Abstract
Intoxication of mammalian cells with the vacuolating toxin (VacA) released by Helicobacter pylori causes the formation of large acidic vacuoles containing the vacuolar ATPase proton pump and Rab7, a late endosome marker. Here, we describe a novel subcellular fractionation procedure, and we show that nanomolar concentrations of VacA induce a clear redistribution of lysosomal membrane glycoproteins among endocytic compartments. This redistribution is an early event in the process of cellular intoxication by VacA and precedes the formation of macroscopic vacuoles. The absence of the cation independent mannose 6-P receptor and the presence of Rab7 and of lysosomal membrane proteins in the newly formed compartment suggest that the vacuolating toxin induces the accumulation of a post-endosomal hybrid compartment presenting both late endosomal and lysosomal features.
Highlights
Helicobacter pylori, a Gram-negative, spiral-shaped bacterium colonizing the stomach, is involved in the pathogenesis of gastritis and gastroduodenal ulcers [1, 2]
Characterization of Vacuoles Originated by vacuolating toxin (VacA) Treatment in Baby hamster kidney (BHK) Cells by Flotation Sucrose Step Gradient—Vacuoles that form in HeLa cells exposed to VacA have components such as Rab7 and the vacuolar Hϩ-ATPase, characteristic of the late endosomal (LE) compartment [14, 17, 28]
VacA treatment of the cells does not lead to significant changes in the distribution of Rab5, transferrin receptor, Rab7, and CI-M6PR as determined by endocytic compartments fractionation with this technique, these preliminary results represent the first evidence that the toxin produced by H. pylori induces changes in the protein content of the LE compartments
Summary
VacA, vacuolating toxin; BHK, baby hamster kidney; CI-M6PR, cation independent mannose 6-P receptor; LE, late endosome(s); Lgp, lysosomal membrane glycoprotein(s); PNS, post-nuclear supernatant; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis. Together with parallel immunofluorescence staining, this procedure allowed us to obtain evidence that vacuoles are enriched in lysosomal membrane markers such as Lgp110, contain low levels of lysosomal hydrolytic activities, are characterized by the presence of the late endocytic marker Rab, but are devoid of another late endosomal marker, the CI-M6PR. These results indicate that VacA induces the formation and pathological accumulation of a mixed endo-lysosomal compartment
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