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J Biol Chem, Vol. 274, Issue 14, 9277-9282, April 2, 1999
From the Department of Biology and Biochemistry, University of
Houston, Houston, Texas 77204-5513
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.
Identification of the Minimal Intracellular Vacuolating
Domain of the Helicobacter pylori Vacuolating
Toxin
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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