However, the yqiC mutant showed complete attenuation in virulence, as all mice QNZ in vitro infected with this strain
survived along the 30-day period of the experiment. The yqiC gene provided in trans fully complemented Compound C concentration the 14028 ΔyqiC::CAT phenotype, causing 100% mice death by day 19. In addition, we determined the LD50 of S. Typhimurium ATCC 14028 and 14028 ΔyqiC::CAT in mice inoculated intraperitoneally as described in Materials and methods. A dramatic increase in the LD50 was observed in the yqiC defective strain (>5 × 105 CFU), as compared with the WT (10-100 CFU) (Table 1). Together, these results clearly show that YqiC is required for S. Typhimurium virulence in the murine infection model. Figure 7 yqiC is essential for virulence in mice. BALB/c mice were orally infected with 1 × 105 CFU of wild-type S. Typhimurium ATCC 14028, 14028 ΔyqiC::CAT or 14028 ΔyqiC::CAT + pBBR-yqiC. The survival of infected mice over time is shown. Table 1 Determination of LD50 of S.Typhimurium strains in mice. Number of dead mice/Number of infected mice (Mean of days to death) Dose (CFU/mouse) S . Typhimurium ATCC 14028 S . Typhimurium 14028 Δ yqiC ::CAT 1 × 101 3/7 (6) 0/7 1 × 102 7/7 (6.7) 0/7 1 × 103 6/6 (5.5) 0/6 1 × 104 6/6 (4.5) 0/6 1 × 105 6/6 (4) 0/6 Groups of the indicated number of mice were inoculated
intraperitoneally with different doses Panobinostat of S. Typhimurium ATCC or S. Typhimurium strain and survival was recorded for up to 30 days. Discussion In this work we have characterized the YqiC protein of S. Typhimurium. YqiC shares common structural and biochemical
characteristics with its previously reported Coproporphyrinogen III oxidase orthologous BMFP protein of Brucella abortus [9], although these proteins share only 22% of sequence identity and Brucella spp and Salmonella are phylogenetically distant bacteria. The common structural characteristics between YqiC and BMFP, namely high alpha helix content, coiled coil C-terminal and amphipathic alpha helix N-terminus, are also predicted by bioinformatics analysis for other proteins of the COG 2960 (such as those encoded by Escherichia coli, Shewanella oneidensis, Legionella pneumophila, Xanthomonas campestris, Pseudomonas aeruginosa, Bordetella pertussis, Agrobacterium tumefaciens, Sinorhizobium meliloti and Rhodopseudomonas palustris). This structural conservation strongly suggests a common function for the members of this COG. In addition, we demonstrated that YqiC has membrane fusogenic activity, like BMFP and other trimeric coiled-coil and/or amphipathic proteins [11, 12]. This activity is higher at acidic pH. A similar fusogenic activity at low pH was observed for B. abortus BMFP (unpublished data). The fusogenic activity could be relevant as many processes that involve bacterial or host cell membrane fusion events are important for pathogenic bacteria to successfully establish host infection. In this regard, both S. Typhimurium and B.