Figure 3. SM is Important for VacA Cell Binding.

(A) HeLa cells were treated in two different ways. First, cells were incubated for 1 h with SMase C (200 mU/mL; black bar), SM (50 μM; black bar), or mock-pretreated with PBS pH 7.2 (shaded bar) as indicated. The monolayers were then incubated for 4 h with VacA (200 nM) at 4 °C, and washed with PBS. Alternatively, cells were first incubated for 4 h with VacA (200 nM) at 4 °C and then incubated for 1 h with SM (50 μM; white bar) or SMase C (200 mU/mL; white bar), as indicated, and washed with PBS. For both cases, the monolayers were then incubated for an additional 24 h at 37 °C, and vacuolation was evaluated by measuring neutral red uptake. (B–D) HeLa cells were pretreated for 1 h with or without SM (50 μM) (B, panels 1–4) or SMase C (100 mU/mL) (B, panels 5–8; C–D). The monolayers were then incubated at 4 °C with 10 nM (B, panels 1–4) or 100 nM (B, panels 5–8; C–D), respectively, of Alexa Fluor 488 labeled VacA. For (D), cells were also incubated with Alexa Fluor 488-labeled cholera toxin B fragment (CTB) (100 nM), Alexa Fluor 488-labeled transferrin (60 nM), or Venus-lysenin (1 μM). After 1 h, the cells were analyzed by DIC/epifluorescence microscopy (B) or flow cytometry (C, D). For (C), MF is the geometric mean fluorescence. (A) VacA-mediated vacuolation in cells treated with SM or SMase C, as indicated, before (black bars) or after (white bars) exposure to toxin, relative to non-pretreated cells (shaded bar) exposed to VacA. Vacuolation data were normalized for neutral red uptake in mock-pretreated cells incubated with VacA. P values were calculated to determine statistically significant differences between cells pretreated with SM or SMase C and mock-pretreated cells. (B) Fluorescence micrographs (left-hand panels) and DIC images (right-hand panels) of VacA binding to cells that had been pretreated with (panels 3–4) or without (panels 1–2) SM, or, with (panels 7–8) or without SMase (panels 5–6). (C) Flow cytometry histograms of cells alone, cells incubated with VacA, and cells that had been pretreated with SMase prior to exposure to VacA, as indicated. (D) Relative binding of VacA, CTB, Venus-lysenin, and Tf, as indicated, to cells that had been preincubated with (black bars) or without (white bars) SMase. Binding data were normalized for geometric mean fluorescence of each fluorescently-labeled protein bound to mock-pretreated cells. For each protein, P values were calculated to determine statistically significant differences in cell binding between cells pretreated with SMase C and mock-pretreated cells.