| Chronic infection with the gastric pathogen Helicobacter pylori is a risk factor for the development of gastric ulcer disease or gastric adenocarcinoma in humans. The success of H. pylori in the harsh stomach environment is due largely to pathogen-mediated remodeling of gastric tissue by virulence factors produced by the pathogen. One of the most important virulence factors, the vacuolating cytotoxin (VacA), which is the only known exotoxin released by H. pylori into the stomach of infected individuals, contributes to the pathogenesis of infection by directly modulating several important properties of host cells. To be effective, VacA, must bind and enter gastric cells, but the molecular basis of toxin binding and entry are complex, and have been poorly understood. In particular, the identity of the receptor that VacA binds on the surface of host cells, as well as the importance of the receptor for toxin entry into human cells, were the major gaps in knowledge addressed within this dissertation research. The toxin-receptor interaction is the first step in the intoxication pathway of essentially all bacterial toxins, and provides the basis for development of novel drugs to block the action of toxins. Here, we identify VacA as the first bacterial virulence factor that exploits the important and abundant plasma membrane sphingolipid, sphingomyelin, as a cellular receptor. Cellular sphingomyelin levels correlated strongly to sensitivity to VacA. Modulation of sphingomyelin levels specifically at the plasma membrane altered VacA-mediated intoxication. However, cells with prebound VacA were no longer sensitive to alterations in cellular sphingomyelin levels, suggesting the importance of this lipid in an early step in the intoxication pathway. Cell binding and internalization of VacA into host cells are modulated by sphingomyelin in a manner correlated to the toxin's cellular activity. Importantly, VacA was demonstrated to bind sphingomyelin in vitro, supporting the idea that VacA directly binds sphingomyelin on the cell surface. To differentiate whether sphingomyelin functions only to bind VacA to the surface of gastric cells or, alternatively, plays an active role in toxin entry into host cells, we took advantage of recent advances in sphingolipid chemistry to evaluate toxin entry and trafficking in cells. Cells were reconstituted with synthetic sphingomyelin derivatives that retain the capacity to bind VacA to the cell surface, but differed in their cellular entry and trafficking properties. These studies revealed that when exposed to VacA, sphingomyelin is important for not only binding the toxin to the cell surface, but also for directing VacA to the correct Cdc42-dependent pinocytic uptake pathway. Moreover, we demonstrated that sphingomyelin is important for trafficking VacA to late-endosomal/lysosomal compartments required for toxin activity. Additional studies into the mechanism of sphingomyelin-dependent cellular entry and trafficking revealed that sphingomyelin functions to partition VacA into specialized signaling domains on the cell surface called membrane rafts. Identifying the VacA receptor provides a new extracellular target for blocking the action of the toxin on cells, which when used in concert with antibiotics to control H. pylori infection, may potentially reduce the risk of stomach cancer. |
