Identification of the minimal intracellular vacuolating domain of the Helicobacter pylori vacuolating toxin

Helicobacter pylori secretes a cytotoxin (VacA) that induces the formation of large vacuoles originating from late endocytic vesicles in sensitive mam malian cells. Although evidence is accumulating that VacA is an A-B toxin, distinct A and B fragments have not been identified. To localize the putati ve catalytic A-fragment, we transfected HeLa cells with plasmids encoding t runcated forms of VacA fused to green fluorescence protein. By analyzing tr uncated VacA fragments for intracellular vacuolating activity, we reduced t he minimal functional domain to the amino-terminal 422 residues of VacA, wh ich is less than one-half of the full-length protein (953 amino acids). Vac A is frequently isolated as a proteolytically nicked protein of two fragmen ts that remain noncovalently associated and retain vacuolating activity. Ne ither the amino-terminal 311 residue fragment (p33) nor the carboxyl-termin al 642 residue fragment (p70) of proteolytically nicked VacA are able to in duce cellular vacuolation by themselves. However, cotransfection of HeLa ce lls with separate plasmids expressing both p33 and p70 resulted in vacuolat ed cells. Further analysis revealed that a minimal fragment comprising just residues 312-478 functionally complemented p33. Collectively, our results suggest a novel molecular architecture for VacA, with cytosolic localizatio n of both fragments of nicked toxin required to mediate intracellular vacuo lating activity.