Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Genetic Inactivation of Chlamydia trachomatis Inclusion Membrane Protein CT228 Alters MYPT1 Recruitment, Extrusion Production, and Longevity of Infection

doi: 10.3389/fcimb.2018.00415

Figure Lengend Snippet: Histopathological assessment of reproductive tracts post-infection. Mice ( n = 5 per group) were euthanized at 23 and 64 days post-infection (dpi) and entire reproductive tracts were removed and formalin fixed for histology. Representative images of H&E stained sections of uterine tissue were captured using an Olympus DP70 for mice infected with (A) C. trachomatis L2-wild type, 23 dpi, (B) C. trachomatis L2-ΔCT228, 23 dpi, (C) C. trachomatis L2-wild type, 64 dpi, (D) C. trachomatis L2-ΔCT228, 64 dpi. See Table for pathological clinical scoring of reproductive tracts at 64 dpi.

Article Snippet: Chlamydia trachomatis serovar L2-wild type and L2-ΔCT228 (LGV 434/Bu, originating from the Hackstadt Lab at Rocky Mountain Laboratories, Hamilton, MT) were propagated in HeLa 229 cells and purified by Renografin density gradient centrifugation as previously described (Caldwell et al., ).

Techniques: Infection, Staining

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Genetic Inactivation of Chlamydia trachomatis Inclusion Membrane Protein CT228 Alters MYPT1 Recruitment, Extrusion Production, and Longevity of Infection

doi: 10.3389/fcimb.2018.00415

Figure Lengend Snippet: TargeTron inactivation of CT228 . (A) TargeTron insertion site in CT228 . (B) Schematic of CT228 containing GII( aadA ). (C) PCR verification of TargeTron insertion in L2-ΔCT228. PCRs were used to amplify incA, CT228 and the Intron in the L2-wild type, L2-ΔCT228 (Mut), and TargeTron vector (pDFTT295). (D) Immunofluorescence images of L2-wild type and L2-ΔCT228 at 18 h post-infection stained with anti-CT228 and anti- Chlamydia LPS followed by fluorescent secondary antibodies. Scale bar, 10 μm.

Article Snippet: Chlamydia trachomatis serovar L2-wild type and L2-ΔCT228 (LGV 434/Bu, originating from the Hackstadt Lab at Rocky Mountain Laboratories, Hamilton, MT) were propagated in HeLa 229 cells and purified by Renografin density gradient centrifugation as previously described (Caldwell et al., ).

Techniques: Plasmid Preparation, Immunofluorescence, Infection, Staining

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Genetic Inactivation of Chlamydia trachomatis Inclusion Membrane Protein CT228 Alters MYPT1 Recruitment, Extrusion Production, and Longevity of Infection

doi: 10.3389/fcimb.2018.00415

Figure Lengend Snippet: TargetTron inactivation of Chlamydia trachomatis CT228 and subsequent CT229-CT224 gene linkage group transcription. (A) Physical map of C. trachomatis CT229-CT224 gene linkage group. Solid arrows represent CT229-CT224 ORFs. Gene designations are indicated above each ORF. Green and red arrows indicate forward and reverse primers, respectively, used for RT-PCR analysis. Red arrowhead indicates the point of TargeTron insertion within CT228 . (B) Agarose gel image(s) show RT-PCR products corresponding to groEL (control), and CT229-CT224 gene linkage group expression (correlating to the indicated primers above). Total RNA (L2-wild type or L2-ΔCT228) is indicated above corresponding gel lanes. + indicates RT added to reaction. – Indicates RT not added to reaction. L2-wild type and L2-ΔCT228 DNA act as PCR controls. (C) Growth curves of L2-wild type and L2-ΔCT228 were performed in triplicate, the entire experiment was repeated on three separate occasions and a representative growth curve was selected. Error bars represent standard deviation.

Article Snippet: Chlamydia trachomatis serovar L2-wild type and L2-ΔCT228 (LGV 434/Bu, originating from the Hackstadt Lab at Rocky Mountain Laboratories, Hamilton, MT) were propagated in HeLa 229 cells and purified by Renografin density gradient centrifugation as previously described (Caldwell et al., ).

Techniques: Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Expressing, Standard Deviation

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Genetic Inactivation of Chlamydia trachomatis Inclusion Membrane Protein CT228 Alters MYPT1 Recruitment, Extrusion Production, and Longevity of Infection

doi: 10.3389/fcimb.2018.00415

Figure Lengend Snippet: Recruitment of MYPT1 and Myosin phosphatase pathway components and extrusion production by C. trachomatis L2-wild type and L2-ΔCT228. HeLa cell monolayers were infected at a MOI of ~0.5 with L2-wild type and L2-ΔCT228 for 18 h (in technical triplicate). Cells were fixed and stained with primary antibodies to MYPT1, Chlamydia LPS, MLC2 (pS19), Src Y474, MLCK (pY471), non-muscle Myosin IIa and IIb followed by fluorescent secondary antibodies. Experiments were repeated on three separate occasions and representative images were selected. (A,B) Top panel shows individual and merged images of MYPT1 recruitment (green) and Chlamydia LPS staining (red) in both the L2-wild type and L2-ΔCT228. Lower panel of individual and merged images show MLC2 (pS19), MLCK (pY471), and Mysoin IIa and IIb (green) co-localizing with active Src Y474 kinase (red) in microdomains at the periphery of inclusions in both L2-wild type and L2-ΔCT228. Scale bar, 10 μm. (C) Total protein from L2-wild type and L2-ΔCT228 infected HeLa cells at 24 and 48 h post-infection were assessed for MLC2, MLC2 (pS19), HsP60, and GAPDH levels by western blot analysis. (D) HeLa cells were treated with either Scramble (Scr) or MYPT1 siRNA for 48 h prior to infection with L2-wild type and L2-ΔCT228. Protein samples were assessed for MYPT1 and GAPDH levels by western blot. (E) Extrusions collected and (F) IFUs were assessed for L2 wild-type and L2-ΔCT228 at 48 h post-infection in either Scramble (symbols and solid bars) or MYPT1 (open symbols and white bars) siRNA treated HeLa cells. * p < 0.0001.

Article Snippet: Chlamydia trachomatis serovar L2-wild type and L2-ΔCT228 (LGV 434/Bu, originating from the Hackstadt Lab at Rocky Mountain Laboratories, Hamilton, MT) were propagated in HeLa 229 cells and purified by Renografin density gradient centrifugation as previously described (Caldwell et al., ).

Techniques: Infection, Staining, Western Blot

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Genetic Inactivation of Chlamydia trachomatis Inclusion Membrane Protein CT228 Alters MYPT1 Recruitment, Extrusion Production, and Longevity of Infection

doi: 10.3389/fcimb.2018.00415

Figure Lengend Snippet: Recoverable IFUs shed by mice infected with C. trachomatis L2-wild type and L2-ΔCT228. Female C3H/HeJ mice were intravaginally infected with 1 × 10 6 EBs of either L2-wild type or L2-ΔCT228. Recoverable IFUs were obtained by swabbing vaginal tracts and enumerating on HeLa cell monolayers. Recoverable IFU data are expressed for (A) L2-wild type ( n = 8) and (B) L2-ΔCT228 ( n = 9) on a logarithmic scale from Day 7 to 42 post-infection. Effect of time ( * p = 0.006) was observed in mice infected with L2-wild type (repeated measures one-way ANOVA). For day 28, 6/8 L2-wild type infected mice were clear compared to 3/9 L2- ΔCT228. Triangles represent individual mice, bars represent mean of group for each time point.

Article Snippet: Chlamydia trachomatis serovar L2-wild type and L2-ΔCT228 (LGV 434/Bu, originating from the Hackstadt Lab at Rocky Mountain Laboratories, Hamilton, MT) were propagated in HeLa 229 cells and purified by Renografin density gradient centrifugation as previously described (Caldwell et al., ).

Techniques: Infection

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Genetic Inactivation of Chlamydia trachomatis Inclusion Membrane Protein CT228 Alters MYPT1 Recruitment, Extrusion Production, and Longevity of Infection

doi: 10.3389/fcimb.2018.00415

Figure Lengend Snippet: Systemic and mucosal antibody titers following infection with C. trachomatis L2-wild type and L2-ΔCT228. (A) Sera were collected from mice 31 days post-infection with L2-wild type ( n = 8) or L2-ΔCT228 ( n = 9) and assayed for the presence of anti-chlamydial IgG2a. (B,C) Vaginal lavages were collected 31 days post-infection and assayed for the presence of anti-chlamydial IgA and IgG. Antibody titers are expressed for individual mice as the highest dilution tested that produced >3-fold the absorbance as control/uninfected mice. Bars represent mean antibody titer per group. * p = 0.0412, unpaired two-tailed Student t -test.

Article Snippet: Chlamydia trachomatis serovar L2-wild type and L2-ΔCT228 (LGV 434/Bu, originating from the Hackstadt Lab at Rocky Mountain Laboratories, Hamilton, MT) were propagated in HeLa 229 cells and purified by Renografin density gradient centrifugation as previously described (Caldwell et al., ).

Techniques: Infection, Produced, Two Tailed Test

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Genetic Inactivation of Chlamydia trachomatis Inclusion Membrane Protein CT228 Alters MYPT1 Recruitment, Extrusion Production, and Longevity of Infection

doi: 10.3389/fcimb.2018.00415

Figure Lengend Snippet: Pathological scoring of murine reproductive tracts.

Article Snippet: Chlamydia trachomatis serovar L2-wild type and L2-ΔCT228 (LGV 434/Bu, originating from the Hackstadt Lab at Rocky Mountain Laboratories, Hamilton, MT) were propagated in HeLa 229 cells and purified by Renografin density gradient centrifugation as previously described (Caldwell et al., ).

Techniques:

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: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IncV, a FFAT motif-containing Chlamydia protein, tethers the endoplasmic reticulum to the pathogen-containing vacuole

doi: 10.1073/pnas.1709060114

Figure Lengend Snippet: Two FFAT motifs in the C terminal of IncV mediate IncV interaction with the MSP domain of VAPA. (A) Co-IP of IncV-3xFLAG and GFP-VAPA (WT or KFM/DFD) from HEK293 lysates. (B) IncV amino acid sequence containing two FFAT motifs. FFAT motif (red); essential position 2 of FFAT motif (blue); ncFFAT, noncanonical FFAT motif; cFFAT, canonical FFAT motif; numbers indicate amino acid position. (C) Co-IP of IncV-3xFLAG (WT, F263A, Y287A, FY/AA) and GFP-VAPAWT from HEK293 lysates. (D) GST pull-down of IncV-3xFLAG full-length (1–363) or C terminal (167–363), WT or FY/AA from HEK293 lysates using GST, GST-VAPAMSP-WT or GST-VAPAMSP-KFM/DFD purified from E. coli (Ponceau).

Article Snippet: Experiments presented in were performed with a C. trachomatis LGV L2 incV mutant or its WT parental strain, both obtained from Ted Hackstadt (NIH, Rocky Mountain Laboratories, Hamilton, MT) ( 43 ).

Techniques: Co-Immunoprecipitation Assay, Sequencing, Purification

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IncV, a FFAT motif-containing Chlamydia protein, tethers the endoplasmic reticulum to the pathogen-containing vacuole

doi: 10.1073/pnas.1709060114

Figure Lengend Snippet: IncV–VAPB interaction occurs in cotransfected and C. trachomatis-infected cells, and depends on the FFAT binding domain of VAPB and on the FFAT motifs of IncV. (A) Co-IP of IncV-3xFLAG and GFP-VAPBWT from HEK293 lysates coexpressing the two constructs. (B) Co-IP of IncV-3xFLAG (WT, F263A, Y287A, FY/AA) and GFP-VAPBWT from HEK293 lysates coexpressing the indicated constructs. (C) Co-IP of IncV-3xFLAG constructs from lysates of HEK293 cells expressing GFP-VAPB (WT or KFM/DFD) and infected with C. trachomatis expressing IncV-3xFLAG WT under the control of an aTc inducible promoter. (D) Same as C with C. trachomatis strains expressing IncV-3xFLAG (WT, F263A, Y287A, or FY/AA) and HEK293 cells expressing GFP-VAPBWT.

Article Snippet: Experiments presented in were performed with a C. trachomatis LGV L2 incV mutant or its WT parental strain, both obtained from Ted Hackstadt (NIH, Rocky Mountain Laboratories, Hamilton, MT) ( 43 ).

Techniques: Infection, Binding Assay, Co-Immunoprecipitation Assay, Construct, Expressing, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IncV, a FFAT motif-containing Chlamydia protein, tethers the endoplasmic reticulum to the pathogen-containing vacuole

doi: 10.1073/pnas.1709060114

Figure Lengend Snippet: Characterization of the IncV constructs used in this study. (A) When expressed in eukaryotic cells, the IncV constructs do not form macroscopic aggregates that could lead to unspecific interaction of the proteins. Confocal micrographs of HeLa cells coexpressing GFP-VAPAWT (green) or IncV-3xFLAG (WT, F263A, Y287A, FY/AA), or IncV167–363-WT-3xFLAG (red). The merge is shown on the Right. (Scale bar, 5 µm.) (B) Hydropathy profile of IncV as generated using: web.expasy.org/protscale/. (C) The IncV constructs are aTc inducible and localize to the inclusion when expressed from C. trachomatis. Confocal micrographs of HeLa cells infected with a C. trachomatis strains expressing mCherry constitutively (red) and IncV-3xFLAG WT, F263A, Y287A, or FY/AA (yellow) under the control of an aTc inducible promoter in the absence (−aTc) or the presence (+aTc) of aTc. The merge is shown on the Right. (Scale bar, 5 µm.) ext. foc., extended focus; xy, cross-section through the center of the inclusion.

Article Snippet: Experiments presented in were performed with a C. trachomatis LGV L2 incV mutant or its WT parental strain, both obtained from Ted Hackstadt (NIH, Rocky Mountain Laboratories, Hamilton, MT) ( 43 ).

Techniques: Construct, Generated, Infection, Expressing, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IncV, a FFAT motif-containing Chlamydia protein, tethers the endoplasmic reticulum to the pathogen-containing vacuole

doi: 10.1073/pnas.1709060114

Figure Lengend Snippet: IncV–VAPA interaction occurs in C. trachomatis-infected cells and depends on the FFAT binding domain of VAPA and on the FFAT motifs of IncV. (A) Co-IP of IncV-3xFLAG constructs from lysates of HEK293 cells expressing GFP-VAPA (WT or KFM/DFD) and infected with C. trachomatis expressing IncV-3xFLAG WT under the control of an aTc inducible promoter. (B) Same as A, with C. trachomatis strains expressing IncV-3xFLAG (WT, F263A, Y287A, or FY/AA) and HEK293 cells expressing GFP-VAPAWT.

Article Snippet: Experiments presented in were performed with a C. trachomatis LGV L2 incV mutant or its WT parental strain, both obtained from Ted Hackstadt (NIH, Rocky Mountain Laboratories, Hamilton, MT) ( 43 ).

Techniques: Infection, Binding Assay, Co-Immunoprecipitation Assay, Construct, Expressing, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IncV, a FFAT motif-containing Chlamydia protein, tethers the endoplasmic reticulum to the pathogen-containing vacuole

doi: 10.1073/pnas.1709060114

Figure Lengend Snippet: The IncV-mediated VAP recruitment to the inclusion depends on the FFAT binding domain of VAPA and on the FFAT motifs of IncV. (A–C) Three-dimensional reconstruction of confocal micrographs of HeLa cells expressing YFP-VAPA WT (A and C) or KFM/DFD (B) (yellow) infected with a C. trachomatis strain expressing mCherry constitutively (red) and IncV-3xFLAG WT (A and B) or WT, F263A, Y287A, or FY/AA (C) (blue) under the control of an aTc inducible promoter, in the absence (−aTc) or the presence (+aTc) of aTc. The merge is shown on the Right. (D and E) Quantification of IncV-3xFLAG (blue, hashed bars) and YFP-VAPA (yellow, solid bars) association with the inclusion in arbitrary units (AU). D and E, respectively, correspond to quantification of A and B, and C. The quantification method is described in Fig. S3A. Error bars are SEM. ****P < 0.0001. ND, none detected. (Scale bar, 5 μm; applies to A–C.)

Article Snippet: Experiments presented in were performed with a C. trachomatis LGV L2 incV mutant or its WT parental strain, both obtained from Ted Hackstadt (NIH, Rocky Mountain Laboratories, Hamilton, MT) ( 43 ).

Techniques: Binding Assay, Expressing, Infection, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IncV, a FFAT motif-containing Chlamydia protein, tethers the endoplasmic reticulum to the pathogen-containing vacuole

doi: 10.1073/pnas.1709060114

Figure Lengend Snippet: The IncV-mediated VAP recruitment to the inclusion depends on the FFAT binding domain of VAPB and on the FFAT motifs of IncV. (A–C) Three-dimensional reconstruction of confocal micrographs of HeLa cells expressing YFP-VAPB WT (A and C) or KFM/DFD (B) (yellow) infected with a C. trachomatis strain expressing mCherry constitutively (red) and IncV-3xFLAG WT (A and B) or WT, F263A, Y287A, or FY/AA (C) (blue) under the control of an aTc inducible promoter, in the absence (−aTc) or the presence (+aTc) of aTc. The merge is shown on the Right. (Scale bars, 5 µm; applies to A and B.) (D and E) Quantification of IncV-3xFLAG (blue, hashed bars) and YFP-VAPB (yellow, solid bars) association with the inclusion in arbitrary units (AU). D and E, respectively, correspond to quantification of A and B, and C. The quantification method is described in Fig. S3A. Error bars are SEM. ****P < 0.0001. ND, None detected.

Article Snippet: Experiments presented in were performed with a C. trachomatis LGV L2 incV mutant or its WT parental strain, both obtained from Ted Hackstadt (NIH, Rocky Mountain Laboratories, Hamilton, MT) ( 43 ).

Techniques: Binding Assay, Expressing, Infection, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IncV, a FFAT motif-containing Chlamydia protein, tethers the endoplasmic reticulum to the pathogen-containing vacuole

doi: 10.1073/pnas.1709060114

Figure Lengend Snippet: IncV induces the formation of ER-Inclusion MCS in a VAP-dependent manner. (A) Three-dimensional reconstruction of confocal micrographs of HeLa cells infected with a C. trachomatis strain expressing mCherry constitutively and IncVWT-3xFLAG under the control of an aTc-inducible promoter in the absence (−aTc) or presence (−aTc) of aTc and stained with antibodies against FLAG (blue) and endogenous VAPA (yellow). The merge is shown on the Right. (Scale bar, 10 µm.) (B) Transmission electron micrographs of HeLa cells infected as described in A. ER patches are highlighted in pink and a higher magnification of the +aTc image is shown on the Right. (Scale bars, 2.5 µm.) (C) XY plane confocal micrographs of HeLa cells, depleted (VAP siRNA) or not (No siRNA) of VAPA and VAPB, expressing YFP-Sec61β (yellow) and infected with a C. trachomatis strain expressing mCherry (red) and IncV-3xFLAG WT (blue). The merge is shown on the Right. (Scale bar, 5 µm.) (D and E) Quantification of inclusions presenting a Sec61β+ ring (D) and of the Sec61β signal associated with IncV in arbitrary units (AU) (E) in cells as described in C. The quantification method used for E is described in Fig. S6C. Error bars are SEM. ****P < 0.0001, ***P < 0.001.

Article Snippet: Experiments presented in were performed with a C. trachomatis LGV L2 incV mutant or its WT parental strain, both obtained from Ted Hackstadt (NIH, Rocky Mountain Laboratories, Hamilton, MT) ( 43 ).

Techniques: Infection, Expressing, Control, Staining, Transmission Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IncV, a FFAT motif-containing Chlamydia protein, tethers the endoplasmic reticulum to the pathogen-containing vacuole

doi: 10.1073/pnas.1709060114

Figure Lengend Snippet: IncV induces the formation of ER-inclusion MCS. Transmission electron micrographs as presented in Fig. 5B without the mask highlighting the ER-inclusion MCS. White arrowheads, ER patches in close contact with the inclusion membrane. (Scale bar, 2.5 µm.)

Article Snippet: Experiments presented in were performed with a C. trachomatis LGV L2 incV mutant or its WT parental strain, both obtained from Ted Hackstadt (NIH, Rocky Mountain Laboratories, Hamilton, MT) ( 43 ).

Techniques: Transmission Assay, Membrane

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IncV, a FFAT motif-containing Chlamydia protein, tethers the endoplasmic reticulum to the pathogen-containing vacuole

doi: 10.1073/pnas.1709060114

Figure Lengend Snippet: Quantification of YFP-Sec61β association with the inclusion. (A) Larger representation of the images presented in Fig. 4C to visualize the Sec61β+ ring. (Scale bar, 5 µm.) (B) VAPA/B siRNA treatment prevents YFP-VAPA but not YFP-Sec61β expression. Fluorescence micrographs of VAPA/B siRNA treated or untreated HeLa cells transfected with YFP-VAPAWT or YFP-Sec61β (green) infected with a C. trachomatis strain expressing mCherry constitutively (red) and IncVWT-3xFLAG under the control of an aTc inducible promoter, in the presence of aTc. The merge is shown on the Right. (Scale bars, 50 µm.) (C) Quantification method. Step 1: 1 µm z-slices in the center of the inclusion were used for quantification of Sec61β inclusion association. Step 2: A three-dimensional object from the raw IncV signal was generated using the Imaris imaging software. The object was restricted to the half of the inclusion opposite the nucleus to limit the background due to the signal from the bulk of the ER present between the nucleus and the inclusion. Step 3: First, the volume corresponding to the sum of the pixels was determined for IncV. Then the total intensity of the Sec61β signal within the IncV+ object was determined. Step 4: The inclusion association of Sec61β was determined in arbitrary units by normalizing the YFP-Sec61β total intensity within the IncV-positive object with the corresponding volume of the IncV object. (Scale bar, 10 μm.)

Article Snippet: Experiments presented in were performed with a C. trachomatis LGV L2 incV mutant or its WT parental strain, both obtained from Ted Hackstadt (NIH, Rocky Mountain Laboratories, Hamilton, MT) ( 43 ).

Techniques: Expressing, Fluorescence, Transfection, Infection, Control, Generated, Imaging, Software

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IncV, a FFAT motif-containing Chlamydia protein, tethers the endoplasmic reticulum to the pathogen-containing vacuole

doi: 10.1073/pnas.1709060114

Figure Lengend Snippet: PM targeting of IncV induces the formation of ER-PM MCS. (A) Confocal micrographs of HeLa cells coexpressing YFP-VAPAWT (yellow) and IncV167–363-WT-3xFLAG, PM-IncV167–363-WT-3xFLAG, or PM-IncV167–363-FY/AA-3xFLAG (blue). The merge is shown on the Right. (Scale bar, 10 µm.) PM targeting was achieved by fusion of a C-terminal CAAX motif. (B) Confocal micrograph of HeLa cells coexpressing PM-IncV167–363-WT-3xFLAG (red), YFP-VAPAWT (yellow), and CFP-ER (blue). The merge is shown on the Left and individual channels to the Right. (Scale bars, Left, 10 µm; Right, 5 μm.) In A and B, XY cross-sections corresponding to the cell glass coverslip interface are shown.

Article Snippet: Experiments presented in were performed with a C. trachomatis LGV L2 incV mutant or its WT parental strain, both obtained from Ted Hackstadt (NIH, Rocky Mountain Laboratories, Hamilton, MT) ( 43 ).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IncV, a FFAT motif-containing Chlamydia protein, tethers the endoplasmic reticulum to the pathogen-containing vacuole

doi: 10.1073/pnas.1709060114

Figure Lengend Snippet: PM targeting of IncV and effect of PM-dsRed on VAPA cellular localization. Confocal micrographs of HeLa cells expressing PM-IncV167–363-WT-3xFLAG or PM-IncV167–363-FY/AA-3xFLAG (blue) (A) or coexpressing PM-dsRed (red) and YFP-VAPA WT (yellow) (B). (Scale bars, 5 µm.)

Article Snippet: Experiments presented in were performed with a C. trachomatis LGV L2 incV mutant or its WT parental strain, both obtained from Ted Hackstadt (NIH, Rocky Mountain Laboratories, Hamilton, MT) ( 43 ).

Techniques: Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IncV, a FFAT motif-containing Chlamydia protein, tethers the endoplasmic reticulum to the pathogen-containing vacuole

doi: 10.1073/pnas.1709060114

Figure Lengend Snippet: Characterization of the C. trachomatis incV::bla mutant. (A) PCR analysis of the incV::bla mutant. The incV ORF was amplified from genomic DNA extracted from WT C. trachomatis (WT) or the incV::bla mutant and the corresponding PCR products were resolved on a 1% DNA agarose gel stained with ethidium bromide (lane 2: WT; lane 3: incV::bla mutant). Lane 1: molecular weight marker. The marker sizes are listed in kilo base pairs to the left. (B) The site of insertion of the group II intron was determined by Sanger sequencing. A translation of the resulting IncV truncated peptide is presented. The asterisk denotes the early stop codon introduced by insertion of the group II intron. Blue: IncV, Red: group II intron. (C) HeLa cells were infected with C. trachomatis WT or the incV::bla strain and infectious forming units (IFUs) were measured 48 h postinfection. ns, not significant.

Article Snippet: Experiments presented in were performed with a C. trachomatis LGV L2 incV mutant or its WT parental strain, both obtained from Ted Hackstadt (NIH, Rocky Mountain Laboratories, Hamilton, MT) ( 43 ).

Techniques: Mutagenesis, Amplification, Agarose Gel Electrophoresis, Staining, Molecular Weight, Marker, Sequencing, Infection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IncV, a FFAT motif-containing Chlamydia protein, tethers the endoplasmic reticulum to the pathogen-containing vacuole

doi: 10.1073/pnas.1709060114

Figure Lengend Snippet: An incV mutant displays diminished VAP association with the inclusion. (A) Three-dimensional reconstruction of confocal micrographs of HeLa cells expressing YFP-VAPAWT (yellow), infected with C. trachomatis WT or an incV mutant (incV::bla) and stained with antimajor outer membrane protein (anti-MOMP) antibodies (red). The merge is shown on the Right. (Scale bar, 5 µm.) (B and C) Quantification of the volume (B) and intensity normalized to the bulk of the ER (C) of the YFP-VAP signal associated with WT (●) or incV mutant (incV::bla, ▲) inclusions in arbitrary units (AU). Each circle or triangle represents one inclusion. The quantification method is described in Fig. S3. Error bars are SEM. ns, not significant. ****P < 0.0001.

Article Snippet: Experiments presented in were performed with a C. trachomatis LGV L2 incV mutant or its WT parental strain, both obtained from Ted Hackstadt (NIH, Rocky Mountain Laboratories, Hamilton, MT) ( 43 ).

Techniques: Mutagenesis, Expressing, Infection, Staining, Membrane

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IncV, a FFAT motif-containing Chlamydia protein, tethers the endoplasmic reticulum to the pathogen-containing vacuole

doi: 10.1073/pnas.1709060114

Figure Lengend Snippet: Schematic representation and proposed role of IncV/VAP and IncD/CERT/VAP at ER-inclusion MCS. EB, elementary body; RB, reticulate body.

Article Snippet: Experiments presented in were performed with a C. trachomatis LGV L2 incV mutant or its WT parental strain, both obtained from Ted Hackstadt (NIH, Rocky Mountain Laboratories, Hamilton, MT) ( 43 ).

Techniques:

Journal: Infection and Immunity

Article Title: Eukaryotic SNARE VAMP3 Dynamically Interacts with Multiple Chlamydial Inclusion Membrane Proteins

doi: 10.1128/IAI.00409-20

Figure Lengend Snippet: VAMP3 localizes to C. trachomatis serovar L2 inclusions from mid- to late developmental cycle, regardless of Golgi structure. HeLa cells were seeded on glass coverslips in a 24-well plate and were allowed to grow overnight. Cells then were infected with WT C. trachomatis serovar L2 (WT Ctr L2) at an MOI of 0.5. Two hours prior to fixation at the indicated times, infected cells were either left untreated or treated with 1 μg/ml Brefeldin A (BFA) to collapse the Golgi structure. At 18, 30, or 42 hpi, coverslips were fixed for 15 min in 4% paraformaldehyde (PFA) and then permeabilized for 5 min with 0.5% Triton X-100. Fixed coverslips were processed for indirect immunofluorescence (IF) to detect endogenous VAMP3 or VAMP4 (green), Golgi structure (red), Chlamydia /MOMP (blue), and DNA (gray) (see Table S3). Images were acquired on a Zeiss LSM 800 confocal microscope at ×63 magnification and compiled using Photoshop 21.1. White stars denote chlamydial inclusions. Scale bar, 10 μm. In samples treated with BFA, there is an absence of Golgi structure IF staining. In untreated samples, the Golgi structure is fragmented around the inclusion. (A) Endogenous VAMP3 localizes most strongly to C. trachomatis serovar L2 inclusions at 18 hpi, and this localization is independent of an intact Golgi structure, as treatment with BFA did not change VAMP3’s localization at the inclusion. VAMP3 still localizes to the inclusion at 30 and 42 hpi, with less localization over time. (B) Endogenous VAMP4 also strongly localizes to C. trachomatis serovar L2 inclusions at 18 hpi, but this localization is dependent upon an intact Golgi structure, as BFA treatment greatly decreases the amount of VAMP4 detected at the inclusion.

Article Snippet: The transfected HeLa cells were then infected with C. trachomatis serovar L2 transformed with pBOMB4 plasmids with incA -, incF -, incG -, ct005 -, ct179 -, ct222 -, ct223 -, ct226 -, ct442 -, ct449 -, and ct813 - flag at an MOI of 2 for 6-well plates or MOI of 1 for 100-mm dishes.

Techniques: Infection, Immunofluorescence, Microscopy, Staining

Journal: Infection and Immunity

Article Title: Eukaryotic SNARE VAMP3 Dynamically Interacts with Multiple Chlamydial Inclusion Membrane Proteins

doi: 10.1128/IAI.00409-20

Figure Lengend Snippet: Localization of VAMP3 to chlamydial inclusions requires de novo chlamydial protein synthesis. HeLa cells seeded on glass coverslips of a 24-well plate and infected with C. trachomatis serovar L2 (MOI of 0.5) then were treated with 200 μg/ml chloramphenicol (CM) at either 15.5 or 23.5 hpi to halt chlamydial protein synthesis during the mid-developmental cycle. Twenty-four hours later, coverslips were fixed and processed, as previously described, for indirect immunofluorescence to detect endogenous VAMP3 (green), Golgi structure (red), Chlamydia /MOMP (blue), and DNA (gray). See Table S3 for antibody details. Images were acquired on a Zeiss LSM 800 confocal microscope at ×63 magnification and compiled using Photoshop v21.1. White stars denote chlamydial inclusions. Scale bar, 10 μm. In cells treated with CM at 15.5 hpi, VAMP3 no longer localizes to the chlamydial inclusion. Instead, it localizes diffusely all over the cell. In cells treated with CM at 23.5 hpi, VAMP3 still localizes to the chlamydial inclusion but in a more polar fashion than untreated cells . Thus, VAMP3 localization to the inclusion is dependent upon de novo chlamydial protein synthesis at around 15.5 hpi.

Article Snippet: The transfected HeLa cells were then infected with C. trachomatis serovar L2 transformed with pBOMB4 plasmids with incA -, incF -, incG -, ct005 -, ct179 -, ct222 -, ct223 -, ct226 -, ct442 -, ct449 -, and ct813 - flag at an MOI of 2 for 6-well plates or MOI of 1 for 100-mm dishes.

Techniques: Infection, Immunofluorescence, Microscopy

Journal: Infection and Immunity

Article Title: Eukaryotic SNARE VAMP3 Dynamically Interacts with Multiple Chlamydial Inclusion Membrane Proteins

doi: 10.1128/IAI.00409-20

Figure Lengend Snippet: Determination of VAMP3-Inc interactions in chlamydia-infected HeLa cells. HeLa cells seeded in a 6-well plate were transfected with 6×His-VAMP3 followed by infection with C. trachomatis serovar L2 expressing a specific Inc-FLAG and induced for expression at 7 hpi for IncF-, IncG-, CT449-, and CT813-FLAG or 20 hpi for CT442-FLAG with aTc (see Materials and Methods for specific details). At the indicated time points postinfection, the cells were collected, solubilized, and affinity purified using anti-FLAG magnetic beads. The eluate fractions were immunoblotted for construct expression using FLAG and 6×His antibodies. Bands for examining proteins were detected at their predicted molecular weights: IncF-FLAG, 11.3 kDa; IncG-FLAG, 18.4 kDa; CT442-FLAG, 17.0 kDa; CT449-FLAG, 13.0 kDa; CT813-FLAG, 30.6 kDa; and 6×His-VAMP3, 16.6 kDa. Within the summary table, a minus sign indicates no interaction; one or two plus signs indicate interaction; and blank indicates not determined. The data shown are representative of three independent experiments. Full results are available in Fig. S7.

Article Snippet: The transfected HeLa cells were then infected with C. trachomatis serovar L2 transformed with pBOMB4 plasmids with incA -, incF -, incG -, ct005 -, ct179 -, ct222 -, ct223 -, ct226 -, ct442 -, ct449 -, and ct813 - flag at an MOI of 2 for 6-well plates or MOI of 1 for 100-mm dishes.

Techniques: Infection, Transfection, Expressing, Affinity Purification, Magnetic Beads, Construct

Journal: Infection and Immunity

Article Title: Eukaryotic SNARE VAMP3 Dynamically Interacts with Multiple Chlamydial Inclusion Membrane Proteins

doi: 10.1128/IAI.00409-20

Figure Lengend Snippet: VAMP3 inclusion localization is altered during infection with C. trachomatis serovar L2 Δ incA and ct813 :: bla inc mutant strains. (A) HeLa cells seeded on glass coverslips in a 24-well plate were infected by centrifugation (400 × g for 15 min room temperature) with either WT C. trachomatis serovar L2 or the indicated C. trachomatis serovar L2 Inc mutant strain at an MOI of 0.5 for 18, 30, or 42 h. Two hours prior to indicated fixation times, cells were treated with 1 μg/ml Brefeldin A (BFA) to collapse the Golgi structure, as shown by the absence of red staining in the merged imaged. At the specified time postinfection, cells were fixed and processed for indirect immunofluorescence as previously described to detect endogenous VAMP3 (green), Golgi structure (red), Chlamydia /MOMP (blue), and DNA (gray) (see Table S3 for specific antibodies used). Images were acquired on a Zeiss LSM 800 confocal microscope at ×63 magnification and compiled using Photoshop v21.1. White stars denote chlamydial inclusions. Scale bar, 10 μm. Images are representative of three independent experiments. (B) VAMP3 intensity around inclusions was quantified for each strain at 30 hpi using images acquired from panel A from three biological replicates with and without BFA treatment. The intensity of VAMP3 inclusion localization was measured in Fiji as described in Materials and Methods. We measured the following numbers of inclusions: WT C. trachomatis serovar L2, 175 inclusions; C. trachomatis serovar L2 Δ incA , 125 inclusions; C. trachomatis serovar L2 ct813 :: bla , 124 inclusions; and C. trachomatis serovar L2 ct005 :: bla , 205 inclusions. Refer to Table S3 for raw data and calculations. Data were plotted as arbitrary units using GraphPad Prism. Statistical significance was determined by an ordinary one-way ANOVA with Dunnett’s post hoc test for multiple comparisons to compare each inc mutant strain to WT C. trachomatis serovar L2, where P < 0.0001 (****) and ns is not significant. At 30 hpi, VAMP3 inclusion localization is increased during infection with C. trachomatis serovar L2 Δ incA mutant, decreased during infection with C. trachomatis serovar L2 ct813 :: bla mutant, and unchanged during infection with C. trachomatis serovar L2 ct005 :: bla mutant compared to WT C. trachomatis serovar L2.

Article Snippet: The transfected HeLa cells were then infected with C. trachomatis serovar L2 transformed with pBOMB4 plasmids with incA -, incF -, incG -, ct005 -, ct179 -, ct222 -, ct223 -, ct226 -, ct442 -, ct449 -, and ct813 - flag at an MOI of 2 for 6-well plates or MOI of 1 for 100-mm dishes.

Techniques: Infection, Mutagenesis, Centrifugation, Staining, Immunofluorescence, Microscopy