Persistence of Helicobacter pylori VacA toxin and vacuolating potential in cultured gastric epithelial cells.
The vacuolating toxin A (VacA) is one of the most important virulence factors in Helicobacter pylori-induced damage to human gastric epithelium. Using human gastric epithelial cells in culture and broth culture filtrate from a VacA-producing H. pylori strain, we studied 1) the delivery of VacA to cells, 2) the localization and fate of internalized toxin, and 3) the persistence of toxin inside the cell. The investigative techniques used were neutral red dye uptake, ultrastructural immunocytochemistry, quantitative immunofluorescence, and immunoblotting. We found that VacA 1) is delivered to cells in both free and membrane-bound form (i.e., as vesicles formed by the bacterial outer membrane), 2) localizes inside the endosomal-lysosomal compartment, in both free and membrane-bound form, 3) persists within the cell for at least 72 h, without loss of vacuolating power, which, however, becomes evident only when NH4Cl is added, and 4) generally does not degrade into fragments smaller than approximately 90 kDa. Our findings suggest that, while accumulating inside the endosomal-lysosomal compartment, a large amount of VacA avoids the main lysosomal degradative processes and retains its apparent molecular integrity.
Sommi
P
P
Institute of Human Physiology, University of Pavia and Istituto Ricovero e Cura a Carrattere Scientifico Policlinico San Matteo, 27100 Pavia, Italy.
Ricci
V
V
Fiocca
R
R
Necchi
V
V
Romano
M
M
Telford
J L
JL
Solcia
E
E
Ventura
U
U
D000230:Adenocarcinoma; D000643:Ammonium Chloride / Q000494:pharmacology; D001426:Bacterial Proteins / Q000032:analysis / Q000493:pharmacokinetics*; D005753:Gastric Mucosa / Q000502:physiology / Q000648:ultrastructure*; D016480:Helicobacter pylori; D006801:Humans; D016253:Microscopy, Immunoelectron; D008871:Microvilli / Q000187:drug effects / Q000648:ultrastructure; D013274:Stomach Neoplasms; D014407:Tumor Cells, Cultured; D014617:Vacuoles / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure
D001426:Bacterial Proteins; C074689:VacA protein, Helicobacter pylori; D000643:Ammonium Chloride