Abstract
Gastric infection by Helicobacter pylori is considered a risk factor for gastric and duodenal cancer, and extragastric diseases. Previous data have shown that, in a non-enzymatic way, H. pylori urease (HPU) activates neutrophils to produce ROS and also induces platelet aggregation, requiring ADP secretion modulated by the 12-lipoxygenase pathway, a signaling cascade also triggered by the physiological agonist collagen. Here we investigated further the effects on platelets of recombinant versions of the holoenzyme HPU, and of its two subunits (HpUreA and HpUreB). Although HpUreA had no aggregating activity on platelets, it partially inhibited collagen-induced aggregation. HpUreB induced platelet aggregation in the nanomolar range, and also interfered dose-dependently on both collagen- and ADP-induced platelet aggregation. HPU-induced platelet aggregation was inhibited by antibodies against glycoprotein VI (GPVI), the main collagen receptor in platelets. Flow cytometry analysis revealed exposure of P-selectin in HPU-activated platelets. Anti-glycoprotein IIbIIIa (GPIIbIIIa) antibodies increased the binding of FITC-labeled HPU to activated platelets, whereas anti-GPVI did not. Evaluation of post-transcriptional events in HPU-activated platelets revealed modifications in the pre-mRNA processing of pro-inflammatory proteins, with increased levels of mRNAs encoding IL-1β and CD14. We concluded that HPU activates platelets probably through its HpUreB subunit. Activation of platelets by HPU turns these cells into a pro-inflammatory phenotype. Altogether, our data suggest that H. pylori urease, besides allowing bacterial survival within the gastric mucosa, may have an important, and so far overlooked, role in gastric inflammation mediated by urease-activated neutrophils and platelets.
Highlights
Diseases caused by Helicobacter pylori have a great impact on public health, since this bacterium colonizes the gastric mucosa of half of the world’s population, with a higher prevalence in the poorer countries (Parkin, 2004)
We have demonstrated that a recombinant Helicobacter pylori urease (HPU) activated platelets through a lipoxygenase-mediated pathway, leading to exocytosis of dense granules and release of adenosine diphosphate (ADP), which promoted platelet aggregation (Wassermann et al, 2010)
To investigate whether the isolated subunits could interfere in the aggregation triggered by the physiological agonists collagen or ADP, the platelets were previously incubated without stirring with HpUreA or HpUreB, and challenged with the agonists (Figure 2)
Summary
Diseases caused by Helicobacter pylori have a great impact on public health, since this bacterium colonizes the gastric mucosa of half of the world’s population, with a higher prevalence in the poorer countries (Parkin, 2004). Urease produced by H. pylori enables bacterial colonization of the gastric mucosa by catalyzing the hydrolysis of urea into carbon dioxide and ammonia, thereby causing a local pH increase and alterations of the mucus properties that favor the pathogen’s survival (Perrais et al, 2014). A monopartite nuclear localization signal is present in HpUreA (sequence 21KKRKEK26), and the protein is able to target the nuclei of COS-7 (Lee et al, 2012) and of AGS gastric epithelial cells, causing alterations of the cellular morphology (Lee et al, 2015). Incubation of AGS gastric epithelial cells with H. pylori OMVs promoted the translocation of HpUreA into the cell cytoplasm and nuclear localization of the protein (Olofsson et al, 2010)
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