colonizes the gastric mucosa and secretes a pore-forming toxin (VacA). Two main types of VacA, m1 and m2, can be distinguished by phylogenetic analysis. Type m1 forms of VacA have been extensively studied, but there has been relatively little study of m2 forms. In this study, we generated strains producing chimeric proteins in which VacA m1 segments of a parental strain were replaced by corresponding m2 sequences. In comparison to the parental m1 VacA protein, a chimeric protein (designated m2/m1) containing m2 sequences in the N-terminal portion of the m region was less potent in causing vacuolation of HeLa cells, AGS gastric cells, and AZ-521 duodenal cells and had reduced capacity to cause membrane depolarization or death of AZ-521 cells. Consistent with the observed differences in activity, the chimeric m2/m1 VacA protein bound to cells at reduced levels compared to the binding levels of the parental m1 protein. The presence of two strain-specific insertions or deletions within or adjacent to the m region did not influence toxin activity. Experiments with human gastric organoids grown as monolayers indicated that m1 and m2/m1 forms of VacA had similar cell-vacuolating activities. Interestingly, both forms of VacA bound preferentially to the basolateral surface of organoid monolayers and caused increased cell vacuolation when interacting with the basolateral surface compared to the apical surface. These data provide insights into functional correlates of sequence variation in the VacA midregion (m region).
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