Motility ring diameters of the wild type 14028s strain and a nega

Motility ring diameters of the wild type 14028s strain and a negative control (fliA) were compared to preA, preB, and preAB strains. Signaling molecules were tested for see more possible affects on motility. (A) 20 μM AI-2 (dark bars) or an equal volume of buffer (light bars) GSK1838705A chemical structure were added to the medium. (B) 50 μM epinephrine (dissolved in acidified water, dark bars) or an equal volume of acidified water (light bars)

was added to medium. An asterisk (*) denotes statistical significance with a p-value < 0.02 as determined with a student t-test. The asterisk in (A) is in comparision of ΔpreB to the wild type strain. Overexpression of mdaB [16] and mutation of preB (ygiY; [17]) were previously shown to affect drug resistance in E. coli and oxidative stress response in Helicobacter spp. [18–20]. In addition, catalase genes appear PreA-regulated (Additional check details file 1). preAB mutant strains were therefore analyzed for resistance

to various chemicals and antibiotics, including nalidixic acid, pyrazinoic acid, H2O2, paraquat, adriamycin, and tetracycline. None of the mutants showed increased sensitivity when compared to the wild type strain (data not shown). To determine if the PreA/PreB system affects virulence, mutant and wild type strains were perorally inoculated in mice and mortality was recorded over two weeks. The preA mutant showed no virulence defect while mice infected with the preAB strain showed a consistent two day delay in mortality, but eventually all mice succumbed to infection (Fig. 4). The preAB mutant strain also demonstrated a consistent competition G protein-coupled receptor kinase infection defect (competitive index: spleen, 0.344; liver, 0.326) when co-inoculated by oral gavage with the wild type strain, which was not observed with strains containing single mutations in preA or preB (data not shown). Thus, the PreA/PreB TCS has a slight but reproducible effect on virulence in mice. Figure 4 Female BALB/c mice were inoculated with 10 6 bacteria via oral gavage and animals were monitored over a period of 13 days. Given

that invasion of the small intestine is a prerequisite to systemic infection upon oral inoculation, we also evaluated the ability of various preA/preB mutants to invade HeLa cells grown in vitro. Again, the response regulator (preA) mutant did not show any defect in invasion of HeLa cells. The preB strain showed a marginal and non-significant reduction in invasion upon 2 hours co-cultivation at a MOI = 100 (invasion ~80% of wild type), while a larger defect was observed for the preAB double mutant (~30% of WT) (Fig. 5). Therefore, the PreA/PreB TCS has a direct or indirect effect on host cell invasion. Figure 5 HeLa cell invasion assays were performed for wild type, prgH (negative control), preA, preB, and preAB strains. HeLa cells were grown to monolayer in DMEM with 10% FBS at 37°C and 5% CO2. Cells were then infected with bacteria at an MOI of 100 in 24-well plates. Data is presented as percent of wild type CFUs.

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