Journal: bioRxiv

Article Title: Increased response to immune checkpoint inhibitors with dietary methionine restriction

doi: 10.1101/2023.04.05.535695

Figure Lengend Snippet: STING was increased with MR at the gene and protein expression levels (A and insert, T-test with mean and SEM). Inhibiting STING with C-176 led to a reduction in the increase in HLAA gene expression (B) and in its cell surface representation (C). CD274 gene expression was increased by C-176 and did not increase further with MR (D). 2-way ANOVA with mean and SEM.

Article Snippet: For cGAS-STING inhibition, the STING inhibitor C-176 was used at a final concentration of 5 μM (Tocris Bioscience (Bio-Techne), Bristol, UK) and the cGAS inhibitor RU.521 was used at a concentration of 1 μM (MedChemExpress, Monmouth Junction, NJ) for 24 hours in both cases.

Techniques: Expressing, Gene Expression

Journal: Biomolecules

Article Title: Cj0683 Is a Competence Protein Essential for Efficient Initialization of DNA Uptake in Campylobacter jejuni

doi: 10.3390/biom13030514

Figure Lengend Snippet: Visualization of import of multiple DNA molecules within one DNA uptake location. Fluorescein (F) and Cy3 labelled DNA was either added for 30 min to separate C. jejuni 81–176 suspensions (( A ), n ≥ 5) or in parallel to one bacterial competent culture (( B , C ), n = 4) and the fraction of cells with F and/or Cy3 fluorescent foci were analyzed. In ( A ) the fraction of active cells relative to the total number of bacteria is shown, in ( B ) the fraction of cells with fluorescent foci relative to the overall fraction of competent cells is depicted. Green bars, cells with only fluorescein labelled DNA foci; orange bars, cells with only Cy3 labelled DNA foci; yellow bar, cells with both fluorescein and Cy3 DNA in one/or two single focus/foci; green/orange hatched bar, cells with at least two separate foci, with either fluorescein or Cy3 DNA. ( C ), overlay image of differential phase contrast (DIC), Cy3 and F channel. Green, fluorescein focus; orange, Cy3 focus.

Article Snippet: C. jejuni 81–176 [ ] from −80 °C stock cultures (MAST Group Ltd., Bootle, UK) was grown on Columbia blood agar plates supplemented with 5% defibrinated sheep blood (ColbA, Oxoid, Thermo Fisher Scientific Inc., Waltham, MA, USA) under microaerobic conditions (5% O 2 , 10% CO 2 , rest N 2 ; Binder, Tuttlingen, Germany).

Techniques: Bacteria

Journal: Biomolecules

Article Title: Cj0683 Is a Competence Protein Essential for Efficient Initialization of DNA Uptake in Campylobacter jejuni

doi: 10.3390/biom13030514

Figure Lengend Snippet: The fraction of competent C. jejuni and of DNA uptake locations per cell increased in time of contact with fluorescent DNA. Competent C. jejuni 81–176 were exposed to fluorescent DNA for different time periods and the fraction of cells with active DNA uptake was monitored in comparison to 10 min DNA uptake in H. pylori J99 ( A ). Proportion of cells with distinct number of DNA uptake locations ( B ). In ( B ), fraction of cells relative to total amount of competent cells are depicted. Mean and standard deviations were derived from five independent experiments.

Article Snippet: C. jejuni 81–176 [ ] from −80 °C stock cultures (MAST Group Ltd., Bootle, UK) was grown on Columbia blood agar plates supplemented with 5% defibrinated sheep blood (ColbA, Oxoid, Thermo Fisher Scientific Inc., Waltham, MA, USA) under microaerobic conditions (5% O 2 , 10% CO 2 , rest N 2 ; Binder, Tuttlingen, Germany).

Techniques: Comparison, Derivative Assay

Journal: Biomolecules

Article Title: Cj0683 Is a Competence Protein Essential for Efficient Initialization of DNA Uptake in Campylobacter jejuni

doi: 10.3390/biom13030514

Figure Lengend Snippet: The median amount of DNA within single foci of C. jejuni was constant over time and ~4-fold less than in H. pylori . C. jejuni 81–176 and H. pylori J99 were incubated with the same batch of fluorescent genomic C. jejuni DNA labelled with Cy3. Uptake was followed in time for C. jejuni and compared to 10 min uptake in H. pylori . ( A ) Analysis of DNA foci in C. jejuni (red; light to dark corresponds to incubation times) and in H. pylori (blue), sorted according to fluorescence intensity. ( B ) ( C. jejuni ) and ( C ) ( H. pylori ) distribution of fluorescence intensities as boxplots; the boxplot length corresponds to the interquartile range (IQR; 50%) of data, the horizontal bar indicates the median value; whiskers represent 1.5 × IQR or the maximum/minimum value of the dataset; dots, outliers. Upper panel, merged DIC and Cy3-channel example images of C. jejuni and H. pylori cells after import of DNA. Note that Cy3 exposure times were 25 ms for H. pylori and 100 ms for C. jejuni . Scale bar of 2 µm. One representative experiment (out of three) is shown.

Article Snippet: C. jejuni 81–176 [ ] from −80 °C stock cultures (MAST Group Ltd., Bootle, UK) was grown on Columbia blood agar plates supplemented with 5% defibrinated sheep blood (ColbA, Oxoid, Thermo Fisher Scientific Inc., Waltham, MA, USA) under microaerobic conditions (5% O 2 , 10% CO 2 , rest N 2 ; Binder, Tuttlingen, Germany).

Techniques: Incubation, Fluorescence

Journal: Biomolecules

Article Title: Cj0683 Is a Competence Protein Essential for Efficient Initialization of DNA Uptake in Campylobacter jejuni

doi: 10.3390/biom13030514

Figure Lengend Snippet: Biofilm formation is independent of active DNA transport or natural transformation in C. jejuni . Upon growth in microtiter plates for 72 h and suitable washing of unbound bacteria, crystal violet staining indicated the intensity of biofilm formation, measured as absorbance at 595 nm. C. jejuni 81–176 (wt) and its isogenic mutants Δ pilQ , Δ comE , Δ comEC and the respective complemented strains were tested, using Pseudomonas aeruginosa as reference for a typical biofilm forming bacterium. Horizontal bar, mean of ≥3 independent experiments; n.s., not significant; *, p < 0.05; ***, p < 0.001.

Article Snippet: C. jejuni 81–176 [ ] from −80 °C stock cultures (MAST Group Ltd., Bootle, UK) was grown on Columbia blood agar plates supplemented with 5% defibrinated sheep blood (ColbA, Oxoid, Thermo Fisher Scientific Inc., Waltham, MA, USA) under microaerobic conditions (5% O 2 , 10% CO 2 , rest N 2 ; Binder, Tuttlingen, Germany).

Techniques: Transformation Assay, Bacteria, Staining

Journal: Biomolecules

Article Title: Cj0683 Is a Competence Protein Essential for Efficient Initialization of DNA Uptake in Campylobacter jejuni

doi: 10.3390/biom13030514

Figure Lengend Snippet: Cj0683 plays a pivotal role for DNA uptake into the periplasm of C. jejuni . DNA uptake ( A ) and transformation rate ( B ) in 81–176 (wt) and the mutant strains Δ cj0683, Δ cj0683-compl and Δ cj0683 Δ comE . ( A ) Cells were either incubated with fluorescein- (green bars, except for Δ cj0683 Δ comE ) or Cy3-labelled (orange bars) genomic DNA of 81–176 for 30 min. ( B ) Transformation rate was determined using unlabeled genomic DNA containing streptomycin resistance. Error bars indicate standard deviation ( n ≥ 4).

Article Snippet: C. jejuni 81–176 [ ] from −80 °C stock cultures (MAST Group Ltd., Bootle, UK) was grown on Columbia blood agar plates supplemented with 5% defibrinated sheep blood (ColbA, Oxoid, Thermo Fisher Scientific Inc., Waltham, MA, USA) under microaerobic conditions (5% O 2 , 10% CO 2 , rest N 2 ; Binder, Tuttlingen, Germany).

Techniques: Transformation Assay, Mutagenesis, Incubation, Standard Deviation

Journal: BMC Microbiology

Article Title: Roles of RpoN in the resistance of Campylobacter jejuni under various stress conditions

doi: 10.1186/1471-2180-11-207

Figure Lengend Snippet: Growth of the rpoN mutant under different aeration conditions . The C. jejuni strains were microaerobically cultured in MH broth at 42°C with shaking at 180 rpm (A) and without shaking (B). At the described time intervals, the optical density at 600 nm was measured, and viable cells were counted in static culture condition (without shaking) by plating serially-diluted C. jejuni cultures on MH agar plates. The results are the mean ± standard deviation of three independent experiments. ***: P < 0.001; the significance of results was statistically analyzed by two-way ANOVA analysis of variance with Bonferroni's post-tests at a 95% confidence interval using Prism software (version 5.01; GraphPad Software Inc., USA).

Article Snippet: We thank Dr. Qijing Zhang (Iowa State University, USA) for providing C. jejuni 81-176.

Techniques: Mutagenesis, Cell Culture, Standard Deviation, Software

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