c trachomatis major outer membrane protein  (Bio-Rad)

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: bioRxiv

Article Title: Single-cell atlas of patient-derived cervical organoids uncovers epithelial immune heterogeneity and intercellular crosstalk during Chlamydia infection

doi: 10.1101/2025.04.13.648603

Figure Lengend Snippet: a) Schematic of the experimental workflow modeling Chlamydia infection in patient-derived ecto- and endocervical organoids. b) Uniform Manifold Approximation and Projection (UMAP) of ecto- and endocervical epithelial cell clusters. Each dot represents a single cell color-coded by tissue type. c-d) UMAP projections highlighting squamous (c) and columnar (d) epithelial subclusters, with cells coloured by cluster. e) URD differentiation tree of ectocervical squamous epithelial cells; each dot represents a single cell, colored by subcluster. Cells ordered based on pseudotime values starting from early (top) to late (bottom). f) Gene expression dynamics of selected squamous markers along the pseudotime; lines represent expression trend of a particular gene. g) Differentiation trajectory of endocervical columnar epithelial cells, colored by subcluster and ordered by pseudotime (early to late). h) Expression dynamics of key columnar epithelial marker genes along pseudotime. i) UMAP visualization of organoid and tissue-derived cell clusters after data integration; cells are color-coded based on their dataset of origin. j) UMAP shows six major cell populations across the integrated dataset. k) UMAP depicting nine epithelial subclusters identified post-integration, with cells color-coded by cluster. l) Bar plot depicting the epithelial cell proportions from different datasets across integrated subclusters. m) Heatmap showing gene set enrichment scores for gene ontology (GO) biological processes across integrated epithelial clusters; scale bar denotes the z-scored enrichment values ranging from high (deep pink) to low (blue).

Article Snippet: The following primary antibodies were used for immunofluorescence: mouse anti-acetylated tubulin-Alexa-647 (Santa Cruz Biotechnology, sc-23950-AF647), mouse anti-E-cadherin-Alexa-488 (BD Biosciences, 560061), mouse anti-E-cadherin (BD Biosciences, 610181), rabbit anti-KRT5-Alexa488 (Abcam, ab193894 mouse anti-MUC5B (Abcam, ab77995), rabbit anti-MUC21 (ProteinAtlas, HPA052028), rabbit anti-KRT8 (1:200, Abcam, abab59400), mouse-anti-KRT6 (Abcam, ab18586), recombinant rabbit anti-PAX8 (Abcam, ab239363), goat anti- Chlamydia trachomatis Major Outer Membrane Protein (MOMP) (1:500, BIO-RAD, 1990-0804) and for labeling the DNA, DAPI (Roche, 10236276001) was used.

Techniques: Infection, Derivative Assay, Gene Expression, Expressing, Marker

Journal: bioRxiv

Article Title: Single-cell atlas of patient-derived cervical organoids uncovers epithelial immune heterogeneity and intercellular crosstalk during Chlamydia infection

doi: 10.1101/2025.04.13.648603

Figure Lengend Snippet: a) IHC images of ectocervix (upper panel) and endocervix (lower panel) organoids, either uninfected (left) or infected (right) with Chlamydia for 48 hours, stained with KRT5 (green), MOMP (red), KRT8 (gray). Nuclei are stained with DAPI (blue). b) UMAP projection of uninfected (UI), infected (Inf), and bystander (Bstd) epithelial cells from ecto- and endocervical organoids; each dot represents a single cell, colored by infection status. c-d) UMAP visualization of the re-clustered ectocervical squamous epithelial population from (b), colored by infection status (c) and subtypes (d). e) Bar plot showing the proportion of UI, Inf, and Bstd cells across squamous epithelial subclusters of the ectocervix. f-g) UMAP depicting re-clustered endocervical columnar epithelia from (b), colored by infection status (f) and subtypes (g). h) Bar plot shows the proportion of UI, Inf, and Bstd cells across columnar epithelial subclusters of endocervix. 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 showing the gene set enrichment scores for GO term corresponding to defense response to bacterium across epithelial compartments and infection states. k) Heatmap showing hallmark pathway enrichment scores across epithelial subtypes and infection conditions. Columns represent individual cells color-coded by tissue and infection status. l) Dot plot showing the relative expression of interferon-related genes across ecto- and endocervical subclusters; circle size represents the percentage of cells expressing a particular gene, the color bar indicates the intensity of scaled mean expression levels ranging from high (red) to low (blue). m) Heatmap of TFs with highly variable activities across ecto- and endocervical samples and infection states, with subcluster annotations. n) Gene-weighted density UMAP projections showing expression of STAT1 , STAT2 , and IRF9 across epithelial cells in (b).

Article Snippet: The following primary antibodies were used for immunofluorescence: mouse anti-acetylated tubulin-Alexa-647 (Santa Cruz Biotechnology, sc-23950-AF647), mouse anti-E-cadherin-Alexa-488 (BD Biosciences, 560061), mouse anti-E-cadherin (BD Biosciences, 610181), rabbit anti-KRT5-Alexa488 (Abcam, ab193894 mouse anti-MUC5B (Abcam, ab77995), rabbit anti-MUC21 (ProteinAtlas, HPA052028), rabbit anti-KRT8 (1:200, Abcam, abab59400), mouse-anti-KRT6 (Abcam, ab18586), recombinant rabbit anti-PAX8 (Abcam, ab239363), goat anti- Chlamydia trachomatis Major Outer Membrane Protein (MOMP) (1:500, BIO-RAD, 1990-0804) and for labeling the DNA, DAPI (Roche, 10236276001) was used.

Techniques: Infection, Staining, Activity Assay, Expressing

Journal: bioRxiv

Article Title: Single-cell atlas of patient-derived cervical organoids uncovers epithelial immune heterogeneity and intercellular crosstalk during Chlamydia infection

doi: 10.1101/2025.04.13.648603

Figure Lengend Snippet: a) Dot plot showing mRNA profiles of PRRs, MHC genes, AMPs and cytokines across ecto- and endocervical epithelial subtypes and UI/Bstd/Inf conditions. The size of each dot reflects the percentage of cells expressing a specific gene, and the color bar signifies the intensity of scaled mean expression levels, ranging from high (red) to low (blue). b-c) qRT-PCR analysis of MHC genes ( HLA-B, HLA-C, HLA-DMB, HLA-DRA ) (b); PRRs ( TLR1, TLR2, TLR5 ) (c) expression in uninfected and infected ecto- and endocervical organoids at 48hpi. d) qRT-PCR analysis of DEFB1 expression in FACS sorted uninfected, infected, and bystander epithelial cells after 36hpi. Data represent mean ± s.d. from three technical replicates normalized to ectocervix control, and p-values were calculated using Student’s T-test. e) Infectivity assay of Chlamydia lysates from ectocervical organoids, with or without pre-treatment with human β-defensins (HBD1, HBD2, or both) at 5 days post-infection (dpi). Data represent mean ± s.d. from 8 non-overlapping regions across two replicates. f) Violin plot showing gene set enrichment scores for the GO term related to the adaptive immune response across epithelial subclusters under different infection conditions. g) Trend plot showing changes in mean expression levels of selected proliferation markers MKI67 and CDK1 under different infection conditions; Lines are colored by gene; point shapes distinguish ectocervical (circles) and endocervical (triangles) samples.

Article Snippet: The following primary antibodies were used for immunofluorescence: mouse anti-acetylated tubulin-Alexa-647 (Santa Cruz Biotechnology, sc-23950-AF647), mouse anti-E-cadherin-Alexa-488 (BD Biosciences, 560061), mouse anti-E-cadherin (BD Biosciences, 610181), rabbit anti-KRT5-Alexa488 (Abcam, ab193894 mouse anti-MUC5B (Abcam, ab77995), rabbit anti-MUC21 (ProteinAtlas, HPA052028), rabbit anti-KRT8 (1:200, Abcam, abab59400), mouse-anti-KRT6 (Abcam, ab18586), recombinant rabbit anti-PAX8 (Abcam, ab239363), goat anti- Chlamydia trachomatis Major Outer Membrane Protein (MOMP) (1:500, BIO-RAD, 1990-0804) and for labeling the DNA, DAPI (Roche, 10236276001) was used.

Techniques: Expressing, Quantitative RT-PCR, Infection, Control

Journal: bioRxiv

Article Title: Single-cell atlas of patient-derived cervical organoids uncovers epithelial immune heterogeneity and intercellular crosstalk during Chlamydia infection

doi: 10.1101/2025.04.13.648603

Figure Lengend Snippet: a-b) Chord diagrams showing upregulated (a) and downregulated (b) ligand-receptor (L-R) signaling pairs in ectocervical squamous epithelial cells upon Chlamydia infection; Outer bars indicate signal sending (ligand-expressing) cell groups; inner bars colored by receiving (receptor-expressing) cell groups; edges colored by signaling source/senders. c-d) River plots depicting the inferred outgoing (c) and incoming (d) communication patterns in ectocervical squamous epithelia, highlighting the associations between latent patterns, cell groups, and enriched signaling pathways; thickness of the flow reflects the contribution of cell groups or signaling pathways to each pattern, with pathway labels colored by signaling category. e) Heatmap showing the relative importance and signaling role of each cell group in the MIF, SEMA6, and CADM signaling networks in the ectocervix; scale bar color denotes the criticality of a cell group in driving the communication network ranging from high (dark green) to low (gray). f-g) Chord diagrams illustrating upregulated (f) and downregulated (g) L–R signaling interactions across endocervical columnar epithelial subpopulations after infection. h) Scatter plot visualization of predominant signal senders and receivers among endocervical epithelial subsets in 2D space; Circle size represents the total inferred links (outgoing and incoming) associated with each cell type, colored by subclusters. i-j) Inferred outgoing (i) and incoming (j) communication patterns in endocervical epithelia, illustrating the links between latent patterns, cell types, and signaling pathways. k) Heatmap visualization of the relative importance and signaling role of each cell group in the endocervix for IL-1, MIF, and TNF signaling networks. Scale bar color as in (e).

Article Snippet: The following primary antibodies were used for immunofluorescence: mouse anti-acetylated tubulin-Alexa-647 (Santa Cruz Biotechnology, sc-23950-AF647), mouse anti-E-cadherin-Alexa-488 (BD Biosciences, 560061), mouse anti-E-cadherin (BD Biosciences, 610181), rabbit anti-KRT5-Alexa488 (Abcam, ab193894 mouse anti-MUC5B (Abcam, ab77995), rabbit anti-MUC21 (ProteinAtlas, HPA052028), rabbit anti-KRT8 (1:200, Abcam, abab59400), mouse-anti-KRT6 (Abcam, ab18586), recombinant rabbit anti-PAX8 (Abcam, ab239363), goat anti- Chlamydia trachomatis Major Outer Membrane Protein (MOMP) (1:500, BIO-RAD, 1990-0804) and for labeling the DNA, DAPI (Roche, 10236276001) was used.

Techniques: Infection, Expressing, Protein-Protein interactions

Journal: bioRxiv

Article Title: Single-cell atlas of patient-derived cervical organoids uncovers epithelial immune heterogeneity and intercellular crosstalk during Chlamydia infection

doi: 10.1101/2025.04.13.648603

Figure Lengend Snippet: (a) Anatomical overview of the human uterine cervix highlighting the ectocervix and endocervix, their distinct epithelial compositions, and convergence at the transition zone. Patient-derived 3D organoids derived from adult epithelial stem cells preserve region-specific epithelial architecture and identity. (b) Integration of organoid and cervical tissue scRNA-seq datasets reveals high transcriptional fidelity and molecular congruence across corresponding epithelial subsets. Gene signature and similarity analyses demonstrate that organoids recapitulate both cellular heterogeneity and native epithelial programs. (c) Depiction shows the identified lineage-specific epithelial subtypes across squamous and columnar compartments under homeostasis. Pseudotime trajectory analysis reveals hierarchical differentiation from basal/stem-like cells to terminally differentiated states, with key subtype-defining transcriptional markers shown. (d) Depiction highlighting region- and cell-type-resolved expression profiles of innate immune defense genes at steady state. Distinct epithelial subtypes show selective enrichment of mucins, junctional components, PRRs, antimicrobial peptides (AMPs), and cytokines, reflecting compartmentalized mucosal immune strategies. (e) Schematic showing that Chlamydia infection remodels the epithelial landscape. In the endocervix, transcriptional reprogramming of uninfected bystander cells gives rise to IFN-responsive columnar subsets with robust ISG expression, hallmarks of paracrine immune activation. (f) Infection reconfigures epithelial communication networks. Cell-cell interaction modeling reveals that columnar bystander cells function as central signaling hubs mediating immune and regenerative responses through pathways such as CCL, CXCL, MHC-II, MIF, SEMA, TNF and IL-1. Region-specific shifts in ligand-receptor interactions underscore divergent mucosal responses between ecto- and endocervix.

Article Snippet: The following primary antibodies were used for immunofluorescence: mouse anti-acetylated tubulin-Alexa-647 (Santa Cruz Biotechnology, sc-23950-AF647), mouse anti-E-cadherin-Alexa-488 (BD Biosciences, 560061), mouse anti-E-cadherin (BD Biosciences, 610181), rabbit anti-KRT5-Alexa488 (Abcam, ab193894 mouse anti-MUC5B (Abcam, ab77995), rabbit anti-MUC21 (ProteinAtlas, HPA052028), rabbit anti-KRT8 (1:200, Abcam, abab59400), mouse-anti-KRT6 (Abcam, ab18586), recombinant rabbit anti-PAX8 (Abcam, ab239363), goat anti- Chlamydia trachomatis Major Outer Membrane Protein (MOMP) (1:500, BIO-RAD, 1990-0804) and for labeling the DNA, DAPI (Roche, 10236276001) was used.

Techniques: Derivative Assay, Expressing, Infection, Activation Assay