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 mammalian cells. Although evidence is accumulating that VacA is an A-B toxin, distinct A and B fragments have not been identified. To localize the putative catalytic A-fragment, we transfected HeLa cells with plasmids encoding truncated forms of VacA fused to green fluorescence protein. By analyzing truncated VacA fragments for intracellular vacuolating activity, we reduced the minimal functional domain to the amino-terminal 422 residues of VacA, which is less than one-half of the full-length protein (953 amino acids). VacA is frequently isolated as a proteolytically nicked protein of two fragments that remain noncovalently associated and retain vacuolating activity. Neither the amino-terminal 311 residue fragment (p33) nor the carboxyl-terminal 642 residue fragment (p70) of proteolytically nicked VacA are able to induce cellular vacuolation by themselves. However, co-transfection of HeLa cells with separate plasmids expressing both p33 and p70 resulted in vacuolated 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 localization of both fragments of nicked toxin required to mediate intracellular vacuolating activity.
Ye
D
D
Department of Biology and Biochemistry, University of Houston, Houston, Texas 77204-5513, USA.
Willhite
D C
DC
Blanke
S R
SR
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000935:Antifungal Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D003603:Cytotoxins / Q000737:chemistry* / Q000235:genetics; D006367:HeLa Cells; D016480:Helicobacter pylori; D006801:Humans; D018942:Macrolides; D008985:Monensin / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D011401:Promoter Regions, Genetic; D013329:Structure-Activity Relationship; D014617:Vacuoles / Q000737:chemistry* / Q000235:genetics
D000900:Anti-Bacterial Agents; D000935:Antifungal Agents; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003603:Cytotoxins; D018942:Macrolides; D010446:Peptide Fragments; C074689:VacA protein, Helicobacter pylori; C040929:bafilomycin A1; D008985:Monensin