Figure 4. sGAGs are major attachment factors for TcdA.

a. The absence of LDLR expression in two SLC35B2^−/−/LDLR^−/− HeLa cell lines was confirmed by immunoblot analysis. The experiments were repeated three times independently with similar results.

b. The sensitivities of two SLC35B2^−/−/LDLR^−/− HeLa cell lines and their parental cell line SLC35B2^−/− (#5) to TcdA^1–1832 were quantified using the cytopathic cell-rounding assay, and normalized to the level of WT HeLa cells.

c. Ectopic expression of a mouse Ldlr did not restore TcdA^1–1832 (2 nM, 4 h) entry into SLC35B2^−/− cells under our assay conditions. GFP marked transfected cells. Representative images were shown on the left side. Cell rounding was quantified and shown on the right side.

d. Immunofluorescence analysis showed that Alexa555-labelled TcdA^1–1874 (5 nM) robustly bound to HeLa WT and LDLR^−/− (#4) cells, but not SLC35B2^−/− (#5) cells. Cell nuclei were labeled with Hoechst dye. DIC: differential interference contrast image. The experiments were repeated three times.

e. Co-injection of surfen (50 μM) with Alexa555-labelled TcdA^1–1874 (5 nM, red) into ligated colon prevented TcdA^1–1874 binding to the colonic epithelium. Cell nuclei were labeled with DAPI dye (blue). Ep: epithelial cells; SM: smooth muscles.

f. Co-injection of heparin, GM-1111, or sulfated cyclodextrin, but not HA (all at 1 mg/mL) with Alexa555-labelled TcdA^1–1874 (5 nM) into the ligated colon reduced TcdA^1–1874 binding to the colonic epithelium. Cell nuclei were labeled with DAPI dye (blue). Ep: epithelial cells; SM: smooth muscles.

For b and c, n = 6, n.s. (p > 0.05). Mann-Whitney Test (two-sided). For e and f, n = 3, binding of TcdA was quantified using ImageJ and statistical analysis is two-sided Student’s t test. Each data point was also shown as triangle in the bar graph. Centre values represent mean and error bars represent ± s.d..