|Abstract:||The sensitivity or resistance of host cells to bacterial toxins is often dependent on the presence or absence of cell surface toxin receptors required for the binding and subsequent downstream action of the toxin. Our laboratory has identified plasma membrane sphingomyelin (SM) to be important for VacA cell surface binding, direct VacA-SM interactions, and intracellular activity, suggesting that SM is a receptor for VacA. More recent work from our lab has identified three proximally located residues, R552, W603, and R647, each situated in a different flexible loop a series of flexible loops are aligned adjacently in a linear fashion along a single edge of the extended beta-helix structure of VacA. These residues were shown to facilitate VacA interactions with plasma membrane SM. Based on our earlier work, we predict that the residues comprising the SM binding site function to correctly orient VacA on the surface of cells. In addition, we hypothesized that residues within adjacent loops along the same ridge might be involved in additional contacts with the plasma membrane. To evaluate this hypothesis, we examined the consequences of removing each of the loops along the ridge, one at a time, on the toxin cellular activity. Using site-directed mutagenesis, we generated eleven individual mutant forms of VacA, each lacking one of the loops. The entire sequence of each loop was substituted, and the remaining beta strands were reconnected with a flexible linker containing the sequence Gly-Gly-Gly. Among nine mutant VacA tested, six showed attenuation in toxin cellular activity, indicating that one or more residues in these loops are important for VacA-mediated cellular activity. Our data also showed that mutants lacking six loops, individually, had similar VacA-SM direct interactions, suggesting that these loops were not important for SM binding activity of VacA. Furthermore, our studies showed that the lack of two of the six loops showed moderated reduction in VacA binding to the surface of cells. However, there were no loops that individually showed a drastic impact on the cellular binding. Our data suggest that multiple loops are involved in VacA cell surface binding rather than any single loop providing the most binding energy. Our studies suggest a model in which once VacA is bound onto the surface of the cells via R552, W603, and R647, several loops along the same ridge of the toxin bind to the surface of the cell and may contribute to increasing the binding energy of the toxin. The results of this study will provide the framework for evaluating how surface bound VacA-SM complex enter the cell.