Journal: bioRxiv

Article Title: Clostridioides difficile major toxins remodel the intestinal epithelia, affecting spore adherence/internalization into intestinal tissue and their association with gut vitronectin

doi: 10.1101/2025.01.29.635439

Figure Lengend Snippet: Differentiated Caco-2 cells intoxicated with TcdA and TcdB for 3, 6, or 8h in DMEM FBS-free. As a control, cells were treated with DMEM FBS-free. Unpermeabilized cells were stained for accessible Fibronectin or Vitronectin (acc Fn or acc Vn; green), permeabilized, and stained total Fibronectin or Vitronectin (total Fn or total Vn; red) and nuclei (blue). a, b Representative confocal microscopy images 3D projection of control cells (left) and intoxicated cells for 8h (right) immunostained for acc Fn or Vn and total Fn or Vn, below a magnified slide (XY), and the orthogonal view (XZ). Relative fluorescence intensity measured as the sum of raw intensity density/area for each z-step of accFn and total Fn, its abundance in the c apical side or d in the basal side of the cell; in the same way, the relative fluorescence intensity of Vn, its abundance in the e apical side and f the basal side of the cell. g , immunoblotting of anti- nonglucosylated Rac1 and total Rac1 of cell lysates of differentiated Caco-2 cells intoxicated with TcdA and TcdB for 3, 6, or 8 h. Nonglucosylated Rac1 was evaluated with corresponding antibodies, then the membrane was stripped, and subsequently tested for total Rac1. Western blotting is representative of 3 independent experiments. Controls were set at 100%. Error bars indicate the mean ± SEM from at least 9 fields ( n = 3). Statistical analysis was performed by Two- Way ANOVA post-Bonferroni; ns, p > 0.05; * p < 0.05. Scale bar, top panels 20µm; bottom panels 5µm.

Article Snippet: For staining surface-accessible proteins, cells were incubated with primary antibodies (1:200 dilution) including mouse monoclonal antibodies against human fibronectin (sc8422, Santa Cruz Biotechnologies, USA), vitronectin (sc74484, Santa Cruz Biotechnologies, USA), integrin α 5 (ab78614, Abcam, USA), integrin α v (ab16821, Abcam, USA), and integrin β 1 (MAB1959Z, Millipore, USA).

Techniques: Control, Staining, Confocal Microscopy, Fluorescence, Western Blot, Membrane

Journal: bioRxiv

Article Title: Clostridioides difficile major toxins remodel the intestinal epithelia, affecting spore adherence/internalization into intestinal tissue and their association with gut vitronectin

doi: 10.1101/2025.01.29.635439

Figure Lengend Snippet: Ileal ligated loops were intoxicated for 5 h with 0.1, 0.5, 1, or 5µg of TcdB or saline as control. Then loops were removed, washed, fixed, and subjected to immunofluorescence. Unpermeabilized tissues were stained for accessible Fibronectin or Vitronectin (acc Fn or acc Vn; green), and then permeabilized and stained total Fibronectin or Vitronectin (total Fn or total Vn; red) and F-actin (grey). a - b , Representative confocal microscopy images 3D projection of control cells (left) or intoxicated loops with 5µg TcdB (right) immunostained for accessible and total Fn or Vn; right bottom, a magnified 3D projection, next to a z-stack (XY), and then magnified orthogonal view (XZ). Quantification of c, e , accessible or d, f , total Fn or Vn fluorescence intensity per cell measured in the z-projection (sum). For acc Fn or Vn, the analyzed area was Ctrl of 170,360 µm 2 ; 0.1 µg TcdB of 340,720 µm 2 ; 0.5 µg TcdB of 340,720 µm 2 ; 1 µg TcdB of 340,720 µm 2 and 5 µg TcdB of 511,080 µm 2 . n = 3 animal per group. In scatter plots, each dot corresponds to one independent cell. Dots in colors correspond to the average of each analyzed mice/field. Error bars indicate mean or mean ± SEM. Statistical analysis was performed by unpaired Student’s t test; ns, p > 0.05; * p < 0.05; ** p < 0.01; **** p < <0.0001. Scale bar 20 µm.

Article Snippet: For staining surface-accessible proteins, cells were incubated with primary antibodies (1:200 dilution) including mouse monoclonal antibodies against human fibronectin (sc8422, Santa Cruz Biotechnologies, USA), vitronectin (sc74484, Santa Cruz Biotechnologies, USA), integrin α 5 (ab78614, Abcam, USA), integrin α v (ab16821, Abcam, USA), and integrin β 1 (MAB1959Z, Millipore, USA).

Techniques: Saline, Control, Immunofluorescence, Staining, Confocal Microscopy, Fluorescence

Journal: bioRxiv

Article Title: Clostridioides difficile major toxins remodel the intestinal epithelia, affecting spore adherence/internalization into intestinal tissue and their association with gut vitronectin

doi: 10.1101/2025.01.29.635439

Figure Lengend Snippet: Differentiated Caco-2 cells were intoxicated with 600pM of TcdA and TcdB for 8h in DMEM FBS-free media. Controls include non-intoxoicated cells in DMEM FBS-free. Supernatant was collected from untreated and intoxicated cells and subsequently utilized to resuspend C. difficile spores and incubate for 1 h at 37 °C. Spores resuspended in DMEM alone were also included as a control. Spores were washed and strained for immunofluorescence anti- fibronectin and - vitronectin. a-b, Micrographs show representative phase-contrast (phase), fibronectin and vitronectin specific immunofluorescence and fluorescence intensity profiles (Fl. int.). Representative Fl. int. were provided using 3D Surface plotter function of Fiji. c - d , Quantitative analysis of the fluorescence Fl. int. of Fn and Vn in spores Fl. Int of 600 spores. Mean ± SEM are denoted. e -g, Ileal ligated loops were intoxicated with 5µg of TcdB and 5 × 10 8 C. difficile R20291 spores for 5 h. Then loops were removed, washed, fixed, and subjected to immunofluorescence. Unpermeabilized tissues were stained for accessible Fibronectin or Vitronectin (acc Fn or acc Vn; green), and then permeabilized and stained total Fibronectin or Vitronectin (total Fn or total Vn; red) and F-actin (grey). e, g , representative 3D confocal micrograph projection reconstruction of fixed whole-mount small intestine tissue, and magnification of C. difficile spores associated with Fn or Vn. Plot profiles of fluorescence intensity of C. difficile spores (red line) and accessible Fn or Vn (green lines). f,h , quantification of spores that were positive (Fn+) or negative (Fn-) for Fn fluorescence signal in f , or positive (Vn+) or negative (Vn-) for Vn fluorescence signal in h . The average of associated and non-associated spores with f Fn or h Vn for each field. A total of ∼ 500 spores were counted per mice (n = 5 per group). GRUBB’s test was performed to identify outliers, and one point was removed in Vn. Error bars indicate mean ± S.E.M. Statistical analysis was performed by two-tailed unpaired Student’s t test; ns indicates non-significant differences. Scale bar, 20 μm.

Article Snippet: For staining surface-accessible proteins, cells were incubated with primary antibodies (1:200 dilution) including mouse monoclonal antibodies against human fibronectin (sc8422, Santa Cruz Biotechnologies, USA), vitronectin (sc74484, Santa Cruz Biotechnologies, USA), integrin α 5 (ab78614, Abcam, USA), integrin α v (ab16821, Abcam, USA), and integrin β 1 (MAB1959Z, Millipore, USA).

Techniques: Control, Immunofluorescence, Fluorescence, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Clostridioides difficile major toxins remodel the intestinal epithelia, affecting spore adherence/internalization into intestinal tissue and their association with gut vitronectin

doi: 10.1101/2025.01.29.635439

Figure Lengend Snippet: Model of C. difficile toxin-mediated spore interactions and Bezlotoxumab effects on intestinal epithelial cells. Left panel (CDI): During C. difficile infection, toxins A and B (TcdA/TcdB) cause increased accessible (Acc) fibronectin (Fn), vitronectin (Vn), and their associated integrins (α5 and αv) on both apical and basal surfaces of intestinal epithelial cells. This leads to enhanced spore adherence and internalization, ultimately resulting in epithelial cell apoptosis. Right panel (CDI + Bezlotoxumab): Treatment with Bezlotoxumab partially neutralizes TcdB, reducing epithelial damage and spore adherence/internalization. While total Vn levels increase, total Fn levels decrease. The presence of Bezlotoxumab antibodies results in reduced spore adherence and internalization compared to untreated conditions, demonstrating partial protection against TcdB- mediated damage. Arrows indicate increased (↑) or decreased (↓) levels of proteins. A and B represent TcdA and TcdB toxins, respectively. The apical and basal sides of the epithelium are indicated.

Article Snippet: For staining surface-accessible proteins, cells were incubated with primary antibodies (1:200 dilution) including mouse monoclonal antibodies against human fibronectin (sc8422, Santa Cruz Biotechnologies, USA), vitronectin (sc74484, Santa Cruz Biotechnologies, USA), integrin α 5 (ab78614, Abcam, USA), integrin α v (ab16821, Abcam, USA), and integrin β 1 (MAB1959Z, Millipore, USA).

Techniques: Infection