Plasma membrane sphingomyelin (SM) binds the Helicobacter pylori vacuolating toxin (VacA) to the surface of epithelial cells. To evaluate the importance of SM for VacA cellular entry, we characterized toxin uptake and trafficking within cells enriched with synthetic variants of SM, whose intracellular trafficking properties are strictly dependent on the acyl chain lengths of their sphingolipid backbones. While toxin binding to the surface of cells was independent of acyl chain length, cells enriched with 12- or 18-carbon acyl chain variants of SM (e.g. C12-SM or C18-SM) were more sensitive to VacA, as indicated by toxin-induced cellular vacuolation, than those enriched with shorter 2- or 6-carbon variants (e.g. C2-SM or C6-SM). In C18-SM-enriched cells, VacA was taken into cells by a previously described Cdc42-dependent pinocytic mechanism, localized initially to GPI-enriched vesicles, and ultimately trafficked to Rab7/Lamp1 compartments. In contrast, within C2-SM-enriched cells, VacA was taken up at a slower rate by a Cdc42-independent mechanism and trafficked to Rab11 compartments. VacA-associated predominantly with detergent-resistant membranes (DRMs) in cells enriched with C18-SM, but predominantly with non-DRMs in C2-SM-enriched cells. These results suggest that SM is required for targeting VacA to membrane rafts important for subsequent Cdc42-dependent pinocytic cellular entry.
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