Typhimurium strains were highly attenuated and conferred protection from further challenges of wild-type S. Typhimurium by eliciting O-antigen specific serum IgG and secretory Crenolanib mw IgA in C57BL/6 mice [34–36]. In a recent study, the ssaV mutant of S. Typhimurium was found to be virulent in immune compromised C57BL/6 mice devoid of Nos2 and Il-10 gene [37]. These two mice strains were used as they lack key elements

of the antibacterial defense like the inducible nitric oxide (NO) synthase, a reactive oxygen species generating enzyme and interleukin-10 gene [38]. In this study, we have also used CD40L KO mice to screen the attenuation of proposed vaccine strain. This particular mouse model is used as it is partially immunocompromised in terms of generation of different class of antibodies. Virulence of TTSS-2 deficient S. Typhimurium in immunocompromised mice unveils the role of other factors favoring the replication and long-term survival of S. Typhimurium in host tissues. Mig-14, an antimicrobial peptide resistance protein, is one such important factor that supports the long-term persistence of Salmonella in the macrophages [39]. Mig-14 protein binds to the anti-microbial peptides like selleck products CRAMPS to protect Salmonella from antimicrobial peptides

[40]. The EPZ-6438 clinical trial presence of Mig-14 in the periplasmic localization inhibits the entry of antimicrobial peptides to the cytoplasm of the bacterium, eventually making macrophage a good niche for Salmonella to replicate check details and survive. This study proposes a diverse role for mig-14 in the survival of TTSS-2 deficient Salmonella in immunocompromised mice like Nos2 −/− , Il-10 −/− and CD40L −/− and explores the possible potential of S. Typhimurium ssaV and mig-14 double mutant as a safe vaccine carrier strain. Methods Bacterial strains and plasmids Streptomycin resistant S. Typhimurium

SB300 and Salmonella Enteritidis P125109 (S. Enteritidis) strains were taken as the wild-type controls [41, 42]. Mutants MT5 (SB300; ΔssaV) and MT4 (SB300; ΔssaV, Δmig-14) were generated by lambda red-mediated recombinase process [43]. Briefly, the host bacterial strain to be mutated was transformed with plasmid pKD46 and induced with arabinose (10 mM). The kanamycin open reading frame was PCR-amplified from template plasmid pKD4 using gene specific knockout primers (Table 1). The cassette was introduced into host bacterial genome with the help of Exo, Bet and Gam proteins from induced pKD46 plasmid of host bacterial strain. The positive mutants were selected on LB agar plates supplemented with kanamycin (50 μg/ml) and mutation in the target gene was confirmed using gene specific confirmatory primers in combination with respective forward knock-out primer (Table 1). Later, the antibiotic cassette was flipped by plasmid pCP20 [43]. An ampicillin resistant plasmid (pM973) was used to maintain the ampicillin resistant trait in wild-type strain (SB300) while challenging vaccinated mice groups with wild-type S. Typhimurium [44].