Fig. 1: Sulfated glycosaminoglycans and LDLR mediate the cellular entry of Tcnα.
a Schematic of the screening process using Tcnα on HeLa cells with GeCKO v2 library. b Genes identified are ranked and plotted based on fold-enrichment of their gRNA from the beginning (R0) to 3-round post-toxin selection (R3). Genes involved in heparan sulfate biosynthesis are marked. c Sensitivities of the WT, SLC35B2‒/‒, B4GALT7‒/‒, EXTL3‒/‒, EXT1‒/‒, and EXT2‒/‒ HeLa cells to Tcnα were measured using the cytopathic cell-rounding assay. The percentages of rounded cells were quantified, plotted, and fitted. d Resistance of SLC35B2‒/‒, B4GALT7‒/‒, EXTL3‒/‒, EXT1‒/‒, and EXT2‒/‒ HeLa cells to Tcnα were normalized to the level of WT cells based on CR50. (***P < 0.001 vs WT). e Confocal fluorescence images of Rhodamine-labeled Tcnα binding to the WT, EXT1‒/‒, and SLC35B2‒/‒ HeLa cells. Cell nuclei were labeled with Hoechst. f HeLa cells were exposed to either Tcnα (7.5 nM) or Tcnα pre-mixed with the indicated glycans (1 mg/mL). The levels of cell rounding with 6 h incubation were plotted in a bar chart. (***P < 0.001 vs control). g HeLa cells were pre-incubated with surfen of the indicated concentrations and then exposed to Tcnα (10 nM). The levels of cell rounding after 7 h incubation were plotted in a bar chart. h Sensitivities of the WT, LDLR‒/‒, FZD1/2/7‒/‒, and CSPG4‒/‒ HeLa cells to Tcnα were measured using the cytopathic cell-rounding assay. The percentages of rounded cells were quantified, plotted, and fitted. i Resistance of LDLR‒/‒, FZD1/2/7‒/‒, and CSPG4‒/‒ HeLa cells to Tcnα were normalized to the level of WT cells. (***P < 0.001 vs WT). j Confocal fluorescence microscopy showing different binding of Rhodamine-labeled Tcnα or TcdB to the WT, LDLR‒/‒, SLC35B2‒/‒, and CSPG4‒/‒ HeLa cells. Cell nuclei were labeled with Hoechst. k Expression of mouse Ldlr restored Tcnα entry into HeLa LDLR‒/‒ cells, resulting in cell rounding; expression of mouse Ldlr∆C also restored Tcnα entry but less efficiently. Red fluorescence (mCherry) marked transfected cells. l Cell rounding among fluorescence positive cells shown in k was quantified and plotted in a bar chart. (***P < 0.001). m Sensitivities of the HeLa WT, LDLR‒/‒, and LDLR‒/‒/SLC35B2‒/‒ cells to Tcnα were measured using the cytopathic cell-rounding assay. The percentages of rounded cells were quantified, plotted, and fitted. n Resistance of LDLR‒/‒, and LDLR‒/‒/SLC35B2‒/‒ HeLa cells to Tcnα were normalized to the level of WT cells. (***P < 0.005). o LRPAP1 in culture medium further protected HeLa LDLR‒/‒ cells from Tcnα (left panel, 5 nM) but not TcdB (right panel, 2.5 pM) under the molar ratio of 1:1000 (toxin vs LRPAP1). The percentages of rounded cells were quantified and plotted over time. p Anatomy of the mouse tibialis anterior muscles after i.m. injection of saline, Tcnα, or Tcnα + SCD. q Representative images of H&E staining sections from the mice tibialis anterior muscles after i.m. injection of saline, Tcnα alone, or Tcnα + SCD. Asterisks indicate inflammatory cell infiltrate, solid arrowheads indicate hemorrhage, and hollow arrowheads indicate central nuclei. r Histopathological scores were accessed and summarized based on hemorrhage, inflammatory cell infiltration, and muscle fiber injury. Data are means ± SD, n = 6 in c, d, f–i, and l–o, two-sided Student’s t-test. Data are means ± SEM, n = 6 in r, Mann–Whitney test. Scale bar represents 50 μm in e, j, and k, and 100 μm in q.
