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ATCC wild type c trachomatis serovar l2 strain

(A) Under non-stressed conditions, HrcA binds to CIRCE elements and represses transcription from the KP1 and EP promoters. (B) Heat shock disrupts HrcA repression, allowing RNA polymerase (RNAP) to transcribe the hrcA-grpE-dnaK and groES-groEL operons. Arrows denote the extent of transcription. Note that dnaK is regulated by both the operon promoter and its own internal promoter (KP2). (C) Consensus and putative CIRCE sequences upstream of indicated heat shock genes in C. <t>trachomatis</t> (Ct) and Rhizobium meliloti (Rm) . Blue letters indicate nucleotide deviations from the consensus; lowercase letters denote mismatches between inverted repeats. The-35 core promoter elements for C. trachomatis genes are shown. (D) HrcA binds the ctl0271 promoter in chlamydiae. ChIP-qPCR was performed in biological triplicate using <t>L2/NH-HrcA</t> cultures treated with 10 nM ATC for 30 min at 37 °C. Anti-His antibody enrichment of the ctl0271 promoter was comparable to that of known HrcA targets ( groESL and dnaK P1), and significantly higher than enrichment at non-target promoters KP2 and E2P ( groEL2 promoter) , . (E) Expression of ctl0271 , dnaK , and groEL was reduced upon ATC-induced NH-HrcA overexpression in L2/NH-HrcA, as determined by qRT-PCR. (F) ctl0271 expression is induced in wild-type L2 cultured at 45 °C, as measured by qRT-PCR. (D–F) Data represent the mean ± SD of biological triplicates. (G, H) Based on results from panels B–F, the updated HrcA regulon includes ctl0271 (renamed hagF ) as its sixth target gene. (A, B, G, H) These panels were generated using a paid subscription to bioRender.

Wild Type C Trachomatis Serovar L2 Strain, supplied by ATCC, used in various techniques. Bioz Stars score: 96/100, based on 459 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "A lineage-specific heat-induced feedback loop controls HrcA to promote chlamydial fitness under stress"

Article Title: A lineage-specific heat-induced feedback loop controls HrcA to promote chlamydial fitness under stress

Journal: bioRxiv

doi: 10.1101/2025.05.30.657042

Figure Legend Snippet: (A) Under non-stressed conditions, HrcA binds to CIRCE elements and represses transcription from the KP1 and EP promoters. (B) Heat shock disrupts HrcA repression, allowing RNA polymerase (RNAP) to transcribe the hrcA-grpE-dnaK and groES-groEL operons. Arrows denote the extent of transcription. Note that dnaK is regulated by both the operon promoter and its own internal promoter (KP2). (C) Consensus and putative CIRCE sequences upstream of indicated heat shock genes in C. trachomatis (Ct) and Rhizobium meliloti (Rm) . Blue letters indicate nucleotide deviations from the consensus; lowercase letters denote mismatches between inverted repeats. The-35 core promoter elements for C. trachomatis genes are shown. (D) HrcA binds the ctl0271 promoter in chlamydiae. ChIP-qPCR was performed in biological triplicate using L2/NH-HrcA cultures treated with 10 nM ATC for 30 min at 37 °C. Anti-His antibody enrichment of the ctl0271 promoter was comparable to that of known HrcA targets ( groESL and dnaK P1), and significantly higher than enrichment at non-target promoters KP2 and E2P ( groEL2 promoter) , . (E) Expression of ctl0271 , dnaK , and groEL was reduced upon ATC-induced NH-HrcA overexpression in L2/NH-HrcA, as determined by qRT-PCR. (F) ctl0271 expression is induced in wild-type L2 cultured at 45 °C, as measured by qRT-PCR. (D–F) Data represent the mean ± SD of biological triplicates. (G, H) Based on results from panels B–F, the updated HrcA regulon includes ctl0271 (renamed hagF ) as its sixth target gene. (A, B, G, H) These panels were generated using a paid subscription to bioRender.

Techniques Used: ChIP-qPCR, Expressing, Over Expression, Quantitative RT-PCR, Cell Culture, Generated

(A) Effective silencing of hagF in C. trachomatis using deactivated CRISPR-associated protein 12 (dC12). L2/dC12-ntg expresses an ATC-inducible dC12 and a non-targeting guide RNA. L2/dC12-hagF-g1 and L2/dC12-hagF-g2 express dC12 and a guide RNA targeting hagF (g1 or g2). The left panel shows elevated dC12 transcript levels upon ATC induction in all three transformants. The right panel shows reduced hagF expression in ATC-treated L2/dC12-hagF-g1 and L2/dC12-hagF-g2 but not in L2/dC12-ntg. RNA levels were quantified by qRT-PCR. (B) ATC-induced dC12 expression has no effect on genome replication (left) or EB formation (right) in 37 °C cultures of L2/dC12-ntg. (C, D) ATC-induced hagF silencing has no detectable effect on genome replication (left) or EB formation (right) in 37 °C cultures of L2/dC12-hagF-g1 (C) and L2/dC12-hagF-g2 (D). (E) ATC-induced dC12 expression has no effect on genome replication (left) or EB formation (right) in 40.5 °C cultures of L2/dC12-ntg. (F, G) ATC-induced hagF silencing does not affect genome replication (left) but causes a substantial reduction in EB formation (right) in 40.5 °C cultures of L2/dC12-hagF-g1 (F) and L2/dC12-hagF-g2 (G). (B-G) ATC was added at 0 hpi. Genome copy number was quantified at the indicated times. EB yields were measured at 30 hpi for 37 °C cultures and at 40 hpi for 40.5 °C cultures.

Figure Legend Snippet: (A) Effective silencing of hagF in C. trachomatis using deactivated CRISPR-associated protein 12 (dC12). L2/dC12-ntg expresses an ATC-inducible dC12 and a non-targeting guide RNA. L2/dC12-hagF-g1 and L2/dC12-hagF-g2 express dC12 and a guide RNA targeting hagF (g1 or g2). The left panel shows elevated dC12 transcript levels upon ATC induction in all three transformants. The right panel shows reduced hagF expression in ATC-treated L2/dC12-hagF-g1 and L2/dC12-hagF-g2 but not in L2/dC12-ntg. RNA levels were quantified by qRT-PCR. (B) ATC-induced dC12 expression has no effect on genome replication (left) or EB formation (right) in 37 °C cultures of L2/dC12-ntg. (C, D) ATC-induced hagF silencing has no detectable effect on genome replication (left) or EB formation (right) in 37 °C cultures of L2/dC12-hagF-g1 (C) and L2/dC12-hagF-g2 (D). (E) ATC-induced dC12 expression has no effect on genome replication (left) or EB formation (right) in 40.5 °C cultures of L2/dC12-ntg. (F, G) ATC-induced hagF silencing does not affect genome replication (left) but causes a substantial reduction in EB formation (right) in 40.5 °C cultures of L2/dC12-hagF-g1 (F) and L2/dC12-hagF-g2 (G). (B-G) ATC was added at 0 hpi. Genome copy number was quantified at the indicated times. EB yields were measured at 30 hpi for 37 °C cultures and at 40 hpi for 40.5 °C cultures.

Techniques Used: CRISPR, Expressing, Quantitative RT-PCR

Variant Assay:

Article Title: The Chlamydia trachomatis inclusion membrane protein CpoS counteracts STING-mediated cellular surveillance and suicide programs
Article Snippet: .. C. trachomatis strain L2/434/Bu (CTL2; ATCC VR-902B), the rifampin-resistant variant CTL2-R ( Nguyen and Valdivia, 2012 ), and derivatives described in this study were propagated in Vero cells. ..

Infection:

Article Title: The Chlamydia effector CpoS modulates the inclusion microenvironment and restricts the interferon response by acting on Rab35
Article Snippet: .. Chlamydia strains and infection Experiments were carried out with C. trachomatis strain L2/434/Bu (CTL2, ATCC VR-902B) and the derivatives generated in this study (Table S2). ..

Generated:

Article Title: The Chlamydia protein CpoS modulates the inclusion microenvironment and restricts the interferon response by acting on Rab35
Article Snippet: In experiments including infection with Chlamydia , cells were transfected with these plasmids either 2-4 hours before infection or at 1 hpi using jetPRIME transfection reagent (Polyplus), with a medium exchange after 2-3 hours, or lipofectamine 2000 (Invitrogen), without medium exchange. .. Experiments were carried out with C. trachomatis strain L2/434/Bu (CTL2, ATCC VR-902B) and the derivatives generated in this study (Table S1). ..

Purification:

Article Title: Fluorescence lifetime imaging unravels C. trachomatis metabolism and its crosstalk with the host cell.
Article Snippet: .. C. trachomatis strain L2 (ATCC VR-902B) and C. pneumoniae CWL029 strain (ATCC VR-1310) were purified on discontinuous density gradients. ..

Article Title: Fluorescence Lifetime Imaging Unravels C. trachomatis Metabolism and Its Crosstalk with the Host Cell
Article Snippet: .. C. trachomatis strain L2 (ATCC VR-902B) and C. pneumoniae CWL029 strain (ATCC VR-1310) were purified on discontinuous density gradients. ..

Isolation:

Article Title: Methylene Blue-Loaded NanoMOFs: Accumulation in Chlamydia trachomatis Inclusions and Light/Dark Antibacterial Effects.
Article Snippet: Metal−organic framework nanoparticles (nanoMOFs) are promising nanomaterials for biomedical applications.. Some of them, including biodegradable porous iron carboxylates are proposed for encapsulation and delivery of antibiotics.. Due to the high drug loading capacity and fast internalization kinetics, nanoMOFs are more beneficial for the treatment of intracellular bacterial infections compared to free antibacterial drugs, which poorly accumulate inside the cells because of the inability to cross membrane barriers or have low intracellular retention.

Article Title: NanoMOFs with Encapsulated Photosensitizer: Accumulation in Chlamydia trachomatis Inclusions and Antimicrobial Effects
Article Snippet: The cell images were acquired in multitrack mode using confocal microscope Zeiss LSM 710 (Carl Zeiss, Oberkochen, Germany), equipped with Plan-Apochromat ×63/1.4 Oil DIC lens. .. C. trachomatis strain L2/434/Bu (ATCC VR-902B) was routinely propagated in McCoy cells (ATCC CRL-1696) and isolated by urografin gradient ultracentrifugation as described previously. ..

Image Search Results

Journal: bioRxiv

Article Title: A lineage-specific heat-induced feedback loop controls HrcA to promote chlamydial fitness under stress

doi: 10.1101/2025.05.30.657042

Figure Lengend Snippet: (A) Under non-stressed conditions, HrcA binds to CIRCE elements and represses transcription from the KP1 and EP promoters. (B) Heat shock disrupts HrcA repression, allowing RNA polymerase (RNAP) to transcribe the hrcA-grpE-dnaK and groES-groEL operons. Arrows denote the extent of transcription. Note that dnaK is regulated by both the operon promoter and its own internal promoter (KP2). (C) Consensus and putative CIRCE sequences upstream of indicated heat shock genes in C. trachomatis (Ct) and Rhizobium meliloti (Rm) . Blue letters indicate nucleotide deviations from the consensus; lowercase letters denote mismatches between inverted repeats. The-35 core promoter elements for C. trachomatis genes are shown. (D) HrcA binds the ctl0271 promoter in chlamydiae. ChIP-qPCR was performed in biological triplicate using L2/NH-HrcA cultures treated with 10 nM ATC for 30 min at 37 °C. Anti-His antibody enrichment of the ctl0271 promoter was comparable to that of known HrcA targets ( groESL and dnaK P1), and significantly higher than enrichment at non-target promoters KP2 and E2P ( groEL2 promoter) , . (E) Expression of ctl0271 , dnaK , and groEL was reduced upon ATC-induced NH-HrcA overexpression in L2/NH-HrcA, as determined by qRT-PCR. (F) ctl0271 expression is induced in wild-type L2 cultured at 45 °C, as measured by qRT-PCR. (D–F) Data represent the mean ± SD of biological triplicates. (G, H) Based on results from panels B–F, the updated HrcA regulon includes ctl0271 (renamed hagF ) as its sixth target gene. (A, B, G, H) These panels were generated using a paid subscription to bioRender.

Article Snippet: Wild-type C. trachomatis serovar L2 strain 434/BU (L2) was obtained from the American Type Culture Collection (ATCC) and expanded in our laboratory using L929 cells as host cells.

Techniques: ChIP-qPCR, Expressing, Over Expression, Quantitative RT-PCR, Cell Culture, Generated

Journal: bioRxiv

Article Title: A lineage-specific heat-induced feedback loop controls HrcA to promote chlamydial fitness under stress

doi: 10.1101/2025.05.30.657042

Figure Lengend Snippet: (A) Effective silencing of hagF in C. trachomatis using deactivated CRISPR-associated protein 12 (dC12). L2/dC12-ntg expresses an ATC-inducible dC12 and a non-targeting guide RNA. L2/dC12-hagF-g1 and L2/dC12-hagF-g2 express dC12 and a guide RNA targeting hagF (g1 or g2). The left panel shows elevated dC12 transcript levels upon ATC induction in all three transformants. The right panel shows reduced hagF expression in ATC-treated L2/dC12-hagF-g1 and L2/dC12-hagF-g2 but not in L2/dC12-ntg. RNA levels were quantified by qRT-PCR. (B) ATC-induced dC12 expression has no effect on genome replication (left) or EB formation (right) in 37 °C cultures of L2/dC12-ntg. (C, D) ATC-induced hagF silencing has no detectable effect on genome replication (left) or EB formation (right) in 37 °C cultures of L2/dC12-hagF-g1 (C) and L2/dC12-hagF-g2 (D). (E) ATC-induced dC12 expression has no effect on genome replication (left) or EB formation (right) in 40.5 °C cultures of L2/dC12-ntg. (F, G) ATC-induced hagF silencing does not affect genome replication (left) but causes a substantial reduction in EB formation (right) in 40.5 °C cultures of L2/dC12-hagF-g1 (F) and L2/dC12-hagF-g2 (G). (B-G) ATC was added at 0 hpi. Genome copy number was quantified at the indicated times. EB yields were measured at 30 hpi for 37 °C cultures and at 40 hpi for 40.5 °C cultures.

Techniques: CRISPR, Expressing, Quantitative RT-PCR

(A) Schematic view of the use of click chemistry to track nucleotide incorporation in nucleic acids. Cells are incubated with a nucleoside analog linked to an alkyne group (5-EC or N6pA). Nucleoside analogs are metabolized into nucleotides in the cytoplasm prior to incorporation into nucleic acids. After fixation and permeabilization, the click reaction between the alkyne and an azide group coupled to a fluorochrome, catalyzed by copper, allows to label the nucleotides. Created in https://BioRender.com. (B) HeLa cells treated with control (siCtrl) or siRNA targeting UCK2 (siUCK2) were infected with C. trachomatis (LGVL2 mCherry ) for 16 h before adding 5-EC or N6pA. Cells were fixed 6 h later, permeabilized and processed for covalent staining of the probes with Alexa-488 (green) and of DNA with DAPI (blue). Bacteria appear in red. The dotted white lines delimit the inclusions. Bar = 10 μm. (C-D) (Left) Fluorescence intensities of nucleoside analogs and of DAPI (C) or mCherry (D), in the nuclei and inclusions, respectively, were quantified in 50 cells per experiments. a.u. arbitrary units. P-values were determined with a paired t-test. (Right) Fluorescence from the nucleoside analogs normalized to the DAPI or to the mCherry signals, respectively. P-values were determined with a ratio paired t-test. Means ± SD of four independent experiments are shown. (E) HeLa cells treated with siCtrl or siUCK2 were infected with LGV L2 GFP (multiplicity of infection (MOI) ~ 0.3) and the percent of infected cells was determined 24 h later. A duplicate well was used to determine IFUs produced 48 hpi. Three independent experiments are represented. P-values were determined with a paired t-test (primary infection) and a ratio paired t-test (progeny).

Journal: microPublication Biology

Article Title: UCK2-dependent conversion of cytidine to CTP is required for CTP uptake by Chlamydia trachomatis

doi: 10.17912/micropub.biology.001607

Figure Lengend Snippet: (A) Schematic view of the use of click chemistry to track nucleotide incorporation in nucleic acids. Cells are incubated with a nucleoside analog linked to an alkyne group (5-EC or N6pA). Nucleoside analogs are metabolized into nucleotides in the cytoplasm prior to incorporation into nucleic acids. After fixation and permeabilization, the click reaction between the alkyne and an azide group coupled to a fluorochrome, catalyzed by copper, allows to label the nucleotides. Created in https://BioRender.com. (B) HeLa cells treated with control (siCtrl) or siRNA targeting UCK2 (siUCK2) were infected with C. trachomatis (LGVL2 mCherry ) for 16 h before adding 5-EC or N6pA. Cells were fixed 6 h later, permeabilized and processed for covalent staining of the probes with Alexa-488 (green) and of DNA with DAPI (blue). Bacteria appear in red. The dotted white lines delimit the inclusions. Bar = 10 μm. (C-D) (Left) Fluorescence intensities of nucleoside analogs and of DAPI (C) or mCherry (D), in the nuclei and inclusions, respectively, were quantified in 50 cells per experiments. a.u. arbitrary units. P-values were determined with a paired t-test. (Right) Fluorescence from the nucleoside analogs normalized to the DAPI or to the mCherry signals, respectively. P-values were determined with a ratio paired t-test. Means ± SD of four independent experiments are shown. (E) HeLa cells treated with siCtrl or siUCK2 were infected with LGV L2 GFP (multiplicity of infection (MOI) ~ 0.3) and the percent of infected cells was determined 24 h later. A duplicate well was used to determine IFUs produced 48 hpi. Three independent experiments are represented. P-values were determined with a paired t-test (primary infection) and a ratio paired t-test (progeny).

Article Snippet: C. trachomatis serovar LGV L2 strain 434 (obtained from ATCC) stably expressing mCherry (click chemistry) or the green fluorescent protein (progeny) were used (Agaisse & Derré, 2013).

Techniques: Expressing, Incubation, Control, Infection, Staining, Bacteria, Fluorescence, Produced

a Graphical depiction of the protective effects of MDP against ionizing radiation. MDP complex protects proteinaceous components of bacterial outer membranes from ROS-induced damage while allowing for the destruction of the genome. b Dose-response γ-irradiation kill curve for C. trachomatis . Each data point represents the mean of three biological replicates obtained from three separate experiments (presented individually in Supplementary Fig. ). Error bars represent the standard error of the mean. LOD limit of detection. c Transmission electron micrographs of C. trachomatis EBs (dark spheres) subjected to 20× the LD 100 of γ-radiation (20 kGy) in the presence or absence of MDP.

Journal: NPJ Vaccines

Article Title: Irradiated whole cell Chlamydia vaccine confers significant protection in a murine genital tract challenge model

doi: 10.1038/s41541-024-00968-z

Figure Lengend Snippet: a Graphical depiction of the protective effects of MDP against ionizing radiation. MDP complex protects proteinaceous components of bacterial outer membranes from ROS-induced damage while allowing for the destruction of the genome. b Dose-response γ-irradiation kill curve for C. trachomatis . Each data point represents the mean of three biological replicates obtained from three separate experiments (presented individually in Supplementary Fig. ). Error bars represent the standard error of the mean. LOD limit of detection. c Transmission electron micrographs of C. trachomatis EBs (dark spheres) subjected to 20× the LD 100 of γ-radiation (20 kGy) in the presence or absence of MDP.

Article Snippet: C. trachomatis serovar L2 strain 434/Bu was provided by Harlan Caldwell (Rocky Mountain Laboratories, MT, USA).

Techniques: Irradiation, Transmission Assay

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.

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

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.

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.

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

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.

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.

Techniques: Transfection, Control

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.

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.

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

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.

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.

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

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wild type c trachomatis serovar l2 strain (ATCC)

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1) Product Images from "A lineage-specific heat-induced feedback loop controls HrcA to promote chlamydial fitness under stress"

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