Journal: Infection and Immunity

Article Title: Roles of OmpX, an Outer Membrane Protein, on Virulence and Flagellar Expression in Uropathogenic Escherichia coli

doi: 10.1128/IAI.00721-20

Figure Lengend Snippet: Colonization by the parent strain (CFT073) and ompX mutant in the bladders and kidneys of mice with UTIs. The female mice (n = 3 for each group) were infected with the parent strain or ompX mutant. At 48 h postinfection, cell numbers of bacteria isolated from the bladder and kidneys were determined as CFU. We repeated experiments independently three times, but one mouse infected with the parent strain died within 48 h of infection. Each data point represents a sample from an individual mouse (n = 8 for the parent strain and n = 9 for the ompX mutant). Horizontal bars show median values. *, P < 0.05 relative to the value for the parent strain.

Article Snippet: CFT073 , Parent strain (ATCC 700928) , ATCC 700928.

Techniques: Mutagenesis, Infection, Bacteria, Isolation

Journal: Infection and Immunity

Article Title: Roles of OmpX, an Outer Membrane Protein, on Virulence and Flagellar Expression in Uropathogenic Escherichia coli

doi: 10.1128/IAI.00721-20

Figure Lengend Snippet: Adhesion to and internalization in kidney epithelial cells (HTB-44) of the parent strain (CFT073) and the ompX mutant (A and B) or the parent strain and the ompX mutant carrying pTrc99K (empty vector) or pTrc99KompX (ompX expression plasmid) (C). Values are percent CFU of adhered/internalized (A) and internalized (B and C) bacteria relative to total bacterial cell numbers. Data are means from three independent experiments; error bars indicate standard deviations. *, P < 0.05 relative to values for CFT073 (A and B) or CFT073/pTrc99K (C).

Article Snippet: CFT073 , Parent strain (ATCC 700928) , ATCC 700928.

Techniques: Mutagenesis, Plasmid Preparation, Expressing, Bacteria

Journal: Infection and Immunity

Article Title: Roles of OmpX, an Outer Membrane Protein, on Virulence and Flagellar Expression in Uropathogenic Escherichia coli

doi: 10.1128/IAI.00721-20

Figure Lengend Snippet: Aggregation within kidney epithelial cells (HTB-44) for the parent strain and the ompX mutant or the parent strain and the ompX mutant carrying pTH18kr (empty vector) or pTH18krompX (ompX expression plasmid). Bacteria carrying a green fluorescence protein (GFP) expression plasmid, pTurboGFP-B, and HTB-44 cells stained with rhodamine-phalloidin were imaged with green and red fluorescence, respectively, using a 100× objective. Images were taken from above (A), and cross-sectional images (B) correspond to the white boxes in panel A. The experiment was repeated twice, and similar results were obtained. (C) Aggregated bacteria within HTB-44 cells were quantified by representing levels of colonized bacteria as areas (in square pixels) of GFP. Microscopy data are means from three fields of view, and error bars indicate standard deviations. **, P < 0.01 relative to the value for the parent strain CFT073.

Article Snippet: CFT073 , Parent strain (ATCC 700928) , ATCC 700928.

Techniques: Mutagenesis, Plasmid Preparation, Expressing, Bacteria, Fluorescence, Staining, Microscopy

Journal: Infection and Immunity

Article Title: Roles of OmpX, an Outer Membrane Protein, on Virulence and Flagellar Expression in Uropathogenic Escherichia coli

doi: 10.1128/IAI.00721-20

Figure Lengend Snippet: Motilities and flagellar production for the parent strain (CFT073) and the ompX mutant or the parent strain and the ompX mutant carrying pTrc99K (empty vector) or pTrc99KompX (ompX expression plasmid). (A) Bacterial migration on LB medium containing 0.3% agar. (B) Diameters reflecting bacterial migration on the agar. Data are means from three independent experiments; error bars indicate standard deviations. (C) Flagella and bacterial cells were stained with Victoria blue/tannic acid were pictured using a 100× objective. (D) Ratios of bacteria observed with flagella to ∼120 to 150 randomly selected bacteria on microscopy, presented as percentages. Data are means, and error bars indicate standard deviations. **, P < 0.01 relative to the value for CFT073.

Article Snippet: CFT073 , Parent strain (ATCC 700928) , ATCC 700928.

Techniques: Mutagenesis, Plasmid Preparation, Expressing, Migration, Staining, Bacteria, Microscopy

Journal: Infection and Immunity

Article Title: Roles of OmpX, an Outer Membrane Protein, on Virulence and Flagellar Expression in Uropathogenic Escherichia coli

doi: 10.1128/IAI.00721-20

Figure Lengend Snippet: FliC expression in the ompX mutant and contribution of fliC to bacterial adhesion to and internalization within the kidney epithelial cells. (A) Western blots of cell lysates and secreted proteins from the parent strain (CFT073) and the ompX mutant containing a VSVG-tagged FliC expression plasmid (pTH18krfliC-VSVG) and pTrc99A (empty vector) or pTrc99AompX (ompX expression plasmid). Locations of molecular mass standards (in kilodaltons) are shown on the left. VSVG-tagged FliC was visualized by probing with a VSVG antibody. Adhesion to and internalization in kidney epithelial cells (HTB-44) of the parent strain, fliC mutant, and fliC/ompX double mutant (B and C) or the parent strain and the fliC mutant carrying pTrc99A (empty vector) or pTrc99AfliC (fliC expression plasmid) (D). Values are percent CFU of adhered/internalized (B) and internalized (C and D) bacteria relative to total bacterial cell numbers. Data are means from three independent experiments; error bars indicate standard deviations. *, P < 0.05 relative to CFT073 (B and C) or CFT073/pTrc99A (D).

Article Snippet: CFT073 , Parent strain (ATCC 700928) , ATCC 700928.

Techniques: Expressing, Mutagenesis, Western Blot, Plasmid Preparation, Bacteria

Journal: Infection and Immunity

Article Title: Roles of OmpX, an Outer Membrane Protein, on Virulence and Flagellar Expression in Uropathogenic Escherichia coli

doi: 10.1128/IAI.00721-20

Figure Lengend Snippet: Aggregation within kidney epithelial cells (HTB-44) for the parent strain and the fliC mutant or the parent strain and the fliC mutant carrying pTH18kr (empty vector) or pTH18krfliC-VSVG (fliC expression plasmid). Bacteria carrying a GFP expression plasmid, pTurboGFP-B, and HTB-44 cells stained with rhodamine-phalloidin were imaged with green and red fluorescence, respectively, using a 100× objective. Images were taken from above (A), and cross-sectional images (B) correspond to the white boxes in panel A. The experiment was repeated twice, and similar results were obtained. (C) Aggregated bacteria within HTB-44 cells were quantified by determining levels of colonized bacteria as areas (in square pixels) of GFP. Microscopy data are means from three fields of view, and error bars indicate standard deviations. *, P < 0.05, and **, P < 0.01, relative to CFT073.

Article Snippet: CFT073 , Parent strain (ATCC 700928) , ATCC 700928.

Techniques: Mutagenesis, Plasmid Preparation, Expressing, Bacteria, Staining, Fluorescence, Microscopy

Journal: Infection and Immunity

Article Title: Roles of OmpX, an Outer Membrane Protein, on Virulence and Flagellar Expression in Uropathogenic Escherichia coli

doi: 10.1128/IAI.00721-20

Figure Lengend Snippet: Transcript levels and promoter activities of flagellum-related and fimbrial genes in the parent strain (CFT073) and the ompX mutant. (A) Transcript levels were determined relative to that of rpoD. Data are means for two biological replicates; error bars indicate the ranges. (B) β-Galactosidase activities corresponding to flhD, fliA, and fliC promoter activities in the parent strain and the ompX mutant containing pNNflhD-P, pNNfliA-P, or pNNfliC-P, the lacZ reporter plasmid. Data are means from three independent experiments; error bars indicate standard deviations. **, P < 0.01 relative to CFT073.

Article Snippet: CFT073 , Parent strain (ATCC 700928) , ATCC 700928.

Techniques: Mutagenesis, Plasmid Preparation

Journal: Infection and Immunity

Article Title: Roles of OmpX, an Outer Membrane Protein, on Virulence and Flagellar Expression in Uropathogenic Escherichia coli

doi: 10.1128/IAI.00721-20

Figure Lengend Snippet: Strains and plasmids used in this study a

Article Snippet: CFT073 , Parent strain (ATCC 700928) , ATCC 700928.

Techniques: Plasmid Preparation, Mutagenesis, Expressing

Journal: bioRxiv

Article Title: DNA glycosylase NEIL2 prevents Fusobacterium -mediated inflammation and DNA damage in colonic epithelial cells

doi: 10.1101/2020.06.11.147454

Figure Lengend Snippet: (A-D) Human colonic EDMs were infected with Fn at moi 100 for 24 h. The RNA from EDMs was used for qRT-PCR to determine the expression of genes involved in base excision repair, mismatch repair and for non-homologous end joining (NHEJ). (A) Schematic showing the experimental design. (B) The level of BER transcripts, NEIL1, NEIL2, NTH1, OGG1, (C) The level of MMR transcripts, MLH1, MLH3, MSH2, MSH6, PMS2, (D) The transcript level of NHEJ marker Ku70 were determined by qRT-PCR. (E-F) Human colonic EDMs were infected with commensal E. coli -K12 strain (E), or pathogenic IBD-associated adherent invasive E. coli LF-82 (F) to determine the expression level of NEIL2 following infection. In (B-F), the expression level of the transcripts was normalized to the housekeeping gene (18srRNA), and the normalized expression value was compared with the respective uninfected control cells. Data represent the mean ± SEM of three separate experiments. * indicates p≤0.05, and ** indicates p≤0.01 as calculated by the unpaired two-tailed student’s t-test.

Article Snippet: Escherichia coli K12 strain DH10B , (ATCC-PTA¬5105), was cultured on L.B. agar and L.B. broth and used to infect EDM at moi of 100.

Techniques: Infection, Quantitative RT-PCR, Expressing, Non-Homologous End Joining, Marker, Control, Two Tailed Test

Journal: bioRxiv

Article Title: DNA glycosylase NEIL2 prevents Fusobacterium -mediated inflammation and DNA damage in colonic epithelial cells

doi: 10.1101/2020.06.11.147454

Figure Lengend Snippet: (A) APC Min /+ EDMs derived from the uninvolved region of the colon were infected with different microbes; commensal E. coli K12, IBD-associated adherent-invasive E.coli LF82 and colon cancer-associated pathogens (NC101, H. pylori and Fn ). The supernatants were collected from the uninfected and infected EDMs done in the same experiments and assessed for oxidative DNA damage (right). Data represent the mean ± SEM of three separate experiments. * indicates p≤0.05, ** indicates p≤0.01 as assayed by student’s t-test. (B) The relative level of the oxidized bases produced by each microbe was compared with uninfected cells, which is considered as 1. The relative production of the oxidized base was compared between different microbes

Article Snippet: Escherichia coli K12 strain DH10B , (ATCC-PTA¬5105), was cultured on L.B. agar and L.B. broth and used to infect EDM at moi of 100.

Techniques: Derivative Assay, Infection, Produced