Involvement of syntaxin 7 in human gastric epithelial cell vacuolation induced by the Helicobacter pylori-produced cytotoxin VacA.
The Helicobacter pylori-produced cytotoxin VacA induces intracellular vacuolation. The formed vacuole is assumed to be a hybrid of late endosome and lysosome. To elucidate the molecular mechanism of VacA-induced vacuolation, we examined the participation of syntaxin 7 in the human gastric epithelial cell line AGS. Immunocytochemistry revealed that endogenous syntaxin 7 was localized to vacuoles induced by VacA. Northern and Western blotting demonstrated that VacA intoxication increased syntaxin 7 mRNA and protein expression, respectively, in a time-dependent manner. Transient transfection of dominant-negative mutant syntaxin 7, which lacks a carboxyl-terminal transmembrane domain, inhibited VacA-induced vacuolation. In contrast, transient transfection of wild-type syntaxin 7, dominant-negative mutant syntaxin 1a, or dominant-negative mutant syntaxin 4 did not alter VacA-induced vacuolation. Furthermore, under VacA treatment, neutral red dye uptake, a parameter of VacA-induced vacuolation, was inhibited in cells stably transfected with mutant syntaxin 7 but not in cells stably transfected with wild-type syntaxin 7, mutant syntaxin 1a, or mutant syntaxin 4. Sequential immunocytochemical observation confirmed that expression of mutant syntaxin 7 did not affect VacA attachment to or internalization into AGS cells. We suggest that syntaxin 7 is involved in the intracellular vacuolation induced by VacA.
Department of Gastroenterology, University of Tokyo School of Medicine, Tokyo 113-8655, Japan.
Junko
J
Suzuki
Hirohide
H
Ohnishi
Akihiro
A
Wada
Toshiya
T
Hirayama
Hideki
H
Ohno
Namiki
N
Ueda
Hiroshi
H
Yasuda
Taroh
T
Iiri
Yoh
Y
Wada
Masamitsu
M
Futai
Hirosato
H
Mashima
D001426:Bacterial Proteins / Q000737:chemistry*; D015152:Blotting, Northern; D015153:Blotting, Western; D004396:Coloring Agents / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism*; D005799:Genes, Dominant; D049452:Green Fluorescent Proteins; D016480:Helicobacter pylori / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009154:Mutation; D009499:Neutral Red / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D050765:Qa-SNARE Proteins; D012333:RNA, Messenger / Q000378:metabolism; D013270:Stomach / Q000166:cytology*; D013274:Stomach Neoplasms / Q000382:microbiology; D050827:Syntaxin 1; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured; D015854:Up-Regulation; D014617:Vacuoles / Q000378:metabolism
D001426:Bacterial Proteins; D004396:Coloring Agents; D008164:Luminescent Proteins; D008565:Membrane Proteins; D050765:Qa-SNARE Proteins; D012333:RNA, Messenger; C494212:STX1A protein, human; D050827:Syntaxin 1; C074689:VacA protein, Helicobacter pylori; D049452:Green Fluorescent Proteins; D009499:Neutral Red; D004247:DNA