Abstract

The vacuolating cytotoxin (VacA) of the gastric pathogen Helicobacter pylori binds and enters epithelial cells, ultimately resulting in cellular vacuolation. Several host factors have been reported to be important for VacA function, but none of these have been demonstrated to be essential for toxin binding to the plasma membrane. Thus, the identity of cell surface receptors critical for both toxin binding and function has remained elusive. Here, we identify VacA as the first bacterial virulence factor that exploits the important plasma membrane sphingolipid, sphingomyelin (SM), as a cellular receptor. Depletion of plasma membrane SM with sphingomyelinase inhibited VacA-mediated vacuolation and significantly reduced the sensitivity of HeLa cells, as well as several other cell lines, to VacA. Further analysis revealed that SM is critical for VacA interactions with the plasma membrane. Restoring plasma membrane SM in cells previously depleted of SM was sufficient to rescue both toxin vacuolation activity and plasma membrane binding. VacA association with detergent-resistant membranes was inhibited in cells pretreated with SMase C, indicating the importance of SM for VacA association with lipid raft microdomains. Finally, VacA bound to SM in an in vitro ELISA assay in a manner competitively inhibited by lysenin, a known SM-binding protein. Our results suggest a model where VacA may exploit the capacity of SM to preferentially partition into lipid rafts in order to access the raft-associated cellular machinery previously shown to be required for toxin entry into host cells. Author Summary Sensitivity to toxins produced by pathogenic bacteria is largely dictated by the presence or absence of toxin receptors on the plasma membrane of host cells. VacA is an important toxin produced by the pathogenic bacterium Helicobacter pylori, which infects the human stomach and causes gastric ulcer disease and stomach cancer. VacA binds and enters human cells, and induces several changes resulting ultimately in the death of the intoxicated cells. However, the identity of the VacA receptor responsible for toxin binding and function has remained a topic of debate. In this paper, we demonstrate that sphingomyelin, a lipid on the surface of cells with important membrane structural and signaling properties, functions as a VacA receptor. We demonstrate that VacA binds to sphingomyelin, and that presence or absence of sphingomyelin on the plasma membrane dictates how much VacA binds to the cell surface, and therefore, how sensitive cells are to the toxin. The identification of sphingomyelin also provides a conceptual framework for how VacA may enter cells through specialized functional domains on the surface of cells. This is the first example of a bacterial toxin that exploits sphingomyelin as a receptor, and future work will focus on developing strategies to block VacA interactions with sphingomyelin, thereby protecting cells from the downstream consequences of toxin action.