Supplementary figures: S1- Evaluation of attenuation profile of mig14::aphT mutant in comparison to wild-type strain of Salmonella Typhimurium . Methodology: The group of streptomycin pretreated mice (n=5) was infected with mixed inoculum of mig14::aphT mutant and wild-type Salmonella Typhimurium st

Methodology: The group of streptomycin pretreated mice (n=5) was infected with mixed inoculum of mig14::aphT mutant and wild-type Salmonella Typhimurium strain (cfu 10 5 each strain). Mice were kept in IVC under observation for 4 days post infection. Host tissues were excised, homogenised and pleated on streptomycin (Sm; for collective cfu count) and streptomycin-kanamycin (Sm-Km; for mig14::aphT mutant cfu count) supplemented Mac Conkey agar plates. The cfu count for wild-type strain was obtained by subtracting mig14::aphT counts from the collective counts obtained from respective tissue. Obtained individual counts were compared to get competitive index of mig14::aphT single mutant in comparison to wild-type Salmonella Typhimurium strain.

: Competitive index profile of mig-14::aphT mutant when compared against wild-type strain. n.s.= not significant; =P<0.05) Analysis: We tested the competitive index of mig14 mutant in a group of five C57BL/6 mice. Bacterial strains were grown separately and mixed at the time of infection to prepare an inoculum mixture. A significant decrease (P<0.05) in the CI values for mLN, Liver and Spleen colonization was observed for mig14::aphT mutant when compared against wild-type S. Typhimurium strain. This result could possibly explain for the further attenuation of the developed double mutant (ssaV, mig14) when subjected to immunocompromised mice.

Infection profile of mig14::aphT mutant in comparison to wild-type strain of Salmonella
Typhimurium .
Methodology: Groups of streptomycin pretreated mice (n=5) were infected with wild-type strain, mig14::aphT mutant strain and mig14-complemented single mutant strain (cfu 10 5 each strain). {The mig14 wild-type allele was cloned into NcoI-XbaI sites of plasmid pCH112 having pBAD backbone and arabinose inducible promoter with cloned segment of hilA gene. In this plasmid, the hilA gene was replaced with mig-14 allele}. Mice were kept in IVC under observation for 4 days post infection. Host tissues were excised, homogenised and pleated on agr plates supplemented with suitable antibiotics. The cecal sections from each mice group were HE stained and analyzed. Analysis: The mig14::aphT mutant was compared against the wild-type SB300 strain (WT) for its systemic colonization and ability to cause cecal inflammation. Groups of streptomycin pretreated C57BL/6 mice (n=5 each) were infected with 10 5 cfu of wild-type S.Typhimurium WT, mig14::aphT and mig14-complemented strain. The bacterial densities in fecal shedding (day 1 post infection) and the organ loads (day 4 p.i.) were assessed. We observed no significant changes in the ability of mig14::aphT mutant strain in terms of cecal colonization when compared with wild-type Salmonella typhimurium strain. However, a significant reduction in the systemic infiltration was noticed in the mice infected with mig14::aphT mutant as compared to the WT infected mice group. This reduction was restored to much extent on complementing wild-type allele of mig14 to its respective mutant (mig14::aphT). The decrease in the ability of mig14::aphT mutant to colonize systemically was not that sharp as of ssaV mutant MT5 ( Fig.1) but was noticeable, this provided enough hint for adding an additional mutation of mig14 on ssaV mutant to possibly attain complete attenuation in immunocompromised mice group.

S3-Flowcytometric analysis of T-cell population after Salmonella infection.
Methodology: Analysis of the T-cell population was performed by collecting the mesenteric lymph node (mLN) of the vaccinated mice in 500 µl of Rose Park Memorial Institute (RPMI) medium with 10% fetal calf serum (FCS). Collected mLN were homogenized and centrifuged at 1200 rpm. The pellet was suspended in 1ml RPMI with 10% FCS. About 10 6 cells were  Analysis: An ideal live-attenuated vaccine strain should develop enough immunogenicity on reaching the host lymphoid tissues. The T-cytotoxic and T-helper cells play a critical role in the clearance of Salmonella as well as in the production of specific antibodies during the late phase of infection. We analyzed the effect of MT5 and MT4 strains on T-cell population of the mesenteric lymph node. We quantified the CD4 + and CD8 + T-cell population recovered from the mLN of the vaccinated mice after day 30 p.v. The T-cell population were analyzed by flowcytometry and found to be almost equally populated in the vaccinated mice but significantly more in comparison to the PBS treated mice. This clearly indicates that, the MT4 strain has an ability to colonize and induce T-cell mediated innate and adaptive immune response in the wild-type C57BL/6 mice.

S4-Luminal and serum specific antibody responses in mice immunized with MT5 and MT4.
Methodology: The quantification of antibody titer by Fluorescence-activated cell sorter (FACS) was performed as per further description. A single bacterial colony was inoculated in 5ml of LB broth at 37 o C without shaking. 1ml of the overnight culture was gently pelleted and washed twice with sterile PBS (1% BSA, 0.05% sodium azide). Finally the pellet was resuspended in PBS to attain a bacterial density of 10 7 CFU/ml. The serum sample was  Analysis: To validate the immunogenic potency of MT4, the antibody titers from the animals vaccinated with MT4 and MT5 strains was analyzed. Serum and gut wash samples were collected at the end of the day 30 p.v. procedure and antibody responses were quantified via FACS and Western blot analysis. This experiment relies on the specific antibody binding to specific antigens on the surface of the bacterium (wild-type S. Typhimurium) as compared to a bacterium of different serovar (S. Enteritidis). The intestinal wash and serum samples from mice vaccinated with either MT5 or with MT4 exhibited equivalent responses of Salmonella specific serum IgG and luminal secretory IgA.