is tightly associated with the inner membrane of Salmonella
The transmission electron microscopy study has revealed that the primary site of host CRAMP activity is the bacterial cytoplasm. Study of inner membrane localization of Mig-14 and cytoplasmic CRAMP activity, possibly suggests the role of Mig-14 in preventing penetration of CRAMP into the cytoplasm [40
]. Taken together, these reports explain contribution of mig-14
towards pathogen survival by encountering host inflammatory responses and promoting both acute and persistent bacterial infection. Therefore, in the present study, mig-14
was taken as an important virulence factor to be knocked out from the existing live attenuated strain (MT5) with the goal to improve the attenuation attributes in immunocompromised mice.
In this study, we have assessed the degree of attenuation of S. Typhimurium ssaV mutant (MT5) and ssaV, mig-14 double mutant (MT4) in immunocompromised mice, by infecting these two strains to Nos2−/−, Il-10−/−and CD40L−/− C57BL/6 mice. The day 4 p.i. observation showed a high degree of systemic attenuation of MT4 (ssaV, mig-14) strain in Nos2−/−, Il-10−/− mice in comparison to the MT5 (ssaV) strain. On the other hand MT5 and MT4 strains were equally attenuated in CD40L−/− mice. Interestingly, MT4 strain also retained its capacity to colonize the mesenteric lymph node of Nos2−/−, Il-10−/−and CD40L−/− mice, demonstrating its ability to access the mLN but not the systemic sites. The in vivo data showed that the attenuation of MT4 in immunocompromised mice could be due to the absence of mig-14 in ssaV deficient S. Typhimurium. Furthermore, the MT4 and MT5 strains were used to vaccinate the wild-type C57BL/6 mice. Results showed that none of the mice developed cecal inflammation at day 30 p.v. However, both the strains (MT5 and MT4) equally colonized the gut lumen of vaccinated mice groups. Apart from this, at 30 day p. v., neither of the strain was found in the systemic organs which diminishes the possibility of late systemic dissemination and associated disease symptoms. Interestingly, apart from MT5, we also found a small population of MT4 strain in the mesenteric lymph node of the immunized mice, showing the potential of MT4 to stay in the lymphoid tissue for a longer period. In a challenge experiment, the vaccinated mice were protected when challenged with wild-type S. Typhimurium, however, the PBS treated mice developed significant inflammation and systemic dissemination of S. Typhimurium during subsequent Salmonella challenge.
In conclusion, the MT4 live-attenuated S. Typhimurium strain provides an efficient antibody mediated immune response which can protect even immunocompromised hosts from lethal infection of Salmonella. Specific antibody response to any protein antigens requires the involvement of both CD4+ and CD8+ T-cells along with the B-cells. The T-cell dependent antigens require the involvement of T-cells for the adaptive immune response. T helper (CD4+) cells play a vital role in stimulating the B-cells for the production of pathogen specific antibody via clonal propagation. Additionally, the activated CD4+ and CD8+ T-cells are the major producers of INF-γ which further activates the tissue and blood macrophages. As T-cell contributes to the cell mediated immune response, it is important to estimate the T-cell propagation during the course of Salmonella infection. In this study we have additionally estimated CD4+ and CD8+ T-cells from the mLN of the immunized mice. CD4+ and CD8+ T-cell population of the mice immunized with MT4 strain found to be comparable with the mice immunized with MT5 strain. Hence, it concludes that the MT4 strain retains its ability to induce the classical innate and adaptive immune response even after a strong attenuation. Therefore, we propose that incorporating additional “safety” features such as the deletion of mig-14 can be of a general interest for the design of new super live attenuated S. Typhimurium strain. This attenuated strain could also be used for developing the recombinant vaccine against other enteric pathogens.