Journal: Infection and Immunity

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

doi: 10.1128/IAI.00537-19

Figure Lengend Snippet: Localization and biotinylation of proteins proximal to the inclusion membrane in HeLa cells infected with C. trachomatis L2 transformants expressing Inc-APEX2 constructs. Coverslips were placed in two wells of a 6-well tissue culture plate to ensure appropriate biotinylation. HeLa cells that were infected with C. trachomatis serovar L2 transformed with the indicated APEX2 constructs or the C. trachomatis L2 wild type (WT) or that were mock infected were induced for construct expression with the indicated concentrations of anhydrotetracycline (aTc) at 7 hpi. Biotin-phenol was added at 23.5 hpi and biotinylation was catalyzed at 24 hpi by the addition of 3 mM H 2 O 2 for 1 min, after which the reaction was quenched. Coverslips were removed from the 6-well plate and processed for immunofluorescence to visualize biotinylated proteins (the streptavidin-488 conjugate), expression of the construct (anti-FLAG, red), chlamydiae (MOMP) and DNA (DAPI; blue), and the inclusion membrane (anti-CT223; pink). Coverslips were imaged using a Zeiss LSM 800 confocal microscope at a ×63 magnification with a ×2 zoom. Bars = 5 μm.

Article Snippet: To confirm the correct expression of each construct containing APEX2, HeLa cells were infected with the C. trachomatis L2 APEX2, IncF-APEX2, IncA TM -APEX2, or IncA-APEX2 transformant and either not induced or induced (with 0.3 nM aTc for IncF-APEX2 and 5 nM aTc for all other transformants) at 7 hpi.

Techniques: Membrane, Infection, Expressing, Construct, Transformation Assay, Immunofluorescence, Microscopy

Journal: Infection and Immunity

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

doi: 10.1128/IAI.00537-19

Figure Lengend Snippet: Ultrastructural localization of APEX2 activity to the cytosolic face of the inclusion membrane in HeLa cells infected with C. trachomatis L2 transformants expressing Inc-APEX2 constructs, as determined by electron microscopy. 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) and induced with anhydrotetracycline (aTc) at 7 hpi (0.3 nM aTc for the IncF-APEX2 transformants and 5 nM aTc for all others). At 24 hpi, a glutaraldehyde and paraformaldehyde fixing solution was added to each sample and the samples were incubated on ice. Next, the samples were pretreated 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 Materials and Methods. (A) C. trachomatis L2 wild type (WT) treated with DAB; (B) C. trachomatis L2 IncA-APEX2 without DAB; (C) C. trachomatis L2 transformants treated with DAB. DAB polymer staining around the inclusion is indicated by arrowheads. Bars = 2 μm (A) and 500 nm (B and C).

Article Snippet: To confirm the correct expression of each construct containing APEX2, HeLa cells were infected with the C. trachomatis L2 APEX2, IncF-APEX2, IncA TM -APEX2, or IncA-APEX2 transformant and either not induced or induced (with 0.3 nM aTc for IncF-APEX2 and 5 nM aTc for all other transformants) at 7 hpi.

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

Journal: Infection and Immunity

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

doi: 10.1128/IAI.00537-19

Figure Lengend Snippet: Western blot detection of affinity-purified biotinylated proteins. HeLa cells infected with C. trachomatis L2 Inc-APEX2 transformants or the wild type (WT) or mock-infected cells were induced with anhydrotetracycline (aTc) at 7 hpi (0.3 nM aTc for IncF-APEX2 transformants 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 a PVDF membrane for Western blotting. The eluate fraction was probed for biotinylated proteins (streptavidin-680 conjugate), construct expression (anti-FLAG antibody), IncA (anti-IncA antibody), and CT223 (anti-CT223 antibody) and imaged using an Azure c600 system. Asterisks indicate the detected proteins. Numbers on the left indicate molecular masses (in kilodaltons). See Fig. S1 in the supplemental material.

Article Snippet: To confirm the correct expression of each construct containing APEX2, HeLa cells were infected with the C. trachomatis L2 APEX2, IncF-APEX2, IncA TM -APEX2, or IncA-APEX2 transformant and either not induced or induced (with 0.3 nM aTc for IncF-APEX2 and 5 nM aTc for all other transformants) at 7 hpi.

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

Journal: Infection and Immunity

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

doi: 10.1128/IAI.00537-19

Figure Lengend Snippet: Significant C. trachomatis L2 proteins

Article Snippet: To confirm the correct expression of each construct containing APEX2, HeLa cells were infected with the C. trachomatis L2 APEX2, IncF-APEX2, IncA TM -APEX2, or IncA-APEX2 transformant and either not induced or induced (with 0.3 nM aTc for IncF-APEX2 and 5 nM aTc for all other transformants) at 7 hpi.

Techniques:

Journal: Infection and Immunity

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

doi: 10.1128/IAI.00537-19

Figure Lengend Snippet: Visualization of global biological functions of AP-MS-identified and statistically significant eukaryotic proteins from Inc-APEX2 pulldowns. A ClueGO global network visualization of eukaryotic proteins identified by mass spectrometry (SAINT BFDR ≤ 0.05) from the C. trachomatis L2 IncF-APEX2 (A), IncA TM -APEX2 (B), and IncA-APEX2 (C) transformants is shown. See Fig. S2 and S3 in the supplemental material. The asterisks indicate significantly enriched GO terms: *, P < 0.05; **, P < 0.01.

Article Snippet: To confirm the correct expression of each construct containing APEX2, HeLa cells were infected with the C. trachomatis L2 APEX2, IncF-APEX2, IncA TM -APEX2, or IncA-APEX2 transformant and either not induced or induced (with 0.3 nM aTc for IncF-APEX2 and 5 nM aTc for all other transformants) at 7 hpi.

Techniques: Protein-Protein interactions, Mass Spectrometry

Journal: Infection and Immunity

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

doi: 10.1128/IAI.00537-19

Figure Lengend Snippet: Confirmation of LRRF1 biotinylation by Inc-APEX2 proteins and localization of LRRF1 and FLII to the chlamydial inclusion. (A) Western blotting confirmation of LRRF1 in the eluates from streptavidin affinity-purified biotinylated lysate from the C. trachomatis L2 IncF-APEX2, IncA TM -APEX2, and IncA-APEX2 transformants at 24 hpi (BP, biotin-phenol). (B) Confirmation of LRRF1 colocalization with the inclusion of C. trachomatis L2 wild-type-infected HeLa cells. Cells were fixed at 24 hpi in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and then stained for indirect immunofluorescence to visualize the inclusion membrane (CT223; red), LRRF1 (green), and DNA and chlamydiae (DRAQ5 and MOMP; blue). (C) Confirmation of FLII colocalization with the inclusion of C. trachomatis L2 wild-type-infected HeLa cells. Cells were fixed at 24 hpi in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, amd then stained for indirect immunofluorescence to visualize the inclusion membrane (CT223; red), FLII (green), and DNA and chlamydiae (DAPI and MOMP; blue). Coverslips were imaged using a Zeiss ApoTome.2 fluorescence microscope at ×100 magnification. Bars = 10 μm. See Fig. S4 in the supplemental material.

Article Snippet: To confirm the correct expression of each construct containing APEX2, HeLa cells were infected with the C. trachomatis L2 APEX2, IncF-APEX2, IncA TM -APEX2, or IncA-APEX2 transformant and either not induced or induced (with 0.3 nM aTc for IncF-APEX2 and 5 nM aTc for all other transformants) at 7 hpi.

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

Journal: Infection and Immunity

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

doi: 10.1128/IAI.00537-19

Figure Lengend Snippet: Assessment of LRRF1 colocalization with chlamydial Incs in C. trachomatis L2-infected HeLa cells using superresolution microscopy. (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 was used for all other transformants). At 24 hpi, the coverslips were fixed with ice-cold methanol and stained for immunofluorescence to visualize construct expression (FLAG) or CT223 (red), LRRF1 (green), and chlamydiae and DNA (DRAQ5 and MOMP; pink). Coverslips were imaged by structural illumination microscopy (SIM) with a Zeiss Elyra superresolution microscope at a ×63 magnification with a ×2 zoom. Bars = 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 colocalization between the indicated expressed construct and LRRF1.

Article Snippet: To confirm the correct expression of each construct containing APEX2, HeLa cells were infected with the C. trachomatis L2 APEX2, IncF-APEX2, IncA TM -APEX2, or IncA-APEX2 transformant and either not induced or induced (with 0.3 nM aTc for IncF-APEX2 and 5 nM aTc for all other transformants) at 7 hpi.

Techniques: Infection, Microscopy, Expressing, Staining, Immunofluorescence, Construct

Journal: Infection and Immunity

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

doi: 10.1128/IAI.00537-19

Figure Lengend Snippet: Model of Inc-Inc organization in the inclusion membrane and Inc-APEX2 proximity labeling. The proposed model of Inc organization is based on mass spectrometry-identified chlamydial Inc proteins, in which IncA-APEX2 and IncF-APEX2 proximity labeling constructs and bacterial adenylate cyclase two-hybrid (BACTH) assays were used to test protein-protein interactions. Based on these data, we propose four possible scenarios for the spatial organization of Incs and how these Incs were detected using the APEX2 proximity labeling system: scenario 1, IncF interacts with CT226, which binds LRRF1; scenario 2, IncA interacts with CT226, which binds LRRF1; scenario 3, IncA binds IncF and CT226, which binds LRRF1; and scenario 4, IncA, CT223, IncF, and CT226 (which binds LRRF1) all interact with each other. CT223 was statistically significant by SAINT analysis from the mass spectrometry data and was able to interact with IncF and IncA by BACTH.

Article Snippet: To confirm the correct expression of each construct containing APEX2, HeLa cells were infected with the C. trachomatis L2 APEX2, IncF-APEX2, IncA TM -APEX2, or IncA-APEX2 transformant and either not induced or induced (with 0.3 nM aTc for IncF-APEX2 and 5 nM aTc for all other transformants) at 7 hpi.

Techniques: Membrane, Labeling, Mass Spectrometry, Construct, Protein-Protein interactions