Wednesday, February 5, 2025
2/5/2025
SUMMARY This exploratory R21 application seeks to identify and characterize the molecular basis by which the Vacuolating Cytotoxin (VacA), which is the only known intracellular-acting exotoxin secreted by the human gastric pathogen Helicobacter pylori (Hp), recognizes and binds to host cells as a requisite step for host cell intoxication. Chronic infection with Hp is the single most important risk factor for gastric adenocarcinoma, the third leading cause of cancer-related deaths worldwide. Nearly all Hp isolates harbor vacA, which has been demonstrated to be important for colonization in a murine model of Hp infection. Relevant to this application, specific polymorphisms within vacA correlate with both toxin cellular activity and the severity of Hp-associated diseases. In particular, a region located in carboxyl-terminal domain of VacA, known as the “middle (m) region”, segregates into two allelic variants, m1-VacA, which is associated with higher risk of disease, and more potent cellular modulatory activity, and m2-VacA, associated with lower risk of gastric disease, and less potent cellular modulatory activity. Although incompletely understood, several studies have suggested that m1-VacA and m2-VacA may differ in host cell receptor specificity, which is a critical determinant of toxin cell tropism. Moreover, results from extensive phylogenetic analyses suggest that the m1- and m2- forms of VacA may possess different cellular activities that bestow a selective advantage for maintaining both alleles in the human population. Previous studies in the PI’s laboratory identified the abundant plasma membrane surface sphingolipid, sphingomyelin (SM), as an important determinant for m1-VacA binding to the cell surface of host cells as an important determinant of toxin cellular activity. Moreover, in vitro experiments indicate that VacA preferentially binds to SM over other common membrane lipids. These and other findings support our overall hypothesis that SM functions as a cell surface receptor for m1-VacA. Nonetheless, essentially nothing is known about how VacA recognizes and binds to the toxin’s sphingolipid receptor. Moreover, it is not currently known whether SM-dependent toxin cell surface binding and cellular activity, which we discovered to be associated with m1-VacA, are properties that extend to m2-variants of the toxin. This application proposes studies to address these gaps in knowledge in the context of two inter-related, but not co-dependent Specific Aims. In Aim 1, we propose to identify the SM-receptor binding site of m1-VacA, using both biochemical and computational approaches. In Aim 2, we will extend these approaches to evaluate whether the importance of cell-surface SM for m1-VacA, extends also to m2-VacA. Completion of these exploratory studies will address for the first time whether SM-receptor dependent cell binding and activity is a shared characteristic of both m1- and m2-toxin allelic variants, and will provide the framework for future studies to not only better understand both the molecular basis and consequences of VacA allelic variation, but also set the stage for structure-based inhibitor development to block the very earliest stages of VacA cellular intoxication.
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