Journal: Cellular microbiology

Article Title: The Haemophilus influenzae Hap autotransporter mediates microcolony formation and adherence to epithelial cells and extracellular matrix via binding regions in the C-terminal end of the passenger domain.

doi: 10.1046/j.1462-5822.2003.00266.x

Figure Lengend Snippet: Fig. 1. Inhibition of adherence by DB117/pLS88:HapS243A with anti- HapS monoclonal antibodies. Adherence to Chang cells and A549 cells was calculated by dividing the number of adherent bacteria by the number of inoculated bacteria. Strains included DB117/pLS88 (vector control), DB117/pLS88::HapS243A treated with BHI alone (no antibody) and DB117/pLS88::HapS243A treated with BHI plus one of eight monoclonal antibodies. Bars represent the mean ± standard error of the mean of measurements made in triplicate from represen- tative experiments.

Article Snippet: Adherence assays using A549 respiratory epithelial cells (ATCC CCL 185) and Chang conjunctival epithelial cells (Wong-Kilbourne derivative, clone 1–5c-4) were performed as described previously (St. Geme et al., 1993).

Techniques: Inhibition, Bioprocessing, Bacteria, Plasmid Preparation, Control

Journal: Cellular microbiology

Article Title: The Haemophilus influenzae Hap autotransporter mediates microcolony formation and adherence to epithelial cells and extracellular matrix via binding regions in the C-terminal end of the passenger domain.

doi: 10.1046/j.1462-5822.2003.00266.x

Figure Lengend Snippet: Fig. 4. Adherence to epithelial cell monolayers by H. influenzae strains expressing HapS243A or HapD26-725. Adherence to Chang cells and A549 cells was calculated by dividing the number of adher- ent bacteria by the number of inoculated bacteria. Bars represent the mean ± standard error of the mean of measurements made in tripli- cate from representative experiments.

Article Snippet: Adherence assays using A549 respiratory epithelial cells (ATCC CCL 185) and Chang conjunctival epithelial cells (Wong-Kilbourne derivative, clone 1–5c-4) were performed as described previously (St. Geme et al., 1993).

Techniques: Expressing, Bacteria

Journal: Cellular microbiology

Article Title: The Haemophilus influenzae Hap autotransporter mediates microcolony formation and adherence to epithelial cells and extracellular matrix via binding regions in the C-terminal end of the passenger domain.

doi: 10.1046/j.1462-5822.2003.00266.x

Figure Lengend Snippet: Fig. 5. Purification of 6 ¥ His-tagged HapS C- terminal domain and confocal immunofluores- cence micrographs of purified HapS and 6 ¥ His-tagged HapS C-terminal domain after incubation with Chang cells and A549 cells. A. Coomassie-stained SDS-PAGE gel loaded with protein eluted with 100 mM imidazole from Talon beads incubated with concentrated supernatant collected from a culture of DB117/ pHapD1036-99 + pHapD26-725::N¢-6 ¥ His. The proteins migrating at 44, 46, 50 and 52 kDa represent passenger domain fragments released from 6 ¥ His-tagged HapD26-725 after cleavage at each of the four autoproteolytic cleavage sites. B. Purified full-length HapS and Chang cells. C. Purified full-length HapS and A549 cells. D. 6 ¥ His-tagged HapS C-terminal domain and Chang cells. E. 6 ¥ His-tagged HapS C-terminal domain and A549 cells. In B–E, protein bound to epithelial cells was detected using antiserum GP74 against HapS and a Cy-2-conjugated secondary antibody. Samples were examined by confocal laser scanning microscopy, and images represent median optical sections.

Article Snippet: Adherence assays using A549 respiratory epithelial cells (ATCC CCL 185) and Chang conjunctival epithelial cells (Wong-Kilbourne derivative, clone 1–5c-4) were performed as described previously (St. Geme et al., 1993).

Techniques: Incubation, Staining, SDS Page, Confocal Laser Scanning Microscopy

Journal: Cellular microbiology

Article Title: The Haemophilus influenzae Hap autotransporter mediates microcolony formation and adherence to epithelial cells and extracellular matrix via binding regions in the C-terminal end of the passenger domain.

doi: 10.1046/j.1462-5822.2003.00266.x

Figure Lengend Snippet: Fig. 6. Dose-dependent binding of purified HapS and 6 ¥ His-tagged HapS C-terminal domain to Chang cells and A549 cells. Binding of increasing concentrations of purified HapS to Chang cells (triangles) and A549 cells (squares) and binding of increasing concentrations of 6 ¥ His-tagged Hap726–1036 to Chang cells (diamonds) and A549 cells (circles). Binding was quantified by ELISA at an absorbance of 650 nm. Points represent the mean ± standard error of the mean of measurements made in triplicate. Binding curves were calculated by non-linear regression analysis using GRAPHPAD PRISM V3.0.

Article Snippet: Adherence assays using A549 respiratory epithelial cells (ATCC CCL 185) and Chang conjunctival epithelial cells (Wong-Kilbourne derivative, clone 1–5c-4) were performed as described previously (St. Geme et al., 1993).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Medical Virology

Article Title: SARS‐CoV‐2 pseudovirus infectivity and expression of viral entry‐related factors ACE2, TMPRSS2, Kim‐1, and NRP‐1 in human cells from the respiratory, urinary, digestive, reproductive, and immune systems

doi: 10.1002/jmv.27244

Figure Lengend Snippet: Cell lines used in the present study

Article Snippet: A549 , Lung adenocarcinoma , ATCC CRM‐CCL‐185 , DMEM complete medium.

Techniques: Cell Culture

Journal: Molecular Microbiology

Article Title: RrgA is a pilus-associated adhesin in Streptococcus pneumoniae

doi: 10.1111/j.1365-2958.2007.05908.x

Figure Lengend Snippet: Expression of rrgA confers pilus-mediated adherence to human respiratory epithelial cells. A. rrgA , but not rrgB and rrgC , is necessary for pilus-mediated adherence to epithelial cells. Adherence of wild-type piliated TIGR4 (‘T4’) to A549 human respiratory epithelial cells was significantly greater than both non-piliated T4Δ( rrgA-srtD ) compared with T4 deficient in the rrgA gene (‘T4Δ rrgA’ ). Adherence of T4 deficient in both rrgB and rrgC (‘T4Δ rrgB C’) was not notably different from wild-type organisms, but was significantly greater compared with T4Δ rrgA . Repeated-measure anova of data collected from three independent determinations indicates statistically significant differences within experimental conditions. Post hoc Bonferroni analyses identify specific significant differences: ** P < 0.01. B. rrgA expression determines adherence to epithelial cells. T4 deficient in rrgA are significantly deficient in adherence (‘Δ rrgA’ ), compared with wild-type (T4), while trans -complementation restores wild-type adherence [‘Δ rrgA ∇( lacE :: rrgA ’)]. Introduction of a second copy of rrgA inserted in trans in the lacE locus [‘∇( lacE :: rrgA ’] results in significant enhancement of adherence over wild-type levels. Statistical analyses were performed with repeated-measure anova of data collected from three independent determinations. Post hoc Bonferroni analyses identify specific significant differences: * P < 0.01 and ** P < 0.001.

Article Snippet: A549 human respiratory epithelial cells (ATCC number CCL-185) were grown to confluency in polystyrene 24-well plates (Corning) on coverslips in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum and 2 mM glutamine.

Techniques: Expressing

Journal: Molecular Microbiology

Article Title: RrgA is a pilus-associated adhesin in Streptococcus pneumoniae

doi: 10.1111/j.1365-2958.2007.05908.x

Figure Lengend Snippet: Recombinant RrgA and RrgC proteins bind to human respiratory epithelial cells. A–C. Recombinant RrgA binds directly to A549 respiratory epithelial cells. Cells were incubated with 100 μg ml −1 of purified RrgA (A1 and A2, ‘+RrgA’) or GFP (B1 and B2, ‘+GFP’) in DMEM culture medium, or medium alone (A3–4, ‘-RrgA’; B3–4, ‘-GFP’) for 2 h at 4°C. Cells were fixed and stained with anti-RrgA and anti-GFP antibodies (A1–4, ‘αRrgA’; B1–4, ‘αGFP’) and phalloidin, which served as a control to demonstrate the presence of cells (A2, A4, B2 and B4). Imaging was performed with a confocal microscope. Scale bar is 20 μm. C. RrgA and RrgC bind to A549 cells in a dose-dependent manner. A549 cells were mixed in suspension with either medium alone (0 μg ml −1 ) or three concentrations (5, 50 and 100 μg ml −1 ) of pilus subunits RrgA (squares with solid black lines), RrgB (triangles with dashed grey lines), RrgC (upside-down triangles with dashed black lines), or GFP protein (diamond with solid grey lines), incubated for 2 h at 4°C, stained with antisera specific to each protein, and detected with Alexa Fluor 488-conjugated secondaries. Cells were analysed with a FACS-Calibur flow cytometer, and the net mean fluorescence intensity for each population was calculated from three independent experiments. Significant differences were detected by repeated-measure anova ( P < 0.0001), and both RrgA and RrgC binding was significantly different from RrgB and GFP binding at 50 and 100 μg ml −1 by post hoc Bonferroni analysis (* P < 0.001). D–G. Pre-incubation of A549 cells with purified RrgA protein inhibits pilus-mediated adherence. A549 cells were pre-incubated with media alone (D), media containing 100 μg ml −1 of RrgA (E), or 100 μg ml −1 of GFP (F). After pre-incubation, A549 monolayers were infected with S. pneumoniae strain T4. Cells were stained with phalloidin (red) anti- S. pneumoniae capsule antibody (green), and imaged with a confocal microscope. RrgA pre-incubation inhibits the adherence of strain T4 to the A549 cells (E versus D), while the negative control GFP protein does not (F versus D). Scale bar is 20 μm. Adherent bacteria were counted, and the number of bacteria adherent to 100 A549 cells is shown in G ( n = 6 fields, * P = 0.0002, ** P = 0.8).

Article Snippet: A549 human respiratory epithelial cells (ATCC number CCL-185) were grown to confluency in polystyrene 24-well plates (Corning) on coverslips in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum and 2 mM glutamine.

Techniques: Recombinant, Incubation, Purification, Staining, Control, Imaging, Microscopy, Suspension, Flow Cytometry, Fluorescence, Binding Assay, Infection, Negative Control, Bacteria

Journal: Journal of Medical Virology

Article Title: SARS‐CoV‐2 pseudovirus infectivity and expression of viral entry‐related factors ACE2, TMPRSS2, Kim‐1, and NRP‐1 in human cells from the respiratory, urinary, digestive, reproductive, and immune systems

doi: 10.1002/jmv.27244

Figure Lengend Snippet: Cell lines used in the present study

Article Snippet: T47D , Ductal carcinoma , ATCC HTB‐133 , RPMI‐1640 complete medium.

Techniques: Cell Culture