Journal: PLoS ONE

Article Title: Campylobacter jejuni pdxA Affects Flagellum-Mediated Motility to Alter Host Colonization

doi: 10.1371/journal.pone.0070418

Figure Lengend Snippet: Bacterial strains and plasmids used in this study.

Article Snippet: C. jejuni strain 81–176 was grown using routine methods in Mueller-Hinton (MH) broth or on MH agar (Becton-Dickinson, Franklin Lakes, NJ, USA) at 37°C for 24 h in a humidified CO 2 AnaeroPack-Microaero gas system (Mitsubishi Gas Chemicals, Tokyo, Japan).

Techniques: Mutagenesis, Plasmid Preparation, Homologous Recombination

Journal: PLoS ONE

Article Title: Campylobacter jejuni pdxA Affects Flagellum-Mediated Motility to Alter Host Colonization

doi: 10.1371/journal.pone.0070418

Figure Lengend Snippet: (A) A scheme for the PLP production pathway (right box) in C. jejuni in relation to Pse biosynthesis (left box) is illustrated based on in silico pathway analysis performed using PATRIC ( http://patricbrc.vbi.vt.edu/portal/portal/patric/Home ). (B) The pdxA mutant produced no PLP. The C. jejuni 81–176 WT, pdxA mutant, and the complemented strains were grown in 10ml of MH broth to an OD 600 of 0.60. The suspensions were then homogenized, serially diluted, and subjected to ELISA to quantify the amounts of PLP (μg 10 ml −1 ). The data show the mean +/− standard deviations from three independent assays.

Article Snippet: C. jejuni strain 81–176 was grown using routine methods in Mueller-Hinton (MH) broth or on MH agar (Becton-Dickinson, Franklin Lakes, NJ, USA) at 37°C for 24 h in a humidified CO 2 AnaeroPack-Microaero gas system (Mitsubishi Gas Chemicals, Tokyo, Japan).

Techniques: In Silico, Mutagenesis, Produced, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Campylobacter jejuni pdxA Affects Flagellum-Mediated Motility to Alter Host Colonization

doi: 10.1371/journal.pone.0070418

Figure Lengend Snippet: (A) The pdxA mutant shows less glycosylation of FlaA. SDS-PAGE and western blotting were conducted to detect the C. jejuni FlaA protein. Crude extracts and subcellular (cytoplasmic and membrane) fractions were extracted from C. jejuni and visualized using CBB staining in an SDS-polyacrylamide gel (left panel). Western blot analyses were simultaneously performed to detect the FlaA protein (arrow, right panel). (B) The pdxA mutant shows reduced Pse production. The left panel shows an extracted ion chromatogram at m / z 441.0–461.0 obtained through SIM of DMB-labeled Pse from the WT and pdxA mutant strains (arrowed). The extracted ion chromatogram of blank sample (fresh MH broth) was simultaneously subjected to confirm the absence of Pse. AA, peak area in arbitrary units. Each ion signal is expressed as a relative percentage of the WT-derived sample (set to 100%) from two independent tests (right panel). MS n data were shown in Fig. S1, S2, S3. (C) The disruption of the pdxA gene impairs motility of C. jejuni . The WT, pdxA mutant, pdxA -complemented ( pdxA −/+), and flaA mutant (flaA-) strains were spotted and incubated onto 0.4% soft agar. Scale bars represent 3 mm. The motility of pdxA mutant was also assayed in the supplementation of 10 mg l −1 of PLP (pdxA− + PLP). (D) The pdxA mutant is aflagellated. Electron micrographs of the C. jejuni WT, pdxA mutant with or without supplementation of PLP (10 mg l −1 ), pdxA - complemented strains. The scale bars represent 1 μm.

Article Snippet: C. jejuni strain 81–176 was grown using routine methods in Mueller-Hinton (MH) broth or on MH agar (Becton-Dickinson, Franklin Lakes, NJ, USA) at 37°C for 24 h in a humidified CO 2 AnaeroPack-Microaero gas system (Mitsubishi Gas Chemicals, Tokyo, Japan).

Techniques: Mutagenesis, Glycoproteomics, SDS Page, Western Blot, Membrane, Staining, Labeling, Derivative Assay, Disruption, Incubation

Journal: PLoS ONE

Article Title: Campylobacter jejuni pdxA Affects Flagellum-Mediated Motility to Alter Host Colonization

doi: 10.1371/journal.pone.0070418

Figure Lengend Snippet: Representative metabolites that are altered between the C. jejuni WT and pdxA mutant strains.

Article Snippet: C. jejuni strain 81–176 was grown using routine methods in Mueller-Hinton (MH) broth or on MH agar (Becton-Dickinson, Franklin Lakes, NJ, USA) at 37°C for 24 h in a humidified CO 2 AnaeroPack-Microaero gas system (Mitsubishi Gas Chemicals, Tokyo, Japan).

Techniques: Mutagenesis

Journal: PLoS ONE

Article Title: Campylobacter jejuni pdxA Affects Flagellum-Mediated Motility to Alter Host Colonization

doi: 10.1371/journal.pone.0070418

Figure Lengend Snippet: (A) Growth curves of C. jejuni 81–176 WT, pdxA−, and the complemented mutant strains in MH broth not supplemented (left panel) or supplemented (right panel) with PLP (10 mg l −1 ). (B) Intracellular ATP levels of C. jejuni 81–176 WT, pdxA−, and the complemented mutant strains. ATP contents of four serial dilutions of the bacteria (shown as CFU 100 μl −1 ) under investigation were measured. The results are shown as means ± SD of data from triplicate wells of a representative experiment. (C) Focused dynamics of the C. jejuni TCA-cycle pathway. The pathway, the relative mean concentrations of the related metabolites in the WT (blue bars) and the pdxA mutant (red bars) strains, and the genes associated with the enzymatic conversion of each metabolite were illustrated with the PATRIC pathway analysis program.

Article Snippet: C. jejuni strain 81–176 was grown using routine methods in Mueller-Hinton (MH) broth or on MH agar (Becton-Dickinson, Franklin Lakes, NJ, USA) at 37°C for 24 h in a humidified CO 2 AnaeroPack-Microaero gas system (Mitsubishi Gas Chemicals, Tokyo, Japan).

Techniques: Mutagenesis, Bacteria

Journal: PLoS ONE

Article Title: Campylobacter jejuni pdxA Affects Flagellum-Mediated Motility to Alter Host Colonization

doi: 10.1371/journal.pone.0070418

Figure Lengend Snippet: (A) INT407 cells were infected for 1 h with the C. jejuni WT, pdxA−, pdxA−/+, and flaA− strains. The number of cell-adherent bacteria was measured by counting the plates after washing three times with PBS. (B) ERK1/2 activation upon infection. Western blotting was performed to detect the levels of phosphorylated and total ERK1/2 in the lysates from infected cells. (C) IL-8 production in INT407 cells was measured at 4 h and 16 h p.i. via ELISA. The data are presented in sections A and C as the mean values ± standard deviations from samples run in duplicate in at least three experiments. (D) Disruption of the pdxA gene reduces the colonization of the chicken cecum by C. jejuni . Groups of 14-day-old chickens (n = 10 per group) were orally inoculated with approximately 3×10 7 CFU of WT or pdxA mutant C. jejuni . At 1 week and 4 weeks p.i. , the ceca were aseptically removed from the infected animals (n = 5 for each time point) and homogenized. Serial dilutions of the suspensions were plated on mCCDA agar to count CFU numbers. The closed diamonds and open circles represent the numbers of WT and pdxA mutant CFUs recovered from the animals, respectively.

Article Snippet: C. jejuni strain 81–176 was grown using routine methods in Mueller-Hinton (MH) broth or on MH agar (Becton-Dickinson, Franklin Lakes, NJ, USA) at 37°C for 24 h in a humidified CO 2 AnaeroPack-Microaero gas system (Mitsubishi Gas Chemicals, Tokyo, Japan).

Techniques: Infection, Bacteria, Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Disruption, Mutagenesis