RACK1 protein interacts with Helicobacter pylori VacA cytotoxin: The yeasttwo-hybrid approach

The VacA toxin is the major virulence factor of Helicobacter pylori. The st udies on VacA intracellular expression suggest that it interacts with cytos olic proteins and that this interaction contributes significantly to vacuol ization. The aim of this study was to identify the host protein(s) that int eracts with the VacA protein. We used the fragments of VacA protein fused w ith GAL4-BD as the baits in the yeast two-hybrid approach. The yeast transf ormed with plasmids encoding bait proteins were screened with human gastric mucosa cDNA library, encoded C-terminal fusion proteins with GAL4-AD. Thre e independent His-beta -Ga1-positive clones were identified in VacA-b1 scre en; they matched two different lengths of cDNA encoding RACK1 protein. The specific activity of beta -galactosidase found in the yeast expressing both VacA-b1 and RACK1 fusion proteins was 12-19 times higher compared to all n egative controls used. VacA is capable of binding the RACK1 in vitro as was confirmed by the pull-down assay with GST fusion VacA protein and [S-35]Me t-labeled RACK1 protein fragments. (C) 2001 Academic Press.