Journal: Microbiology Spectrum

Article Title: The Chlamydia trachomatis Inc Tri1 interacts with TRAF7 to displace native TRAF7 interacting partners

doi: 10.1128/spectrum.00453-24

Figure Lengend Snippet: Tri1 and TRAF7 interact during infection. ( A ) HeLa cells were transiently transfected with mCh-TRAF7 for 24 h and then infected with L2+pIncG FLAG or L2+pTri1 FLAG for 24 h in the presence of inducer (aTc). Shown are the lysates and FLAG-bead affinity purified eluates immunoblotted with the indicated antibodies. Glyceraldehyde phosphate dehydrogenase (GAPDH)) serves as a loading control, and Chlamydia major outer membrane protein (MOMP) serves as a control for the efficiency of infection. ( B ) FLAG-bead affinity purified eluates prepared from HeLa cells infected with L2 expressing the indicated Incs (pTri1 FLAG , pIncE FLAG , or pDre1 FLAG ) in the presence of inducer (aTc) were analyzed by liquid chromatography (LC)/MS-MS. Shown are selected average spectral counts from biological triplicates, SAINT scores, and Bayesian false discovery rate (BFDR). SAINT scores closer to 1 with a BFDR ≤ 0.05 suggest a high confidence interaction. N/D, not determined because no spectral counts were recorded. SNX5, sorting nexin 5. DCTN4, dynactin 4.

Article Snippet: The C. trachomatis L2 strains overexpressing Tri1 FLAG (originally “CT224-FLAG”) and Dre1 FLAG were generous gifts from Drs. Mary Weber (University of Iowa) and Ted Hackstadt (Rocky Mountain Laboratories).

Techniques: Infection, Transfection, Affinity Purification, Control, Membrane, Expressing, Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy

Journal: Microbiology Spectrum

Article Title: The Chlamydia trachomatis Inc Tri1 interacts with TRAF7 to displace native TRAF7 interacting partners

doi: 10.1128/spectrum.00453-24

Figure Lengend Snippet: TRAF7 is recruited to the inclusion. ( A ) Confocal immunofluorescence microscopy of HeLa cells transfected with mCh-TRAF7 for 24 h and then infected with L2+pTri1 FLAG for 24 h with or without aTc induction. Cells were fixed and stained with α-FLAG to visualize Tri1 FLAG . Merged images show mCh-TRAF7 (pseudo-colored magenta), Tri1 FLAG (pseudo-colored green), and DAPI (4′,6-diamidino-2-phenylindole) (blue) staining. ( B ) Confocal immunofluorescence microscopy of HeLa cells infected with L2+pTri1 FLAG for 24 h with or without aTc induction. Cells were fixed and stained with antibodies to FLAG (to detect Tri1), TRAF7 (pseudo-colored magenta in merge), and IncA (blue in merge, to delineate the inclusion membrane). The merge panel only includes TRAF7 and IncA. The small amount of transfected mCh-TRAF7 present at the inclusion in the absence of inducer likely represents recruitment by chromosomally encoded Tri1. Shown are single z-slices. I, inclusion. N, nucleus. Scale bar = 10 µm. Cell outlines are included for clarity.

Article Snippet: The C. trachomatis L2 strains overexpressing Tri1 FLAG (originally “CT224-FLAG”) and Dre1 FLAG were generous gifts from Drs. Mary Weber (University of Iowa) and Ted Hackstadt (Rocky Mountain Laboratories).

Techniques: Immunofluorescence, Microscopy, Transfection, Infection, Staining, Membrane

Journal: Microbiology Spectrum

Article Title: The Chlamydia trachomatis Inc Tri1 interacts with TRAF7 to displace native TRAF7 interacting partners

doi: 10.1128/spectrum.00453-24

Figure Lengend Snippet: The coiled-coil domain of Tri1 interacts with TRAF7. ( A ) Schematic of Strep-tagged Tri1 variants. Variants that interact with TRAF7 by co-AP are indicated with a “+” sign, and variants that do not interact are indicated with a ”−“ sign. ( B ) Lysates and eluates from affinity purifications of HEK293T cells co-transfected with the indicated Tri1-Strep or Strep-sfGFP-tagged variants (indicated with *) and with FLAG-TRAF7 were immunoblotted with the indicated antibodies. The control condition in which cells were transfected only with FLAG-TRAF7 is designated “−.” GAPDH serves as a loading control for the lysates. Only Tri1 variants containing a complete coiled-coil domain (Tri1-Strep, Tri1- Strep-sfGFP, and Tri1 84-147 - Strep-sfGFP) co-AP’d with TRAF7.

Article Snippet: The C. trachomatis L2 strains overexpressing Tri1 FLAG (originally “CT224-FLAG”) and Dre1 FLAG were generous gifts from Drs. Mary Weber (University of Iowa) and Ted Hackstadt (Rocky Mountain Laboratories).

Techniques: Transfection, Control

Journal: Microbiology Spectrum

Article Title: The Chlamydia trachomatis Inc Tri1 interacts with TRAF7 to displace native TRAF7 interacting partners

doi: 10.1128/spectrum.00453-24

Figure Lengend Snippet: The WD40 of TRAF7 is necessary and sufficient to interact with Tri1. ( A ) Schematic of TRAF7 constructs. Zn, zinc. CC, coiled-coil. RING, RING finger ubiquitin ligase domain. Variants that interact with TRAF7 by co-AP are indicated with a “+” sign, and variants that do not interact are indicated with a ”−“ sign. ( B ) Lysates and eluates of HEK293T cells co-transfected with Tri1-Strep and the indicated FLAG-TRAF7 variants (“−” indicates the control with no TRAF7 added) were affinity purified with Strep-Tactin beads and immunoblotted with the indicated antibodies. GAPDH serves as a loading control for lysates. Only variants containing the WD40 domain of TRAF7 co-affinity purified with Tri1. The slower migrating band present in some of the TRAF7 samples likely represents stable dimers. ( C ) Confocal immunofluorescence microscopy of HeLa cells transfected with the indicated mCh-TRAF7 variants for 24 h followed by infection L2+pTri1 FLAG in the presence of aTc for 24 h. Cells were fixed and stained with α-FLAG and DAPI and imaged by confocal microscopy. Cell membranes are outlined. Tri1 is pseudocolored green and TRAF7 is pseudocolored magenta in the merged image. Shown are single z-slices. Scale bar = 10 µm. I, inclusion. N, nucleus.

Article Snippet: The C. trachomatis L2 strains overexpressing Tri1 FLAG (originally “CT224-FLAG”) and Dre1 FLAG were generous gifts from Drs. Mary Weber (University of Iowa) and Ted Hackstadt (Rocky Mountain Laboratories).

Techniques: Construct, Ubiquitin Proteomics, Transfection, Control, Affinity Purification, Immunofluorescence, Microscopy, Infection, Staining, Confocal Microscopy

Journal: Microbiology Spectrum

Article Title: The Chlamydia trachomatis Inc Tri1 interacts with TRAF7 to displace native TRAF7 interacting partners

doi: 10.1128/spectrum.00453-24

Figure Lengend Snippet: Tri1 displaces MEKK2 and MEKK3 binding to TRAF7. ( A ) Schematic of displacement AP-MS analysis with a potential displaced TRAF7 native interactor represented by “X.” ( B ) HEK293T cells co-transfected with FLAG-TRAF7 WD40 (bait) and either Tri1 1-128 -Strep or Tri1-Strep. Lysates were affinity purified over FLAG beads and analyzed by LC/MS-MS. Shown are the average spectral counts from three biological replicates, SAINT scores, and BFDR of selected TRAF7 WD40 interacting partners in the presence of Tri1 1-128 -Strep or Tri1-Strep. SAINT scores closer to 1 with a BFDR ≤ 0.05 suggest a high confidence interaction. Tri1 displaces MEKK2 binding to TRAF7 WD40 but not to TCPD, TCPG, or DNJA2. ( C–E ) Validation of Tri1-mediated displacement of MEKK2 and MEKK3 binding to the TRAF7 WD40 domain by co-transfection studies. HEK293T cells were co-transfected with either Tri1-Strep or Tri1 84-147 - Strep ( C–E ), FLAG-TRAF7 WD40 ( C and E ), FLAG-TRAF7 ( D ), and Myc-MEKK3 ( E ). Cells only transfected with FLAG-TRAF7 WD40 ( C and E ) and FLAG-TRAF7 ( D ) were designated “−” and served as a control. Lysates were affinity purified using FLAG beads and analyzed by immunoblot with the indicated antibodies. GAPDH serves as a loading control for the lysates. Full-length Tri1, but not Tri1 1-128 , disrupts TRAF7 binding to MEKK2 and to MEKK3.

Article Snippet: The C. trachomatis L2 strains overexpressing Tri1 FLAG (originally “CT224-FLAG”) and Dre1 FLAG were generous gifts from Drs. Mary Weber (University of Iowa) and Ted Hackstadt (Rocky Mountain Laboratories).

Techniques: Binding Assay, Protein-Protein interactions, Transfection, Affinity Purification, Liquid Chromatography with Mass Spectroscopy, Biomarker Discovery, Cotransfection, Control, Western Blot

Journal: Infection and Immunity

Article Title: Inclusion Membrane Growth and Composition Are Altered by Overexpression of Specific Inclusion Membrane Proteins in Chlamydia trachomatis L2

doi: 10.1128/IAI.00094-21

Figure Lengend Snippet: Overexpression of CT813-FLAG from C. trachomatis L2 negatively impacts inclusion growth and progeny production. (A) HeLa cells seeded on coverslips were infected with C. trachomatis L2 CT813-FLAG, CT226-FLAG, or CT483-FLAG transformed strains or wild-type L2 and induced at 7 hpi with 5 or 20 nM aTc or not induced. Coverslips were methanol fixed at 36 hpi and stained for immunofluorescence to determine the inclusion area. The inclusion areas (μm 2 ) with standard deviations were plotted and analyzed for statistical significance by an ordinary one-way ANOVA with Tukey’s multiple-comparison test using GraphPad Prism 8.4.0. These data are combined from three biological replicates. #### & indicates a significant difference between C. trachomatis L2 transformed strains induced with 20 nM aTc. **** & , P < 0.0001; ***, P = 0.0004; all P values reflect comparison to the control (wild-type and uninduced strains); ns, not significant. The red values in the gray box in panel A are the average inclusion area for each C. trachomatis L2 transformed strain. (B) Duplicate wells of HeLa cells were infected as described for panel A. At 36 hpi, infected monolayers were lysed, serially diluted, and infected onto a fresh monolayer of HeLa cells (i.e., secondary infection) in medium containing penicillin to enumerate infectious progeny (inclusion-forming units [IFU]/ml). Infectious progeny (IFU/ml) (normalized to uninduced strains and expressed as a percentage of uninduced from three biological replicates and standard deviation) were plotted and statistically analyzed (ordinary one-way ANOVA with Tukey’s multiple-comparison test using GraphPad Prism 8.4.0). Only inclusions containing normal (not aberrant) bacteria were enumerated. (C and E) HeLa cells were infected with C. trachomatis L2 CT813-FLAG, CT226-FLAG, or CT483-FLAG transformed strains, or wild-type C. trachomatis L2, and either not induced or induced at 7 hpi (5 nM or 20 nM aTc). DNA collected from separate wells of a 6-well plate at 7, 16, 24, and 36 hpi was processed as described in Materials and Methods. The genomic DNA (gDNA; ng) (C) and plasmid DNA (pDNA; ng) (E) data sets from three biological replicates were plotted using GraphPad Prism 8.4.0. The data are representative of three independent experiments. (D) Plasmid loss was indicated by inclusions containing aberrant bacteria in medium containing penicillin (i.e., sensitivity due to the loss of the plasmid-borne bla resistance gene). To enumerate the percentage of inclusions containing aberrant bacteria, the number of inclusions with aberrant bacteria was divided by the total number of inclusions counted (B) from three biological replicates and the standard deviation was plotted and statistically analyzed as described for panels A and B using GraphPad Prism 8.4.0. Differences were not statistically significant.

Article Snippet: The inclusion areas of C. trachomatis L2 IncF-FLAG were 247.0 ± 58.8 μm 2 for uninduced cultures, 171.4 ± 59.0 μm 2 for 1 nM aTc, 57.66 ± 18.1 μm 2 for 5 nM aTc, and 27.5 ± 15.5 μm 2 for 20 nM (Fig. S1A).

Techniques: Over Expression, Infection, Transformation Assay, Staining, Immunofluorescence, Comparison, Control, Standard Deviation, Bacteria, Plasmid Preparation

Journal: Infection and Immunity

Article Title: Inclusion Membrane Growth and Composition Are Altered by Overexpression of Specific Inclusion Membrane Proteins in Chlamydia trachomatis L2

doi: 10.1128/IAI.00094-21

Figure Lengend Snippet: The overexpression of CT813-FLAG from C. trachomatis L2 results in decreased detectable IncA, CT223, and IncE. HeLa cells were infected with C. trachomatis L2 CT813-FLAG, CT226-FLAG, or CT483-FLAG transformed strains, or wild-type C. trachomatis L2, and either not induced or induced at 7 hpi (5 nM or 20 nM aTc). At 48 hpi, infected monolayers were lysed in 8 M urea supplemented with 1% SDS, 10 mM Tris-HCl (pH 7.4), 2.5% β-mercaptoethanol, and nuclease. Lysates were normalized for equal protein content, separated by SDS-PAGE, and transferred to PVDF to blot for chlamydial proteins, MOMP, IncA, CT223, and IncE. GAPDH was used as a loading control. These data are representative of three independent experiments.

Article Snippet: The inclusion areas of C. trachomatis L2 IncF-FLAG were 247.0 ± 58.8 μm 2 for uninduced cultures, 171.4 ± 59.0 μm 2 for 1 nM aTc, 57.66 ± 18.1 μm 2 for 5 nM aTc, and 27.5 ± 15.5 μm 2 for 20 nM (Fig. S1A).

Techniques: Over Expression, Infection, Transformation Assay, SDS Page, Control

Journal: Infection and Immunity

Article Title: Inclusion Membrane Growth and Composition Are Altered by Overexpression of Specific Inclusion Membrane Proteins in Chlamydia trachomatis L2

doi: 10.1128/IAI.00094-21

Figure Lengend Snippet: The overexpression of CT813-FLAG from C. trachomatis L2 results in loss of detectable endogenous IncE in the inclusion membrane. (A) HeLa cells infected with C. trachomatis L2 transformed strains or wild-type L2 were induced with 5 or 20 nM aTc or not induced at 7 hpi. Coverslips were methanol fixed at 36 hpi and stained for immunofluorescence to observe expression of the constructs (FLAG; red), IncE (green), IncA (pink), or DNA (DAPI; blue) and imaged at ×63 magnification using the same exposure for each sample. Scale bar, 10 μm. Individual panels were converted to black and white and inverted to visualize the loss of IncE staining. These data are from three biological replicates. (B) C. trachomatis L2-infected HeLa cells were fixed at 18 hpi and stained for immunofluorescence to observe IncE (green), MOMP (red), and DNA (blue). Coverslips were imaged using the same exposure for each sample at ×63 magnification. Scale bar, 10 μm. (C) The intensity of IncE was measured using ImageJ from a minimum of 80 inclusions per experiment (see Materials and Methods). For each image, the background integrated density was subtracted from individual images, and the intensity was normalized to the inclusion perimeter (integrated density/μm). The mean integrated density/μm is reported in red for each sample measured. The intensity (integrated density/μm) and standard deviation were plotted using GraphPad Prism 8.4.0. Samples were analyzed for statistical significance using a one-way ANOVA and Tukey’s multiple-comparison test. ****, P < 0.0001 between C. trachomatis L2 transformed strains; ####, P < 0.0001 between C. trachomatis L2 transformed strains.

Article Snippet: The inclusion areas of C. trachomatis L2 IncF-FLAG were 247.0 ± 58.8 μm 2 for uninduced cultures, 171.4 ± 59.0 μm 2 for 1 nM aTc, 57.66 ± 18.1 μm 2 for 5 nM aTc, and 27.5 ± 15.5 μm 2 for 20 nM (Fig. S1A).

Techniques: Over Expression, Membrane, Infection, Transformation Assay, Staining, Immunofluorescence, Expressing, Construct, Standard Deviation, Comparison

Journal: Infection and Immunity

Article Title: Inclusion Membrane Growth and Composition Are Altered by Overexpression of Specific Inclusion Membrane Proteins in Chlamydia trachomatis L2

doi: 10.1128/IAI.00094-21

Figure Lengend Snippet: The overexpression of CT813-FLAG from C. trachomatis L2 results in reduced localization of endogenous SNX6 to the inclusion membrane. HeLa cells infected with C. trachomatis L2 transformed strains or wild-type L2 were induced at 7 hpi with 5 or 20 nM aTc or were not induced. Coverslips were methanol fixed at 30 hpi and stained for immunofluorescence to observe expression of the Inc-FLAG constructs (FLAG; red), SNX6 (green), IncA (pink), or DNA (DAPI; blue). Coverslips were imaged at ×63 magnification using the same exposure (scale bar, 10 μm). Arrows indicate C. trachomatis L2 CT813-FLAG inclusions that do not have SNX6. These data are representative of three independent experiments.

Article Snippet: The inclusion areas of C. trachomatis L2 IncF-FLAG were 247.0 ± 58.8 μm 2 for uninduced cultures, 171.4 ± 59.0 μm 2 for 1 nM aTc, 57.66 ± 18.1 μm 2 for 5 nM aTc, and 27.5 ± 15.5 μm 2 for 20 nM (Fig. S1A).

Techniques: Over Expression, Membrane, Infection, Transformation Assay, Staining, Immunofluorescence, Expressing, Construct

Journal: Infection and Immunity

Article Title: Inclusion Membrane Growth and Composition Are Altered by Overexpression of Specific Inclusion Membrane Proteins in Chlamydia trachomatis L2

doi: 10.1128/IAI.00094-21

Figure Lengend Snippet: The overexpression of CT813-FLAG from C. trachomatis L2 results in reduced transcription of early and midcycle genes. HeLa cells were infected with C. trachomatis L2 CT813-FLAG, CT226-FLAG, or CT483-FLAG transformed strains, or wild-type C. trachomatis L2, and either not induced or induced at 7 hpi (5 nM or 20 nM aTc). RNA and DNA collected from separate wells of a 6-well plate at 7, 16, 24, and 36 hpi were processed as described in Materials and Methods. Normalized RNA was reverse transcribed to cDNA, and inc expression was measured by quantitative PCR. cDNA (ng) was normalized to genomic DNA (ng) for three biological replicates (except for clpP2 , n = 2), and then these data were plotted using GraphPad Prism 8.4.0. Transcript profiles of early gene euo (A), midcycle gene clpP2 (B), late gene omcB (C), an early expressed inc gene, incE (D), and a midcycle inc gene, incA (E), are shown. Normalized transcripts from wild-type and C. trachomatis L2 CT813-FLAG were log transformed and then analyzed for statistical significance using a paired two-tailed Student's t test. Samples that were statistically analyzed were wild type and CT813 at 16 hpi (for omcB only), 24 hpi (all genes), and 36 hpi (all genes). Asterisks denote statistical significance: **, P ≤ 0.01; *, P ≤ 0.05.

Article Snippet: The inclusion areas of C. trachomatis L2 IncF-FLAG were 247.0 ± 58.8 μm 2 for uninduced cultures, 171.4 ± 59.0 μm 2 for 1 nM aTc, 57.66 ± 18.1 μm 2 for 5 nM aTc, and 27.5 ± 15.5 μm 2 for 20 nM (Fig. S1A).

Techniques: Over Expression, Infection, Transformation Assay, Reverse Transcription, Expressing, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: Infection and Immunity

Article Title: Inclusion Membrane Growth and Composition Are Altered by Overexpression of Specific Inclusion Membrane Proteins in Chlamydia trachomatis L2

doi: 10.1128/IAI.00094-21

Figure Lengend Snippet: The overexpression of CT813 and IncF from C. trachomatis L2 results in fewer organisms per inclusion. HeLa cells were infected with C. trachomatis L2, CT226-FLAG, CT813-FLAG, or IncF-FLAG transformed strains and either not induced or induced at 7 hpi (5 nM or 20 nM aTc). At 36 hpi, cells were fixed and processed for transmission electron microscopy as previously described . The graphed results are the mean with standard error of the mean and are combined data from two biological replicates with the following total numbers of inclusions analyzed: 14 for CT226–0 nM aTc, 12 for CT226–20 nM aTc, 18 for CT813–0 nM aTc, 27 for CT813–20 nM aTc, 20 for IncF–0 nM aTc, and 23 for IncF–20 nM aTc. The data were statistically analyzed in GraphPad Prism (version 8.4.3) using an ordinary one-way ANOVA with Tukey’s multiple-comparison test.

Article Snippet: The inclusion areas of C. trachomatis L2 IncF-FLAG were 247.0 ± 58.8 μm 2 for uninduced cultures, 171.4 ± 59.0 μm 2 for 1 nM aTc, 57.66 ± 18.1 μm 2 for 5 nM aTc, and 27.5 ± 15.5 μm 2 for 20 nM (Fig. S1A).

Techniques: Over Expression, Infection, Transformation Assay, Transmission Assay, Electron Microscopy, Comparison