The Helicobacter pylori VacA toxin is a urea permease that promotes urea diffusion across epithelia
Francesco Tombola, … , Mario Zoratti, Emanuele Papini
Francesco Tombola, … , Mario Zoratti, Emanuele Papini
Published September 15, 2001
Citation Information: J Clin Invest. 2001;108(6):929-937. https://doi.org/10.1172/JCI13045.
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Article

The Helicobacter pylori VacA toxin is a urea permease that promotes urea diffusion across epithelia

Abstract

Urease and the cytotoxin VacA are two major virulence factors of the human pathogen Helicobacter pylori, which is responsible for severe gastroduodenal diseases. Diffusion of urea, the substrate of urease, into the stomach is critically required for the survival of infecting H. pylori. We now show that VacA increases the transepithelial flux of urea across model epithelia by inducing an unsaturable permeation pathway. This transcellular pathway is selective, as it conducts thiourea, but not glycerol and mannitol, demonstrating that it is not due to a loosening of intercellular junctions. Experiments performed with different cell lines, grown in a nonpolarized state, confirm that VacA permeabilizes the cell plasma membrane to urea. Inhibition studies indicate that transmembrane pores formed by VacA act as passive urea transporters. Thus, their inhibition by the anion channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid significantly decreases toxin-induced urea fluxes in both polarized and nonpolarized cells. Moreover, phloretin, a well-known inhibitor of eukaryotic urea transporters, blocks VacA-mediated urea and ion transport and the toxin’s main biologic effects. These data show that VacA behaves as a low-pH activated, passive urea transporter potentially capable of permeabilizing the gastric epithelium to urea. This opens the novel possibility that in vivo VacA may favor H. pylori infectivity by optimizing urease activity.

Authors

Francesco Tombola, Laura Morbiato, Giuseppe Del Giudice, Rino Rappuoli, Mario Zoratti, Emanuele Papini

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Figure 3

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Effect of VacA on the plasma membrane permeability to urea in different ...
Effect of VacA on the plasma membrane permeability to urea in different nonpolarized cell types. (a and b) In the representative experiments shown (n = 10), AGS cells were grown on plastic for 2 days and treated (filled circles) or not (filled squares) for 3 hours with activated VacA (125 nM) at 37°C in DMEM, 10% FCS, washed with PBS-BSA at 4°C and further incubated in the same medium. Influx of [14C]urea into cells was determined at the desired time intervals, after cell solubilization with 0.5% SDS (a). Alternatively, cells were equilibrated with [14C]urea in DMEM, 10% FCS for 1 hour at 37°C, and cell-associated radioactivity was determined as above after washing with PBS-BSA at 4°C and further incubation (b). (c and d) The rate of cell urea influx or efflux (expressed as percent of maximal content after the first 5 minutes of assay) was determined in control (filled bars) or VacA-intoxicated (open bars) nondifferentiated cell lines, as specified. Values are the mean from at least three experiments run in duplicate ± SE.