Article

Persistence of Helicobacter pylori VacA toxin and vacuolating potential in cultured gastric epithelial cells.

Institute of Human Physiology, University of Pavia and Istituto Ricovero e Cura a Carrattere Scientifico Policlinico San Matteo, 27100 Pavia, Italy.
The American journal of physiology 11/1998; 275(4 Pt 1):G681-8. pp.G681-8
Source: PubMed

ABSTRACT 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.

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    Article: Fluorescence resonance energy transfer microscopy of the Helicobacter pylori vacuolating cytotoxin within mammalian cells.
    David C Willhite, Dan Ye, Steven R Blanke
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    ABSTRACT: The Helicobacter pylori vacuolating cytotoxin (VacA) binds and enters mammalian cells to induce cellular vacuolation. To investigate the quaternary structure of VacA within the intracellular environment where toxin cytotoxicity is elaborated, we employed fluorescence resonance energy transfer (FRET) microscopy. HeLa cells coexpressing full-length and truncated forms of VacA fused to cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP) were analyzed for FRET to indicate direct associations. These studies revealed that VacA-CFP and VacA-YFP interact within vacuolated cells, supporting the belief that monomer associations at an intracellular site are important for the toxin's vacuolating activity. In addition, the two fragments of proteolytically nicked VacA, p37 and p58, interact when coexpressed within mammalian cells. Because p37 and p58 function in trans when expressed separately within mammalian cells, these data suggest that the mechanism by which these two fragments induce vacuolation requires direct association. FRET microscopy also demonstrated interactions between mutant forms of VacA, as well as wild-type VacA with mutant forms of the toxin within vacuolated cells. Finally, a dominant-negative form of the toxin directly associates with wild-type VacA in cells where vacuolation was not detectable, suggesting that the formation of complexes comprising wild-type and dominant-negative forms of toxin acts to block intracellular toxin function.
    Infection and Immunity 08/2002; 70(7):3824-32. · 4.16 Impact Factor
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.

Keywords

apparent molecular integrity
 
bacterial outer membrane
 
broth culture filtrate
 
endosomal-lysosomal compartment
 
fragments smaller
 
Helicobacter pylori-induced damage
 
human gastric epithelial cells
 
human gastric epithelium
 
internalized toxin
 
investigative techniques
 
large amount
 
main lysosomal degradative processes
 
membrane-bound form
 
NH4Cl
 
VacA
 
VacA 1
 
VacA avoids
 
VacA-producing H. pylori strain
 
vacuolating power
 
vacuolating toxin