chlamydia lipopolysaccharide  (Santa Cruz Biotechnology)

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: Rheumatology (Oxford, England)

Article Title: A potential role for chlamydial infection in rheumatoid arthritis development

doi: 10.1093/rheumatology/kead682

Figure Lengend Snippet: Prevalence of anti-MOMP IgG antibodies in serum of RA-FDR defined by their preclinical status

Article Snippet: To explore the robustness of our primary exposure, we performed a nested case–control study in a subset of RA-FDRs for a secondary exposure of interest, the serology against C. trachomatis major outer membrane protein (MOMP) IgG (ELISA, Vircell, Granada, Spain, cat. no. G/M1017).

Techniques:

Journal: iScience

Article Title: CT135 mediates the resistance of Chlamydia trachomatis to primate interferon gamma stimulated immune defenses

doi: 10.1016/j.isci.2024.110143

Figure Lengend Snippet: Genetic screen of C. trachomatis chimera library mutants susceptible to IFNγ-activated, cell-autonomous immune defenses (A) Approach used to generate the CT × CM chimera mutant library, and linear genome maps depicting the 13-strain library used for screening (CT DNA, blue; CM DNA, orange). (B) Visual screen for cell-autonomous immunity-susceptible strains in IFNγ-activated human A549 cells by measuring recruitment of ubiquitin (red) to Chlamydia -containing inclusions (green) at 20 hpi. Host nuclei labeled with DAPI (blue). Arrows indicate ubiquitinated inclusions. Scale bar, 20 μm. (C) Screen of wild type CT-L2, CM, and 17 CT chimera mutants for susceptibility to IFNγ-activated targeting of ubiquitin to inclusion membranes (mean ± SEM, n = 3, unpaired Student’s t test). (D) Hit locus identified from comparative genomic analysis of sensitive and resistant chimera strains, with enlarged map of the 11 candidate genes contained in this locus. Gene colors denote amino acid sequence identity between CT and CM reference genomes. (E) Tabulated summary of candidate genes.

Article Snippet: Mouse anti C. trachomatis MOMP , Virostat , Cat# 1621.

Techniques: Mutagenesis, Ubiquitin Proteomics, Labeling, Sequencing

Journal: iScience

Article Title: CT135 mediates the resistance of Chlamydia trachomatis to primate interferon gamma stimulated immune defenses

doi: 10.1016/j.isci.2024.110143

Figure Lengend Snippet: CT strains with frameshift mutations in CT135 are susceptible to cell-autonomous immune defenses in human cells (A) Recruitment of ubiquitin (anti-ubiquitin, red) to inclusions containing CT-D strains with frameshift mutations in CT135 (CT-D/EC and CT-D/LC; anti-MOMP, green), and CM inclusions, in IFNγ-primed A549 cells at 20 hpi. Arrows indicate ubiquitinated inclusions. Scale bar, 20 μm. (B) Quantitative assessment of inclusion ubiquitination to CT, CT135 frameshift mutant, and CM inclusions in unstimulated and IFNγ-activated cells (mean ± SEM, n = 3, unpaired Student’s t test). (C) Abrogation of CT135 mutant and CM growth in IFNγ-activated A549 cells was determined by assaying the amount of infectious Chlamydia EBs (IFUs) at 27 hpi. Cultures were supplemented with tryptophan to mitigate the effects of IFNγ-induced IDO. Values represent the ratio (%) of IFUs produced under +IFNγ to unstimulated conditions (mean ± SEM, n = 3, one-way ANOVA).

Article Snippet: Mouse anti C. trachomatis MOMP , Virostat , Cat# 1621.

Techniques: Ubiquitin Proteomics, Mutagenesis, Produced

Journal: iScience

Article Title: CT135 mediates the resistance of Chlamydia trachomatis to primate interferon gamma stimulated immune defenses

doi: 10.1016/j.isci.2024.110143

Figure Lengend Snippet: CT135 mutants lose virulence in a nonhuman primate model of Chlamydia infection (A) Overview of CT-D and CT-J challenge of macaques ( n = 6) and weekly sampling schedule for NHP infections. (B and C) Data from animals challenged with CT-D/6319 or CT-J/p225. Top: infection positivity at weeks 0–5 as measured by culture (mean ± SEM) and NAAT analysis of cervical swabs. Middle: seroconversion by longitudinal anti-CT serum IgG titers (mean ± SEM). Bottom: SNPs in the CT135 gene identified by whole genome sequencing of day 0 inoculum and week 5 cervical samples. (D) In vitro cell-autonomous immunity phenotypes (A549 cells ± IFNγ, 20 hpi) of the day 0 inoculum and week 5 cervical samples from CT-D/6319 NHP experiments (anti-ubiquitin, red; anti-MOMP, green; DAPI, blue). Arrows indicate ubiquitinated inclusions. Scale bar, 20 μm. (E) Evaluation of susceptibility of CT-D/6319 in vivo samples to cell-autonomous immune defenses in A549 cells (mean ± SEM, n = 3, unpaired Student’s t test). (F) Longitudinal bacterial burdens of weekly cervical swabs from animals infected with CT-D/6319 (top, mean ± SEM) or a pure inoculum of ΔCT135 (bottom, mean ± SEM); hollow red circles, single infection with ΔCT135 ( n = 1); filled red circles, animals serially infected 5x with ΔCT135 ( n = 2). NAAT results for ΔCT135 infected animals also shown.

Article Snippet: Mouse anti C. trachomatis MOMP , Virostat , Cat# 1621.

Techniques: Infection, Sampling, Sequencing, In Vitro, Ubiquitin Proteomics, In Vivo

Journal: iScience

Article Title: CT135 mediates the resistance of Chlamydia trachomatis to primate interferon gamma stimulated immune defenses

doi: 10.1016/j.isci.2024.110143

Figure Lengend Snippet: The inclusion membrane protein CT135/GarD is required for CT evasion from cell-autonomous immune defenses (A) Human cell-autonomous immune defenses target inclusions containing CTΔCT135 at 20 hpi (anti-ubiquitin, red; anti-MOMP, green; DAPI, blue). Restoration of CTΔCT135 with a wild type CT135 allele rescues the defect. Arrows indicate ubiquitinated inclusions. Scale bar, 20 μm. (B) Quantitative assessment of IFNγ-mediated targeting of ubiquitin to CTΔCT135 inclusions in A549 cells (mean ± SEM, n = 3, unpaired Student’s t test). (C) p62/SQSTM (anti-p62, red) is recruited to CTΔCT135 and CM inclusions in a IFNγ-dependent manner (anti-MOMP, green; DAPI, blue). Arrows indicate cells with pronounced p62 recruitment to inclusion membranes. Scale bar, 20 μm. (D) Quantitative analysis of p62 recruitment in Chlamydia infected A549 cells (mean ± SEM, n = 3, unpaired Student’s t test). (E) Quantitative evaluation of inclusion ubiquitination in A549 cells ectopically transfected with CT135-GFP and subsequently infected with CT, CTΔCT135, or CM (mean ± SEM, n = 3, unpaired Student’s t test). (F) Subcellular distribution of transfected CT135-GFP (green) in A549 cells subsequently infected with CT or CTΔCT135. (G) Isolation of primary skin fibroblasts from pig-tailed macaques. (H) IFNγ-induced targeting of CTΔCT135 and CM inclusions in primary macaque cells is conserved with the human pathway (anti-ubiquitin, red; anti-MOMP, green; DAPI, blue). Arrows indicate ubiquitinated inclusions. Scale bar, 20 μm. (I) Quantitative summary of ubiquitin-mediated targeting of CTΔCT135 and CM inclusions in macaque cells (mean ± SEM, n = 3, unpaired Student’s t test). (J) Model of human and NHP CAI evasion by CT135.

Article Snippet: Mouse anti C. trachomatis MOMP , Virostat , Cat# 1621.

Techniques: Membrane, Ubiquitin Proteomics, Infection, Transfection, Isolation

Journal: iScience

Article Title: CT135 mediates the resistance of Chlamydia trachomatis to primate interferon gamma stimulated immune defenses

doi: 10.1016/j.isci.2024.110143

Figure Lengend Snippet:

Article Snippet: Mouse anti C. trachomatis MOMP , Virostat , Cat# 1621.

Techniques: Virus, Isolation, Recombinant, Modification, DNA Library Preparation, Software

Journal: Veterinary World

Article Title: Coinfection of Chlamydia spp. and herpesvirus in juvenile farmed Siamese crocodiles ( Crocodylus siamensis ) in Thailand

doi: 10.14202/vetworld.2021.1908-1914

Figure Lengend Snippet: Histopathology of the liver sections. (a) Hematoxylin and eosin (H&E)-stained section showed the area of hepatocellular necrosis and cord disruption with associated infiltrates of mixed leukocytes. The intracytoplasmic clumps of granular basophilic coccoid bacteria were seen in the occasional hepatocytes and Kupffer cells (arrowhead). (b) The immunohistochemistry against monoclonal antibody to Chlamydia lipopolysaccharide on liver section (red color). (c) H&E-stained section showed rare hepatocytes containing large eosinophilic intranuclear inclusion bodies with marginating nuclear chromatin (arrowhead). Photos were taken using VetScan HDmicroscope (Abaxis, UK) at ×400 ( and ) and 1000× .

Article Snippet: The formalin-fixed samples were subsequently processed according to the standard procedure and stained using hematoxylin and eosin (H and E) and monoclonal antibody to Chlamydia lipopolysaccharide (sc-58108, Santa Cruz Biotechnology, USA) for further pathological analysis.

Techniques: Histopathology, Staining, Disruption, Bacteria, Immunohistochemistry

Journal: Veterinary World

Article Title: Coinfection of Chlamydia spp. and herpesvirus in juvenile farmed Siamese crocodiles ( Crocodylus siamensis ) in Thailand

doi: 10.14202/vetworld.2021.1908-1914

Figure Lengend Snippet: Phylogenetic trees constructed from the amino acid sequences of OMP protein (a) and DNA polymerase protein (b). The phylogenetic trees were constructed using the maximum likelihood method implemented in MEGA7.0.21. The percentage of tree in which the associated taxa clustered together is shown next to the branches. Each tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The number shown in each species of Chlamydia and herpesvirus is the protein ID. Bold letters represented the sequences obtained from this study.

Article Snippet: The formalin-fixed samples were subsequently processed according to the standard procedure and stained using hematoxylin and eosin (H and E) and monoclonal antibody to Chlamydia lipopolysaccharide (sc-58108, Santa Cruz Biotechnology, USA) for further pathological analysis.

Techniques: Construct