s pneumoniae strain r6  (ATCC)

Journal: EMBO Reports

Article Title: Streptococcus pneumoniae hijacks host autophagy by deploying CbpC as a decoy for Atg14 depletion

doi: 10.15252/embr.201949232

Figure Lengend Snippet: A Pneumococcal CBPs used in (B). B Confocal images of HeLa cells transiently expressing GFP‐CBPs or GFP. The dotted lines show each cell shape. Scale bars, 10 μm. C Confocal images of HeLa cells transiently expressing GFP‐CbpC T4 and mCherry‐LC3 (upper). The fluorescence intensities of GFP‐CbpC T4 (green) and mCherry‐LC3 (red) along the arrow are shown in the graph at the bottom. D Lysates from 293T cells transiently expressing GFP‐Cbps or GFP were subjected to SDS–PAGE and analyzed by immunoblotting using antibodies against LC3, GFP, or actin. E Lysates from 293T cells transiently expressing GFP‐CbpC T4 , GFP‐LytR, or GFP in the presence or absence of chloroquine were subjected to SDS–PAGE and analyzed by immunoblotting using antibodies against LC3, GFP, or actin. F MEFs were infected with S. pneumoniae R6 WT or Δ cbpF for the indicated periods, and the intracellular survival of bacteria expressed as the number of CFUs. G p62‐KO MEF cells infected with S. pneumoniae R6 Δ cbpF /pCbpF R6 ‐FLAG for 2 h were fixed and stained with DAPI and an anti‐FLAG antibody, and representative epifluorescence images are shown. Scale bars, 10 μm. Data information: In (F), data represent mean ± SEM of 3 biological replicates. Student's t ‐test was used to calculate statistical significance. * P < 0.05, ** P < 0.01. Source data are available online for this figure.

Article Snippet: The S. pneumoniae strains R6 (ATCC BAA‐255) and TIGR4 (ATCC BAA‐334) were purchased from the American Type Culture Collection.

Techniques: Expressing, Fluorescence, SDS Page, Western Blot, Infection, Bacteria, Staining

Journal: EMBO Reports

Article Title: Streptococcus pneumoniae hijacks host autophagy by deploying CbpC as a decoy for Atg14 depletion

doi: 10.15252/embr.201949232

Figure Lengend Snippet: A Quantification of the band intensities shown in Figure D. B MEFs were infected with S. pneumoniae TIGR4 WT or Δ cbpC for the indicated periods, and intracellular survivability of bacteria was determined as CFU (colony‐forming units, n = 3). C MEFs were infected with S. pneumoniae R6 WT or Δ cbpC, and invasion efficiency of bacteria was determined by CFU. D p62‐KO MEF cells infected with S. pneumoniae R6 ΔcbpF for 2 h were fixed and stained with DAPI and an anti‐FLAG antibody. Representative epifluorescence images are shown. Scale bars, 10 μm. E Lysates from S. pneumoniae R6 Δ cbpF or Δ cbpF expressing CbpF R6 ‐FLAG were subjected to SDS–PAGE and analyzed by immunoblotting using an anti‐FLAG antibody. Data information: In (B, C), data represent mean ± SEM of 3 biological replicates. Student's t ‐test was used to calculate statistical significance. * P < 0.01. N.S., not significant. Source data are available online for this figure.

Article Snippet: The S. pneumoniae strains R6 (ATCC BAA‐255) and TIGR4 (ATCC BAA‐334) were purchased from the American Type Culture Collection.

Techniques: Infection, Bacteria, Staining, Expressing, SDS Page, Western Blot

Journal: EMBO Reports

Article Title: Streptococcus pneumoniae hijacks host autophagy by deploying CbpC as a decoy for Atg14 depletion

doi: 10.15252/embr.201949232

Figure Lengend Snippet: A MEFs were infected with S. pneumoniae R6 WT or Δ cbpF , and the number of internalized bacteria per cell was determined. B Atg5‐KO MEFs were infected with S. pneumoniae R6 WT or Δ cbpF for the indicated periods, and the intracellular survival of bacteria was determined and expressed as CFUs. C A549 cells infected with the indicated S. pneumoniae strains for 2 h were fixed and stained with DAPI and an anti‐Atg14 antibody, and the percentages of perinuclear‐localizing Atg14 containing cells were quantified. D Representative epifluorescence images of the data presented in (C) are shown. Scale bars, 10 μm E Knockdown effects of the indicated siRNAs were evaluated by RT–PCR and visualized by agarose gel electrophoresis. F Lysates from 293T cells transiently expressing the indicated proteins were subjected to SDS–PAGE and analyzed by immunoblotting using the indicated antibodies. G Quantification of NanoBRET signals in 293A cells transiently expressing Nanoluc‐Beclin1 and HaloTag‐Atg14 in the presence or absence of GFP‐CbpC or GFP. H Lysates from 293T cells transiently expressing the indicated proteins were subjected to IP assays using anti‐HA beads, and bound proteins were analyzed by Western blotting using the indicated antibodies. I Schematic diagram of p62–CbpC–Atg14‐driven autophagy subversion in pneumococcal infection. Data information: In (A, B, C, G), data represent mean ± SEM of 3 biological replicates. Student's t ‐test was used to calculate statistical significance. * P < 0.01, N.S., not significant. Source data are available online for this figure.

Article Snippet: The S. pneumoniae strains R6 (ATCC BAA‐255) and TIGR4 (ATCC BAA‐334) were purchased from the American Type Culture Collection.

Techniques: Infection, Bacteria, Staining, Knockdown, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Expressing, SDS Page, Western Blot

Journal: EMBO Reports

Article Title: Streptococcus pneumoniae hijacks host autophagy by deploying CbpC as a decoy for Atg14 depletion

doi: 10.15252/embr.201949232

Figure Lengend Snippet: A Lysates from MEFs stably expressing HA‐Atg14 infected with S. pneumoniae TIGR4 WT or Δ cbpC for 1, 2, or 3 h in the presence or absence of cycloheximide were subjected to SDS–PAGE and analyzed by immunoblotting with the indicated antibodies. B MEFs/HA‐Atg14 cells were infected with S. pneumoniae TIGR4 WT or Δ cbpC for 1, 2, or 3 h in the presence or absence of Bafilomycin A1 (BafA1). The lysates were subjected to SDS–PAGE and analyzed by immunoblotting with the indicated antibodies. C–F (C, E) A549 cells were infected with S. pneumoniae R6 WT or Δ cbpC for 2 h and fixed and stained with DAPI and an anti‐Atg14 or anti‐GM130 antibody. Representative epifluorescence images are shown. Scale bars, 10 μm. (D, F) The percentages of perinuclear‐localizing Atg14 containing cells in (C) or intact Golgi apparatus‐containing cells in (E) were quantified. G Streptococcus pneumoniae (Sp) invasion‐permissive A549 cells (marked with GFP) and Sp invasion‐non‐permissive (pIgR knocked down) A549 cells were co‐cultured at a 1:3 ratio. Atg14‐disappearance experiments were conducted, and representative epifluorescence images are shown. Scale bars, 10 μm. The lines show each cell shape, and the arrows show invasion‐permissive A549 cells marked with GFP. H The percentages of perinuclear‐localizing Atg14 containing cells in (G) were quantified. I Lysates from 293T cells transiently expressing GFP‐CbpC or GFP, and HA‐Atg14 were subjected to SDS–PAGE and analyzed by immunoblotting using antibodies against HA or GFP. J Lysates from 293T cells transiently expressing GFP‐CbpC T4 or GFP were immunoprecipitated using GST‐GFP‐Nanobody. Additionally, beads were mixed with lysates from 293T cells transiently expressing p62‐3Myc and HA‐Atg14, and bound proteins were analyzed by immunoblotting. K A549 cells treated with the indicated siRNAs were infected with S. pneumoniae R6 WT or Δ cbpF for the indicated durations. The cells were fixed and stained with DAPI and an anti‐Atg14 antibody, and percentages of perinuclear‐localizing Atg14 containing cells were quantified. L Lysates from MEFs stably expressing GFP‐CbpC T4 or GFP in the presence or absence of rapamycin or chloroquine were subjected to SDS–PAGE and immunoblotted using antibodies against LC3 or p62. M Quantification of NanoBRET signals in 293A cells transiently expressing HaloTag‐Stx17 and Nanoluc‐Atg14 in the presence or absence of GFP‐CbpC or GFP. Data information: In (D, F, H, K, M), data represent mean ± SEM of 3 biological replicates. Student's t ‐test was used to calculate statistical significance. * P < 0.01, N.S., not significant. Source data are available online for this figure.

Article Snippet: The S. pneumoniae strains R6 (ATCC BAA‐255) and TIGR4 (ATCC BAA‐334) were purchased from the American Type Culture Collection.

Techniques: Stable Transfection, Expressing, Infection, SDS Page, Western Blot, Staining, Cell Culture, Immunoprecipitation

Journal: EMBO Reports

Article Title: Streptococcus pneumoniae hijacks host autophagy by deploying CbpC as a decoy for Atg14 depletion

doi: 10.15252/embr.201949232

Figure Lengend Snippet: A Schematic representation of the CbpC T4 variants used in (B), (E), (H), and (I). B Lysates from 293T cells transiently expressing p62‐3Myc and GFP‐CbpC T4 variants were immunoprecipitated using a GST‐GFP‐Nanobody fusion protein. Bound proteins were analyzed by immunoblotting. C Diagram of the ∆DD mutation in dp5 loop domain. D Structures of the dp5 and dp6 domains in CbpF R6 . The side chains of D167–D168 are shown. E Lysates from 293T cells transiently expressing p62‐3Myc and GFP‐CbpC T4 variants were immunoprecipitated using the GST‐GFP‐Nanobody protein. Bound proteins were analyzed by immunoblotting. F Diagram of the p62 variants used in (G). G Lysates of 293T cells transiently expressing GFP‐CbpC T4 and p62‐3Myc variants were immunoprecipitated with the GST‐GFP‐Nanobody protein. Bound proteins were analyzed by immunoblotting using the indicated antibodies. H A549 cells infected with the indicated S. pneumoniae strains for 2 h were fixed and stained with DAPI and an anti‐Atg14 antibody, and the percentages of perinuclear‐localizing Atg14 containing cells were quantified. I Representative epifluorescence images in (H) are shown. Scale bars, 10 μm. Data information: In (H), data represent mean ± SEM of 3 biological replicates. Student's t ‐test was used to calculate statistical significance. * P < 0.01. Source data are available online for this figure.

Article Snippet: The S. pneumoniae strains R6 (ATCC BAA‐255) and TIGR4 (ATCC BAA‐334) were purchased from the American Type Culture Collection.

Techniques: Expressing, Immunoprecipitation, Western Blot, Mutagenesis, Infection, Staining

Journal: Frontiers in Immunology

Article Title: Streptococcal Extracellular Membrane Vesicles Are Rapidly Internalized by Immune Cells and Alter Their Cytokine Release

doi: 10.3389/fimmu.2020.00080

Figure Lengend Snippet: Characterization of streptococcal membrane vesicles (MVs) using electron microscopy. (A) Scanning electron micrograph (SEM) of Streptococcus pneumoniae reference strain R6 cells, during shedding of vesicles from their surface. (B–F) Cryogenic transmission electron micrographs (cryo-TEM) of isolated pneumococcal MVs, showing heterogeneous morphology, tiny vesicles budding from large ones (white arrows), chain-like structures (white arrows), and some vesicles with darker content (red arrows).

Article Snippet: S. pneumoniae reference strain R6 (ATCC® BAA255TM, USA), whose genome is fully sequenced and annotated was selected ( ).

Techniques: Membrane, Electron Microscopy, Transmission Assay, Isolation