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