full-length tcda and tcdb toxins  (List Biological Laboratories)

Journal: Nature Communications

Article Title: Inhibition of Clostridium difficile TcdA and TcdB toxins with transition state analogues

doi: 10.1038/s41467-021-26580-6

Figure Lengend Snippet: a (top) Schematic of the Tcd toxin domain structure: N-terminal glucosyltransferase domain GTD (red), cysteine protease domain CPD (blue), delivery domain (orange) and receptor binding domain RBD (green). (Bottom) Schematic of mechanism of TcdA and TcdB-induced toxicity of mammalian cells (see “Introduction” for details), adapted from reference 25. b Schematic of TcdA-GTD and TcdB-GTD catalyzed reactions. Glucosyltransferase reaction catalyzed by TcdB-GTD and TcdA-GTD (left). Glucohydrolase reaction catalyzed by TcdB-GTD and TcdA-GTD with water acting as the nucleophile (right).

Article Snippet: Full-length TcdA and TcdB toxins were purchased from List Biological Laboratories Inc. FITC-Annexin V staining kit was purchased from Biolegend.

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Inhibition of Clostridium difficile TcdA and TcdB toxins with transition state analogues

doi: 10.1038/s41467-021-26580-6

Figure Lengend Snippet: Chemical structures of transition state analogues that were tested for inhibition of TcdA-GTD and TcdB-GTD in this study. Positions of relevant carbon atoms in isofagomine and noeuromycin are indicated.

Article Snippet: Full-length TcdA and TcdB toxins were purchased from List Biological Laboratories Inc. FITC-Annexin V staining kit was purchased from Biolegend.

Techniques: Analogues, Inhibition

Journal: Nature Communications

Article Title: Inhibition of Clostridium difficile TcdA and TcdB toxins with transition state analogues

doi: 10.1038/s41467-021-26580-6

Figure Lengend Snippet: a Representative image from at least 3 experiments of Vero cells 2 h after treatment with TcdB and isofagomine (isof). Scale bar represents 10 µM. b Representative Western blot images for intracellular Rac1 glucosylation (n=3). Human IMR90 cells were treated with isofagomine (isof) or noeuromycin (noe) (doses indicated) for 30 min, followed by treatment with buffer control, 1 nM TcdA or 0.1 nM TcdB for 6 h. Cell lysates were prepared as described in Methods. Mab102 was used to detect unglucosylated Rac1 in cell lysates. Anti-Rac1 (23A8) was used to detect total Rac1 levels and anti-GAPDH served as the loading control. Uncropped Western blots are shown in Supplementary Figs. , . c Flow cytometry analysis of AnnexinV in HT-29 cells from 5 independent experiments (n=5). HT-29 cells were pre-treated with isofagomine (isof) or noeuromycin (noe) (250 µM or 500 µM) for 30 min before treatment with buffer control or 1 nM TcdA for 24 h. Cells were harvested and stained with AnnexinV as described in Methods. AnnexinV positivity (%) was normalized to untreated HT-29 cells. Untreated cells are shown as white circles, TcdA treated cells are shown in black circles, isofagomine and TcdA treated cells are shown as red circles and noeuromycin and TcdA treated cells are shown as blue circles. Ordinary one-way ANOVA with Tukey’s multiple comparison test, significance ****p < 0.0001, *** p < 0.001. Error bars show mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: Full-length TcdA and TcdB toxins were purchased from List Biological Laboratories Inc. FITC-Annexin V staining kit was purchased from Biolegend.

Techniques: Western Blot, Control, Flow Cytometry, Staining, Comparison

Journal: Nature Communications

Article Title: Inhibition of Clostridium difficile TcdA and TcdB toxins with transition state analogues

doi: 10.1038/s41467-021-26580-6

Figure Lengend Snippet: Efficacy of isofagomine and noeuromycin against TcdA and TcdB-induced cell rounding of Vero cells.

Article Snippet: Full-length TcdA and TcdB toxins were purchased from List Biological Laboratories Inc. FITC-Annexin V staining kit was purchased from Biolegend.

Techniques: