WT and arcA mutant Salmonella were grown in LB-MOPS-X broth to stationary phase for about 20 h.
check details For intraperitoneal (i.p.) challenge, two groups of five mice per strain (WT and arcA mutant) were inoculated with 250 CFU in 500 μl PBS/mouse. Mortality was scored over a 15- to 30-day period. Competitive infection assays were carried-out as described [33] with modifications. The strains were separately grown overnight in LB broth at 37°C with shaking at 200 rpm. Tetracycline (10 μg/ml) was used to propagate and isolate the arcA mutant. Bacterial (i. e.: WT and arcA mutant) cultures were diluted in phosphate-buffered saline (PBS) and mixed to produce a 1:1 inoculum ratio. Groups of mice were infected either i.p. or orally (p.o.). Prior to oral infection, food and water were withheld from the mice for 4 h and the bacterial cocktail was administered to the mice by allowing them to drink 20 μl from the end Tamoxifen research buy of a pipette tip. On day 4 or day 6 after i.p. or p.o. infection, respectively, mice were euthanized and mesenteric lymph nodes (MLN), liver and spleen collected for bacterial enumeration. The tissues were homogenized in sterile PBS and 10-fold serial dilutions were plated
on LB agar medium with or without 10 μg/mL tetracycline to distinguish the WT (Tets) from the arcA mutant (Tetr). The number of CFUs of S. Typhimurium 14028 s per organ was calculated by subtracting the number of CFU/ml on the LB-Tet plates from the number of CFU/ml on the corresponding LB plates. The competitive index (CI) was calculated as the ratio of the CFU of arcA mutant to the CFU of the WT strain recovered from the spleen, liver, and mesenteric lymph nodes (i.e.; CI = [arcA mutant/WT]output/[arcA mutant/WT]input). Results Bacterial growth kinetics The growth kinetics of the WT and the arcA mutant strains were determined under anaerobic conditions in LB-MOPS-X. The arcA mutant strain grew at a slower rate than the WT strain. The doubling-times of the WT and arcA mutant were 37.0 ± 0.4 and 55.4 ± 0.1 min under anaerobic
conditions. Due to the difference in the doubling-times of the two strains, cells used for RNA isolation and subsequent transcriptome profiling were allowed to grow for an equal number of generations (~five generations: OD600 = 0.30-0.35) instead of an equal length of time. Anaerobic transcriptome profiling Anidulafungin (LY303366) Out of 4,579 genes, the two-tailed Student’s t test, produced a set of 2,026 coding sequences showing a significant difference between the arcA mutant and the WT (p < 0.05). We restricted the analyses to only include highly affected genes (i.e., has a ratio ≥ 2.5-fold) as previously described [20]. Under this constraint, 392 genes were differentially expressed in the arcA mutant relative to the WT and, therefore, regulated directly or indirectly by ArcA. Of these, 147 genes were up-regulated and 245 genes were down-regulated (Additional file 1: Table S1).