Journal: bioRxiv

Article Title: Proximity Labeling to Map Host-Pathogen Interactions at the Membrane of a Bacteria Containing Vacuole in Chlamydia trachomatis Infected Human Cells

doi: 10.1101/616896

Figure Lengend Snippet: HeLa cells seeded onto electron microscopy grade, cell culture treated coverslips were infected with C. trachomatis serovar L2 transformed with the indicated constructs, or C. trachomatis serovar L2 wild-type (WT) were induced with anhydrotetracycline (aTc) at 7 hpi (IncF-APEX2 0.3 nM aTc, all others 5 nM aTc). At 24 hpi, a glutaraldehyde and paraformaldehyde fixing solution was added to each sample and incubated on ice. Next, samples were pre-treated with DAB (or not, as indicated) 30 min prior to labeling by the addition of H 2 O 2 solution (also containing DAB) to catalyze DAB polymerization. The reaction was quenched with glycine and processed for electron microscopy as indicated in Methods . (A) C. trachomatis L2 wild-type (WT) treated with DAB; scale bar= 2 µm, (B) C. trachomatis L2 IncA-APEX2 without DAB; scale bar= 500 nm, (C) C. trachomatis L2 transformants treated with DAB; DAB polymer staining around the inclusion is indicated by arrows; scale bar= 500 nm.

Article Snippet: HeLa cells were infected with C. trachomatis L2 wild-type (i.e., non-transformed) or the transformants and induced for construct expression at 20 hpi (1 nM aTc for IncF-APEX2 and 5 nM for all other transformants).

Techniques: Electron Microscopy, Cell Culture, Infection, Transformation Assay, Construct, Incubation, Labeling, Polymer, Staining

Journal: bioRxiv

Article Title: Proximity Labeling to Map Host-Pathogen Interactions at the Membrane of a Bacteria Containing Vacuole in Chlamydia trachomatis Infected Human Cells

doi: 10.1101/616896

Figure Lengend Snippet: HeLa cells infected with C. trachomatis L2 Inc-APEX2 transformants, wild-type (WT), or mock-infected were induced with anhydrotetracycline (aTc) at 7 hpi (0.3 nM for IncF-APEX2, and 4 nM for all others). Biotin-phenol (BP) was added 30 min prior to the biotinylation reaction at 24 hpi. Biotinylation was catalyzed by the addition of 3 mM H 2 O 2 for 1 min and stopped with a quenching wash solution. Biotinylated proteins were affinity purified from solubilized lysates using streptavidin beads, eluted in sample buffer, separated by SDS-PAGE and transferred to PVDF for western blotting. The eluate fraction was probed for biotinylated proteins (streptavidin-680 conjugate), construct expression (anti-FLAG antibody), IncA (anti-IncA antibody), CT223 (anti-CT223 antibody), and imaged using Azure c600. Asterisks indicate detected proteins. See Supplementary Figure 1.

Article Snippet: HeLa cells were infected with C. trachomatis L2 wild-type (i.e., non-transformed) or the transformants and induced for construct expression at 20 hpi (1 nM aTc for IncF-APEX2 and 5 nM for all other transformants).

Techniques: Infection, Affinity Purification, SDS Page, Western Blot, Construct, Expressing

Journal: bioRxiv

Article Title: Proximity Labeling to Map Host-Pathogen Interactions at the Membrane of a Bacteria Containing Vacuole in Chlamydia trachomatis Infected Human Cells

doi: 10.1101/616896

Figure Lengend Snippet: (A) Western blot confirmation of LRRF1 in the eluates from streptavidin affinity purified biotinylated lysate from C. trachomatis L2 IncF-APEX2, IncA TM -APEX2, and IncA-APEX2 transformants at 24 hpi (BP= biotin-phenol). (B) Confirmation of LRRF1 co-localization with the inclusion of C. trachomatis L2 wild-type infected HeLa cells. Cells were fixed at 24 hpi in 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 then stained for indirect immunofluorescence to visualize the inclusion membrane (CT223; red), LRRF1 (green), DNA and Chlamydiae (DRAQ5 and MOMP; blue). (C) Confirmation of FLI1 co-localization with the inclusion of C. trachomatis L2 wild-type infected HeLa cells. Cells were fixed at 24 hpi in 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 then stained for indirect immunofluorescence to visualize the inclusion membrane (CT223; red), FLI1 (green), and DNA and Chlamydiae (DAPI and MOMP; blue). Coverslips were imaged using Zeiss Apotome.2 with 100x magnification. Scale bar= 10 µm. See Supplementary Figure 4.

Article Snippet: HeLa cells were infected with C. trachomatis L2 wild-type (i.e., non-transformed) or the transformants and induced for construct expression at 20 hpi (1 nM aTc for IncF-APEX2 and 5 nM for all other transformants).

Techniques: Western Blot, Affinity Purification, Infection, Staining, Immunofluorescence, Membrane

Journal: bioRxiv

Article Title: Proximity Labeling to Map Host-Pathogen Interactions at the Membrane of a Bacteria Containing Vacuole in Chlamydia trachomatis Infected Human Cells

doi: 10.1101/616896

Figure Lengend Snippet: (A) Hela cells seeded on glass coverslips were infected with C. trachomatis L2 Inc-APEX2 transformants or CT226 FLAG transformants and induced for expression at 20 hpi (IncF-APEX2 was induced with 1 nM aTc; 5 nM aTc for all other transformants). At 24 hpi, coverslips were fixed with ice cold methanol and stained for immunofluorescence to visualize construct expression (FLAG) or CT223 (red), LRRF1 (green), Chlamydiae and DNA (DRAQ5 and MOMP; pink). Coverslips were imaged by Zeiss Elyra super-resolution microscopy 63×2x with structural illumination (SIM). Scale bar = 5 µm. (B) SIM 3D snapshot of C. trachomatis L2 CT226 FLAG infected HeLa cells with CT226 FLAG and LRRF1 positive fibers. (C) SIM 3D snapshot of C. trachomatis L2 IncA-APEX2 infected HeLa cells with IncA fibers. Arrows indicate co-localization between the indicated expressed construct and LRRF1.

Article Snippet: HeLa cells were infected with C. trachomatis L2 wild-type (i.e., non-transformed) or the transformants and induced for construct expression at 20 hpi (1 nM aTc for IncF-APEX2 and 5 nM for all other transformants).

Techniques: Infection, Expressing, Staining, Immunofluorescence, Construct, Super-Resolution Microscopy