c. jejuni strain 81-176  (Charles River Laboratories)

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

Journal:

Article Title: Campylobacter jejuni Colonization of Mice with Limited Enteric Flora

doi: 10.1128/IAI.01094-05

Figure Lengend Snippet: Time course of GI tract colonization by C. jejuni in wild-type normal flora mice, LF mice, and LF-SCID mice. Groups of 16 normal flora mice of each strain and groups of 19 LF and LF-SCID mice were intragastrically inoculated with approximately 5 × 108 CFU of wild-type strain 81-176. Stool and GI tract tissue were collected and processed as described in Materials and Methods. Fresh stool was collected for all mice at the indicated time points; C. jejuni recovered from stool is denoted by open symbols. Large intestine (cecum and colon) tissue was collected from four to eight mice euthanized at the indicated time points, and C. jejuni recovered is represented by closed symbols. C3H mice are denoted by ovals, triangles, and diamonds; BALB/c mice are denoted by squares. Error bars represent the SEM and may be obscured by symbols denoting the mean value. Pairwise differences in colonization levels between normal flora, LF, and LF-SCID C3H mice at day 7 were statistically significant (defined as P < 0.05, with P values ranging from 0.0015 to 0.040) except for large intestine tissue of LF versus SCID mice (P = 0.095). At day 28, the pairwise differences in colonization were statistically significant (P values ranging from 0.0019 to 0.034) except for tissue and stool of normal flora versus LF mice (P = 0.37 and 0.05, respectively). Statistical analysis of colonization levels was performed with the unpaired two-sample t test with unequal variances.

Article Snippet: As shown in Fig. , colonization establishment was inconsistent (∼50%) in C3H and BALB/c mice (Charles River) following an intragastric inoculum of 5 × 10 8 CFU of C. jejuni strain 81-176.

Techniques:

Journal:

Article Title: Campylobacter jejuni Colonization of Mice with Limited Enteric Flora

doi: 10.1128/IAI.01094-05

Figure Lengend Snippet: Persistence of GI tract colonization by C. jejuni in LF and LF-SCID mice. Results are for a survey of C. jejuni colonization persistence in groups of four to eight LF or LF-SCID mice, as described for Fig. ​Fig.11.

Article Snippet: As shown in Fig. , colonization establishment was inconsistent (∼50%) in C3H and BALB/c mice (Charles River) following an intragastric inoculum of 5 × 10 8 CFU of C. jejuni strain 81-176.

Techniques:

Journal:

Article Title: Campylobacter jejuni Colonization of Mice with Limited Enteric Flora

doi: 10.1128/IAI.01094-05

Figure Lengend Snippet: Histopathology of large intestine tissue from LF and LF-SCID mice 28 days postinoculation. (A) Only mild inflammation was detected in the lamina propria of LF mice colonized by C. jejuni, with preservation of the normal tissue architecture. (B to E) Severe inflammation was evident in the mucosa and submucosa of the cecum and colon tissue of similarly colonized SCID mice, with marked inflammatory infiltrate, including ulceration (B), epithelial hyperplasia and loss of goblet cells (C), and edema and architectural distortion (D). (E) Cryptitis was also frequently appreciated. Magnification, ×200.

Article Snippet: As shown in Fig. , colonization establishment was inconsistent (∼50%) in C3H and BALB/c mice (Charles River) following an intragastric inoculum of 5 × 10 8 CFU of C. jejuni strain 81-176.

Techniques: Histopathology, Preserving

Journal:

Article Title: Campylobacter jejuni Colonization of Mice with Limited Enteric Flora

doi: 10.1128/IAI.01094-05

Figure Lengend Snippet: Histopathology of large intestine tissue from LF-SCID mice 7 days postinoculation. (A) Control cecum tissue from an uninfected SCID mouse revealed normal crypt architecture with only a small number of inflammatory cells in the lamina propria. Magnification, ×100. (B to D) In contrast, severe inflammation of the mucosa and submucosa in the cecum and colon was observed in LF-SCID mice 7 days after inoculation with C. jejuni strain 81-176, with architectural distortion, hyperplasia, and edema evident (B). (C) An ulcerated region of the mucosa revealed an intense inflammatory infiltrate with accompanying hemorrhage. Magnification in panels B and C, ×100. (D) A ×200 magnification confirmed that the infiltrate was composed primarily of neutrophils, although mononuclear cells were also present.

Article Snippet: As shown in Fig. , colonization establishment was inconsistent (∼50%) in C3H and BALB/c mice (Charles River) following an intragastric inoculum of 5 × 10 8 CFU of C. jejuni strain 81-176.

Techniques: Histopathology

Journal:

Article Title: Campylobacter jejuni Colonization of Mice with Limited Enteric Flora

doi: 10.1128/IAI.01094-05

Figure Lengend Snippet: C. jejuni mutants defective in motility or chemotaxis are unable to colonize the GI tract of LF mice. Insertion-deletion mutations were made in motB, cheAWY, and fliI in both the 81-176 (A) and 11168 (B) backgrounds. Groups of four to eight mice were inoculated with 102 to 103 CFU of wild-type or mutant C. jejuni, and stool and tissue samples were collected at the indicated days p.i., with half the group sacrificed at day 7 to assess intestinal colonization. In striking contrast to both wild-type strains, all three mutants failed to colonize the LF C3H mice as demonstrated by analysis of fresh stool and large intestine tissue. Error bars represent the SEM. The limit of detection is 100 CFU/g of stool or tissue. Colonization-level differences between wild-type 81-176 and each of the mutants at day 7 for both stool and large intestine tissue were statistically significant (P values ranging from 0.0014 to 0.0467). For later time points and for analysis in the 11168 background, statistical significance was not achieved despite a lack of colonization by chemotaxis and motility mutants (within the limits of detection), due to smaller sample sizes. Statistical analysis of colonization levels was performed with the unpaired two-sample t test with unequal variances.

Article Snippet: As shown in Fig. , colonization establishment was inconsistent (∼50%) in C3H and BALB/c mice (Charles River) following an intragastric inoculum of 5 × 10 8 CFU of C. jejuni strain 81-176.

Techniques: Chemotaxis Assay, Mutagenesis