Journal: Nature Communications

Article Title: The C. difficile toxin B membrane translocation machinery is an evolutionarily conserved protein delivery apparatus

doi: 10.1038/s41467-020-14306-z

Figure Lengend Snippet: Defining the evolutionarily conserved and minimal LCT translocase region. a Construct design of ADPR-[truncated TcdB T-domain]-DTR chimeras. b Dye release from HPTS/DPX loaded liposomes at pH 4.5, with truncated TcdB T-domains colored as follows: 800–1500 (red), 800–1473 (green), 800–1394 (yellow), and 800–1338 (blue). c Quantification of dye release from HPTS/DPX loaded liposomes after 20 min, at pH 4.5 and pH 8.0 ( N = 3). d Box plot of EC 50 values from cell viability and protein synthesis inhibition of Vero cells ( N = 4), where center line is the median, bounds of box represent the interquartile range, and the wiskers represent the minimum and maximum of data. Panels e – h the same as a – d , respectively, but with truncated TcdB T-domains 851–1500 (red), 851–1473 (green), 851–1394 (yellow), and 851–1338 (blue). Source data are provided as a Source Data File for Fig. 5 c , g , d , h .

Article Snippet: After drying down with N2 , the lipid film was resuspended in 20 mM Tris, 150 mM NaCl, pH 8.0, 35 mM 8-Hydroxypyrene-1,3,6-trisulfonic acid (HPTS) and 50 mM p-xylene-bis-pyridinium bromide (DPX) (Thermo Fischer).

Techniques: Construct, Inhibition

Journal: Nature Communications

Article Title: The C. difficile toxin B membrane translocation machinery is an evolutionarily conserved protein delivery apparatus

doi: 10.1038/s41467-020-14306-z

Figure Lengend Snippet: TcdB 851-1473 is an autonomous protein domain. a TcdA T-domain (pdb: 4R04), with 851–1473 colored in green. b TcdB 851–1473 induced dye release from HPTS/DPX loaded liposomes from pH 4.0 to pH 7.0 in 0.5 pH increments. c Stability of TcdB 851–1473 in aqueous buffer and with d 20 mM DPC from pH 4.0 to pH 7.5 in 0.5 pH increments. e Circular dichroism (CD) spectroscopy of TcdB 851–1473 in aqueous buffer and f 20 mM DPC from pH 4.0 to pH 7.5 in 0.5 pH. Coloring for each pH in f is the same as in e . All experiments are N = 3. Source data are provided as a Source Data File for Fig. 7c, d.

Article Snippet: After drying down with N2 , the lipid film was resuspended in 20 mM Tris, 150 mM NaCl, pH 8.0, 35 mM 8-Hydroxypyrene-1,3,6-trisulfonic acid (HPTS) and 50 mM p-xylene-bis-pyridinium bromide (DPX) (Thermo Fischer).

Techniques: Spectroscopy

Journal: Nature Communications

Article Title: The C. difficile toxin B membrane translocation machinery is an evolutionarily conserved protein delivery apparatus

doi: 10.1038/s41467-020-14306-z

Figure Lengend Snippet: Characterization of S. marcescens (Sm) toxin. a Light microscopy images of HeLa cells transfected with PiggyBac Dual Promoter PB5131B-1 vector, expressing empty plasmid or Sm toxin effector (1–600 or 36–600) after 24 h. b Addition of Insp6 (+) to Sm toxin results in cleavage of the effector (band 3) and the CPD (band 4) from the full-length toxin (band 1). Band 2 represents full-length Sm toxin without the effector and CPD. c Dye release from HPTS/DPX loaded liposomes at pH 4.5 and pH 8.0. d Quantification of dye release from HPTS/DPX loaded liposomes after 20 minutes. N = 3 for all experiments. Source data are provided as a Source Data File for Fig. 3e. e Light microscopy images of HCT-116 cells with no toxin (untreated) or with varying concentrations of Sm toxin after 24 h.

Article Snippet: After drying down with N2 , the lipid film was resuspended in 20 mM Tris, 150 mM NaCl, pH 8.0, 35 mM 8-Hydroxypyrene-1,3,6-trisulfonic acid (HPTS) and 50 mM p-xylene-bis-pyridinium bromide (DPX) (Thermo Fischer).

Techniques: Light Microscopy, Transfection, Plasmid Preparation, Expressing