Lipid rafts are essential for cellular intoxication mediated by Helicobacter pylori vacuolating toxin

Persistent Helicobacter pylori infections can progress to peptic ulcer disease and gastric cancer in humans. Many H. pylori strains secrete a protein toxin (VacA) that causes degenerative vacuolation of mammalian cells both in vitro and in vivo. The mechanism by which VacA interacts with and is trafficked within the target cells subsequent to cellular entry is not well understood. Identification of cellular determinants that are essential for vacuolation will yield critical information regarding the mechanism of VacA intoxication. By disrupting specific steps of two well characterized internalization pathways, the late endosomal and the retrograde pathway, we confirmed and expanded previous observations that these pathways are not important for VacA cellular intoxication. Since these are the only two pathways known to be utilized by intracellularly-acting toxins such as cholera, diphtheria and anthrax toxin, these results suggest that VacA may be internalized by a novel mechanism. Herein, we demonstrate that VacA-mediated vacuolation is dependent on lipid rafts. Lipid rafts can be divided into different subclasses based on protein and lipid composition. We have started to characterize lipid raft components that are important for VacA mediated vacuolation. We discovered that plasma membrane cholesterol and sphingomyelin are important for VacA-mediated vacuolation. Depletion of both cholesterol and sphingomyelin inhibited VacA-mediated vacuolation. However, depleting plasma membrane cholesterol and sphingomyelin had differential effects on VacA interactions with target cells. Cholesterol is important for the cellular activity of VacA by regulating entry of toxin into sensitive cells, and then modulating cellular processes that are essential for vacuole biogenesis whereas sphingomyelin is important for the association of VacA to host cells but not vacuole biogenesis. We also demonstrate that VacA does not bind directly to cholesterol. Collectively, these results suggest that multiple subclasses of lipid rafts may be involved in VacA-mediated cellular intoxication. This would be the first example of an intoxication mechanism that involves multiple subclasses of lipid rafts. Finally, preliminary studies imply that different toxins may utilize different subclasses of lipid raft as a portal of entry into cells. This finding serves as springboard for future studies to identify the role of these subclasses in the intoxication mechanisms of bacterial toxins and as potential targets for the development of efficient therapeutic strategies to prevent infection by several pathogens.