Figure 4. Binding of ErpG-producing B. burgdorferi strain B314 to C6 glial cells is mediated by heparan sulfate.

(A) Heparin, Hep-SO₄, Chon-4-SO₄, Derm SO₄, or Chon-6-SO₄ were added to the indicated B. burgdorferi strain to a final concentration of 6.25mg/ml prior to infection of C6 glial cells in suspension (see Experimental Procedures). PBS alone (“None”) was utilized as a negative control. The bacteria were stained with FITC-conjugated anti-B. burgdorferi antibody. The percentage of B. burgdorferi-bound cells was measured by flow cytometry. Each bar represents the mean of 12 independent determinations ± SEM. Statistically significant reductions in the percentage of cells bound by treated spirochetes compared to PBS-treated spirochetes are indicated (*, P-value <0.05). (B) Radiolabeled strains B31 and B314/pErpG were incubated with 2 mg/ml of the indicated GAG (or PBS alone as a negative control) for 30 min prior to infection of C6 glial cell monolayers, and the percentage of spirochetes stably bound was determined by scintillation counting. Each bar represents the mean of 12 independent determinations ± SEM. Significant reductions in spirochetal binding relative to spirochetes pre-incubated with PBS are indicated (*, P-value < 0.05). (C) The indicated B. burgdorferi strain was added to C6 glial cells in suspension that had been mock-treated (i.e. buffer alone) or treated with heparitinase (Hpt.) chondroitinase AC (Chon. AC), or chondroitinase B (Chon. B), and the percentage of B. burgdorferi-bound cells was measured as above. Note that the experiments in panels A and C were performed concurrently and binding of strain B314/Vector to mock-treated C6 glial cells in the absence of competitor served as a negative control for both experiments. Each bar represents the mean of 12 independent determinations ± SEM. Statistically significant reduction in the percentage of lyase-treated cells bound by spirochetes compared with PBS-treated cells is indicated (*, P-value <0.05). (D) The percentage of radiolabeled strains B31, B314/pErpG, and B314/pJF21 (“B314/vector”) (negative control) that stably bound to wells containing monolayers of C6 glial cells that had been mock-treated or treated with the indicated lyase was determined by scintillation counting. Note that the experiments in panels B and D were performed concurrently, and binding of strain B314/Vector to mock-treated C6 glial cells in the absence of competitor served as a negative control for both experiments. Each bar represents the mean of 12 independent determinations ± SEM. Significant reduction in spirochetal binding relative to PBS-treated cells is indicated (*, P-value < 0.05).