The N-terminal 34 kDa fragment of Helicobacter pylori vacuolating cytotoxin targets mitochondria and induces cytochrome c release.
The pathogenic bacterium Helicobacter pylori produces the cytotoxin VacA, which is implicated in the genesis of gastric epithelial lesions. By transfect ing HEp-2 cells with DNAs encoding either the N-terminal (p34) or the C-terminal (p58) fragment of VacA, p34 was found localized specifically to mitochondria, whereas p58 was cytosolic. Incubated in vitro with purified mitochondria, VacA and p34 but not p58 translocated into the mitochondria. Microinjection of DNAs encoding VacA-GFP and p34-GFP, but not GFP-VacA or GFP-p34, induced cell death by apoptosis. Transient transfection of HeLa cells with p34-GFP or VacA-GFP induced the release of cytochrome c from mitochondria and activated the executioner caspase 3, as determined by the cleavage of poly(ADP-ribose) polymerase (PARP). PARP cleavage was antagonized specifically by co-transfection of DNA encoding Bcl-2, known to block mitochondria-dependent apoptotic signals. The relevance of these observations to the in vivo mechanism of VacA action was supported by the fact that purified activated VacA applied externally to cells induced cytochrome c release into the cytosol.
Galmiche
A
A
INSERM U452, Faculté de Médecine, 28 avenue de Valombrose, 06107 Nice, CNRS-UMR 6543 and Centre Antoine Lacassagne, avenue de Valombrose, 06189 Nice, France.
Rassow
J
J
Doye
A
A
Cagnol
S
S
Chambard
J C
JC
Contamin
S
S
de Thillot
V
V
Just
I
I
Ricci
V
V
Solcia
E
E
Van Obberghen
E
E
Boquet
P
P
D000818:Animals; D017209:Apoptosis; D001426:Bacterial Proteins / Q000378:metabolism*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003574:Cytochrome c Group / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004072:Digitonin / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D008164:Luminescent Proteins / Q000378:metabolism; D008854:Microscopy, Electron; D008928:Mitochondria / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D013272:Stomach Diseases / Q000378:metabolism; D014162:Transfection
D001426:Bacterial Proteins; D003574:Cytochrome c Group; D008164:Luminescent Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; C074689:VacA protein, Helicobacter pylori; D049452:Green Fluorescent Proteins; D011065:Poly(ADP-ribose) Polymerases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D004072:Digitonin