Journal: bioRxiv

Article Title: Reemergence of the Murine Bacterial Pathogen Chlamydia muridarum in Laboratory Mouse Colonies

doi: 10.1101/2022.05.16.491822

Figure Lengend Snippet: Representative histopathology of the lung and cecum from a 1.5-year-old, female, C57Bl/6/Foxp3 CreER R26 tdTomato mouse. A. Multifocally, peribronchiolar and perivascular spaces are infiltrated by small to moderate clusters of lymphocytes and histiocytes (scale bar = 500μm). B. High magnification field shows lymphocytic and histiocytic infiltrates around a bronchiole (scale bar = 100μm). C. IHC of the lung demonstrating focal detection of chlamydial MOMP antigen in bronchiolar epithelial cells (scale bars = 100μm; 20 μm - brown staining in inset). D. Representative section of normal mucosa and gut-associated lymphoid tissue (GALT) in the cecum with low numbers of luminal T. muris (arrowheads; scale bars = 100μm). E. IHC of the cecum demonstrating detection of intracytoplasmic chlamydial inclusions in surface epithelial cells (arrowhead; scale bar = 20 μm). F. ISH demonstrates positive staining (red) for Cm mRNA within the cecal epithelium (arrowhead) and lumen (arrow; scale bar = 20 μm).

Article Snippet: Following deparaffinization and heat-induced epitope retrieval in a citrate buffer at pH 6.0, the primary antibody against Chlamydia MOMP (NB100-65054, Novus Biologicals, Centennial, CO) was applied at a dilution of 1:500.

Techniques: Histopathology, Staining

Journal: bioRxiv

Article Title: Reemergence of the Murine Bacterial Pathogen Chlamydia muridarum in Laboratory Mouse Colonies

doi: 10.1101/2022.05.16.491822

Figure Lengend Snippet: Histopathology of the lung and large intestine from a 1-year-old, female, NSG mouse. A. Representative airway demonstrating a bronchiolar and alveolar inflammation characterized by luminal neutrophilic infiltration mixed with necrotic debris and proteinaceous material. Multifocally, bronchiolar epithelial cells exhibit intracytoplasmic clear vacuoles with pale-basophilic structures compatible with Chlamydial inclusions (arrowhead). Peribronchiolar and alveolar space is infiltrated with moderate numbers of macrophages and neutrophils intermixed with reactive fibroblasts (scale bar = 20 μm). B. IHC of the lung demonstrating detection of chlamydial MOMP antigen in bronchiolar epithelial cells (arrowhead; brown staining) and areas with peribronchiolar inflammation (arrow, scale bar = 20 μm). C. ISH demonstrates positive staining (red) for Cm mRNA in bronchiolar epithelial cells (arrowhead) and areas of peribronchiolar inflammation (arrow; scale bar = 20 μm). D. Representative H&E-stained section of a normal cecal wall (scale bars = 100μm). E. High magnification field demonstrates ISH signal (red staining) in the cecal epithelium (arrow) and lumen (arrowhead; scale bar = 20μm). F. IHC of descending colon demonstrating detection of intracytoplasmic chlamydial MOMP antigen in surface epithelial cells (inset - brown staining; scale bars = 20 −200μm).

Article Snippet: Following deparaffinization and heat-induced epitope retrieval in a citrate buffer at pH 6.0, the primary antibody against Chlamydia MOMP (NB100-65054, Novus Biologicals, Centennial, CO) was applied at a dilution of 1:500.

Techniques: Histopathology, Staining

Journal: bioRxiv

Article Title: Reemergence of the Murine Bacterial Pathogen Chlamydia muridarum in Laboratory Mouse Colonies

doi: 10.1101/2022.05.16.491822

Figure Lengend Snippet: Fluorescent images of Chlamydia -infected cells. HeLa 229 cells were infected with Chlamydia spp. isolated with an NSG mouse cecum sample. At 30 hours post-infection, cells were fixed and stained with FITC-labeled anti -Chlamydia spp. antibody (green) and analyzed using an EVOS™ FL Auto Imaging fluorescent microscope (Life Technologies).

Article Snippet: Following deparaffinization and heat-induced epitope retrieval in a citrate buffer at pH 6.0, the primary antibody against Chlamydia MOMP (NB100-65054, Novus Biologicals, Centennial, CO) was applied at a dilution of 1:500.

Techniques: Infection, Isolation, Staining, Labeling, Imaging, Microscopy

Journal: Science Advances

Article Title: Single-cell atlas of cervical organoids uncovers epithelial immune heterogeneity and intercellular cross-talk during Chlamydia infection

doi: 10.1126/sciadv.ady1640

Figure Lengend Snippet: ( A ) Immunofluorescence images of ectocervical (top) and endocervical (bottom) organoids, uninfected (left) or infected (right) for 48 hours with Chlamydia, stained for KRT5 (green), major outer membrane protein (MOMP) (red), KRT8 (gray), and DAPI (blue). ( B ) UMAP projection of single cells from ecto- and endocervical organoids, colored by infection status: uninfected (UI), infected (Inf), and bystander (Bstd). ( C and D ) UMAP showing reclustered ectocervical squamous epithelial population from (B), colored by infection status (C) and subtype identity (D). ( E ) Proportion of UI, Bstd, and Inf cells in each ectocervical squamous subtype. ( F to G ) UMAP showing reclustered endocervical columnar epithelia from (B), colored by infection status (F) and subtype (G). ( H ) Proportion of UI, Bstd, and Inf cells in each endocervical columnar subtype. ( I ) Heatmap of differentially regulated TFs between ecto- and endocervix across infection conditions; color bar depicts the TF activity scores from high (deep pink) to low (blue). ( J ) Violin plot of gene set enrichment scores for the GO term defense response to bacterium across epithelial compartments and infection states; statistical significance assessed by Wilcoxon rank-sum test with Holm-adjusted P values ( ****P ≤ 0.0001). ( K ) The relative expression of IFN-related genes across ecto- and endocervical subclusters; dot size represents the % of cells expressing a particular gene, and the color bar indicates the intensity of scaled mean expression levels ranging from high (red) to low (blue). ( L ) Gene-weighted density UMAP projections showing expression of STAT1 , STAT2 , and IRF9 across epithelial cells in (B). ( M ) Violin plot showing ISG15 expression across ecto- and endocervical organoids in uninfected, bystander, and infected states. ( N ) IHC images showing CDH1 (green), ISG15 (red), MOMP (gray), and DAPI (blue) in ecto- and endocervical organoids, uninfected (left) or infected (right). Yellow arrows mark infected cells; arrowheads indicate ISG15 + bystander cells.

Article Snippet: The following primary antibodies were used for immunofluorescence: mouse anti–acetylated tubulin–Alexa Fluor 647 (1:300, Santa Cruz Biotechnology, sc-23950-AF647), mouse anti–E-cadherin–Alexa Fluor 488 (1:50, BD Biosciences, 560061), mouse anti–E-cadherin (1:50, BD Biosciences, 610181), rabbit anti–KRT5–Alexa Fluor 488 (1:300, Abcam, ab193894), mouse anti-MUC5B (1:200, Abcam, ab77995), rabbit anti-MUC21 (1:200, ProteinAtlas, HPA052028), rabbit anti-KRT8 (1:200, Abcam, ab59400), mouse-anti-KRT6 (1:50, Abcam, ab18586), recombinant rabbit anti-PAX8 (1:200, Abcam, ab239363), goat anti– C. trachomatis major outer membrane protein (1:500, Bio-Rad, 1990-0804), rabbit anti–HLA-DQA1 antibody (EPR7300) (1:200, Abcam, ab128959), rabbit anti-ISG15 polyclonal antibody (1:200, Proteintech,15981-1-AP), and for labeling the DNA, 4′,6-diamidino-2-phenylindole (DAPI, Roche, 10236276001) were used.

Techniques: Immunofluorescence, Infection, Staining, Membrane, Activity Assay, Expressing

Journal: Immunology

Article Title: Evaluation of intra- and extra-epithelial secretory IgA in chlamydial infections

doi: 10.1111/imm.12317

Figure Lengend Snippet: Purification of antigen-specific dimeric mouse IgA. (a) Potential chlamydial antigen targets for intra- and extra-epithelial IgA. SDS–PAGE gels of purified recombinant Chlamydia muridarum antigens major outer membrane protein (MOMP), inclusion membrane protein A (IncA) (b), and chlamydial protease-like activity factor (CPAF) (c). (d) Serum from MOMP/IncA/CPAF or ovalbumin (OVA) -immunized mice was pooled (n = 10), depleted of IgG and purified with Affiland® Mouse IgA Purification Resin. Samples were separated on SDS–PAGE, blocked and probed with anti-mouse IgA (α chain) horseradish peroxidase-conjugated antibodies. (e) Non-reducing/non-denaturing SDS–PAGE of IgA and IgG elutions. (f) Protein antigens were separated by SDS–PAGE, and Western blotted with corresponding purified IgA. Bound IgA was detected with anti-mouse IgA (α heavy chain)-horseradish peroxidase IgG.

Article Snippet: Recombinant protein production Recombinant C. muridarum MOMP was a generous gift from Harlan Caldwell (Rocky Mountain Labs, Hamilton, MT) and was expressed and purified as previously described.

Techniques: Purification, SDS Page, Recombinant, Membrane, Activity Assay, Western Blot

Journal: Immunology

Article Title: Evaluation of intra- and extra-epithelial secretory IgA in chlamydial infections

doi: 10.1111/imm.12317

Figure Lengend Snippet: A model to evaluate efficacy of intra- and extra-epithelial IgA against chlamydial infection. (a) Schematic showing the in vitro model used to determine intra- and extra-epithelial neutralization. (b) MDCK I-II, HEC-1A, ECC-1, C2Bbe1, Vero E6 and BEAS-2b cells were grown on Transwell® inserts and the transepithelial electrical resistance (TEERs) were recorded. (c) Susceptibility of cell lines to apical infection following 5 days of polarization on Transwell® inserts. (d) Quantitative expression of human polymeric immunoglobulin receptor (pIgR) mRNA in BEAS2b, ECC-1, C2Bbe1, and HEC-1A cells was determined by quantitative RT-PCR. (e) C2Bbe1 cells (± murine pIgR) were fixed and incubated with pIgR−/− mouse sera, and bound IgA was detected with goat anti-mouse IgA-horseradish peroxidase antibody. (f, g) C2Bbe1 cells were grown on Transwell® inserts for 5 days then apically infected with Chlamydia muridarum for 24 hr. (f) TEER of C2Bbe1 cells following 24 hr of infection. (g) Confocal microscopy demonstrating tight junction (ZO-1) expression in mock and C. muridarum-infected C2Bbe1 cells. (h) C2Bbe1 cells (± murine pIgR) were grown on Transwell® inserts for 5 days and then purified mouse IgA was basolaterally loaded. Apical samples were taken and quantified by sandwich ELISA at 1, 3, 6 and 24 hr post inoculation. Error bars represent mean ± SEM (n = 3 or n = 4). Scale = 25 μm. ND = none detected.

Article Snippet: Recombinant protein production Recombinant C. muridarum MOMP was a generous gift from Harlan Caldwell (Rocky Mountain Labs, Hamilton, MT) and was expressed and purified as previously described.

Techniques: Infection, In Vitro, Neutralization, Expressing, Quantitative RT-PCR, Incubation, Confocal Microscopy, Purification, Sandwich ELISA

Journal: Immunology

Article Title: Evaluation of intra- and extra-epithelial secretory IgA in chlamydial infections

doi: 10.1111/imm.12317

Figure Lengend Snippet: The polymeric immunoglobulin receptor (pIgR) mediates delivery of neutralizing IgA to extra- but not intra-epithelial chlamydial antigens. C2Bbe1 cells (± murine pIgR) were seeded on Transwell® inserts for 5 days. One hundred micrograms of purified IgA was loaded basolaterally and allowed to transport for 24 hr. Cells were then apically infected with 105 inclusion-forming units of Chlamydia muridarum for 24 hr. Inclusion-forming units were quantified by fluorescence microscopy. (a) Neutralization of chlamydial infection in polarized epithelia loaded basolaterally with polyclonal IgA from mice immunized with major outer membrane protein (MOMP) or ovalbumin (OVA). (b) Neutralization of chlamydial infection in polarized epithelia loaded basolaterally with polyclonal IgA from mice immunized with inclusion membrane protein A (IncA), chlamydial protease-like activity factor (CPAF) or OVA. (c) Confocal microscopy of OVA and IncA-IgA treated cells staining for DNA (DAPI), Chlamydia (anti-MOMP), and mouse IgA (IgA). Results representative of three individual experiments (n = 4 inserts per group). Error bars showing mean ± SEM. Scale = 10 μm. * = P < 0.05.

Article Snippet: Recombinant protein production Recombinant C. muridarum MOMP was a generous gift from Harlan Caldwell (Rocky Mountain Labs, Hamilton, MT) and was expressed and purified as previously described.

Techniques: Purification, Infection, Fluorescence, Microscopy, Neutralization, Membrane, Activity Assay, Confocal Microscopy, Staining