The bacterium Helicobacter pylori causes peptic ulcers and gastric cancer in human beings by mechanisms yet not fully understood. H. pylori produces urease which neutralizes the acidic medium permitting its survival in the stomach. We have previously shown that ureases from jackbean, soybean or Bacillus pasteurii induce blood platelet aggregation independently of their enzyme activity by a pathway requiring platelet secretion, activation of calcium channels and lipoxygenase-derived eicosanoids. We investigated whether H. pylori urease displays platelet-activating properties and defined biochemical pathways involved in this phenomenon. For that the effects of purified recombinant H. pylori urease (HPU) added to rabbit platelets were assessed turbidimetrically. ATP secretion and production of lipoxygenase metabolites by activated platelets were measured. Fluorescein-labelled HPU bound to platelets but not to erythrocytes. HPU induced aggregation of rabbit platelets (ED50 0.28 μM) accompanied by ATP secretion. No correlation was found between platelet activation and ureolytic activity of HPU. Platelet aggregation was blocked by esculetin (12-lipoxygenase inhibitor) and enhanced ∼3-fold by indomethacin (cyclooxygenase inhibitor). A metabolite of 12-lipoxygenase was produced by platelets exposed to HPU. Platelet responses to HPU did not involve platelet-activating factor, but required activation of verapamil-inhibitable calcium channels. Our data show that purified H. pylori urease activates blood platelets at submicromolar concentrations. This property seems to be common to ureases regardless of their source (plant or bacteria) or quaternary structure (single, di- or tri-chain proteins). These properties of HPU could play an important role in pathogenesis of gastrointestinal and associated cardiovascular diseases caused by H. pylori.

The gastric pathogen Helicobacter pylori produces large amounts of urease, whose enzyme activity enables the bacterium to survive in the stomach. We have previously shown that ureases display enzyme-independent effects in mammalian models, most through lipoxygenases-mediated pathways. Here, we evaluated potential pro-inflammatory properties of H. pylori urease (HPU). Mouse paw edema and activation of human neutrophils were tested using a purified, cell-free, recombinant HPU. rHPU induced paw edema with intense neutrophil infiltration. In vitro 100 nM rHPU was chemotactic to human neutrophils, inducing production of reactive oxygen species. rHPU-activated neutrophils showed increased lifespan, with inhibition of apoptosis accompanied by alterations of Bcl-XL and Bad contents. These effects of rHPU persisted in the absence of enzyme activity. rHPU-induced paw edema, neutrophil chemotaxis and apoptosis inhibition reverted in the presence of the lipoxygenase inhibitors esculetin or AA861. Neutrophils exposed to rHPU showed increased content of lipoxygenase(s) and no alteration of cyclooxygenase(s). Altogether, our data indicate that HPU, besides allowing the bacterial survival in the stomach, could play an important role in the pathogenesis of the gastrointestinal inflammatory disease caused by H. pylori.

Helicobacter pylori is a pathogen involved in gastric diseases such as ulcers and carcinomas. H. pylori’s urease is an important virulence factor produced in large amounts by this bacterium. In previous studies, we have shown that this protein is able to activate several cell types like neutrophils, monocytes, platelets, endothelial cells, and gastric epithelial cells. Angiogenesis is a physiological process implicated in growth, invasion and metastization of tumors. Here, we have analyzed the angiogenic potential of H. pylori urease (HPU) in gastric epithelial cells. No cytotoxicity was observed in AGS, Kato-III, and MKN28 gastric cell lines treated with 300 nM HPU, as evaluated by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. As we previously reported in neutrophils, treatment with 300 nM HPU also had an anti-apoptotic effect in gastric epithelial cells leading to a 2.2-fold increase in the levels of Bcl-XL after 6 h, and a decrease of 80% in the content of BAD, after 48 h, two mitochondrial proteins involved in regulation of apoptosis. Within 10 min of exposure, HPU is rapidly internalized by gastric epithelial cells. Treatment of the gastric cells with methyl-β-cyclodextrin abolished HPU internalization suggesting a cholesterol-dependent process. HPU induces the expression of pro-angiogenic factors and the decrease of expression of anti-angiogenic factors by AGS cells. The angiogenic activity of HPU was analyzed using in vitro and in vivo models. HPU induced formation of tube-like structures by human umbilical vascular endothelial cells in a 9 h experiment. In the chicken embryo chorioallantoic membrane model, HPU induced intense neo-vascularization after 3 days. In conclusion, our results indicate that besides allowing bacterial colonization of the gastric mucosa, H. pylori’s urease triggers processes that initiate pro-angiogenic responses in different cellular models. Thus, this bacterial urease, a major virulence factor, may also play a role in gastric carcinoma development.

In this work we compared two plant ureases, jackbean urease (JBU) and embryo-specific soybean urease (SBU) and a bacterial (Bacillus pasteurii) urease, for kinetic parameters and other biological properties described recently for ureases that are independent of the ureolytic activity. The insecticidal effect of ureases was investigated in feeding trials with the cotton sucker bug, Dysdercus peruvianus (Hemiptera) as an insect model. Contrasting with B. pasteurii urease (PBU), both plant ureases presented potent insecticidal activity, with LD 50 values of 0.017% (w/w) and 0.052% (w/w) for JBU and SBU, respectively. The insecticidal property of JBU or SBU was not affected by treatment with p-hydroxymercuribenzoate, an irreversible inhibitor of ureolytic activity of both proteins. Also, contrasting with canatoxin -a urease isoform from jackbean seeds that displays a toxic effect in mice (LD 50 ¼ 2 mgAEkg ). Similarly to canatoxin, the three enzymes promoted aggregation of blood platelets (EC 50 ¼ 400.0 lgAEmL , 15.8 lgAEmL )1 for BPU, SBU and JBU, respectively). This platelet activating property was also independent of urease activity. Comparison of the kinetic properties indicated that SBU is fivefold less susceptible than JBU to inhibition by acetohydroxamic acid, a chelator of Ni +2 and Zn +2 ions. The ureases also showed different susceptibility to agents that modify cysteine residues, such as p-hydroxymercuribenzoate and p-benzoquinone. Altogether, these data emphasize that biological properties that are independent of ureolytic activity are not restricted to jackbean ureases and that these proteins may have a role in plant defense against insect predators.

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.

Ureases are enzymes from plants, fungi and bacteria that catalyze the hydrolysis of urea to form ammonia and carbon dioxide. While fungal and plant ureases are homo-oligomers of 90-kDa subunits, bacterial ureases are multimers of two or three subunit complexes. We showed that some isoforms of jack bean urease, canatoxin and the classical urease, bind to glycoconjugates and induce platelet aggregation. Canatoxin also promotes release of histamine from mast cells, insulin from pancreatic cells and neurotransmitters from brain synaptosomes. In vivo it induces rat paw edema and neutrophil chemotaxis. These effects are independent of ureolytic activity and require activation of eicosanoid metabolism and calcium channels. Helicobacter pylori, a Gram-negative bacterium that colonizes the human stomach mucosa, causes gastric ulcers and cancer by a mechanism that is not understood. H. pylori produces factors that damage gastric epithelial cells, such as the vacuolating cytotoxin VacA, the cytotoxin-associated protein CagA, and a urease (up to 10% of bacterial protein) that neutralizes the acidic medium permitting its survival in the stomach. H. pylori whole cells or extracts of its water-soluble proteins promote inflammation, activate neutrophils and induce the release of cytokines. In this paper we review data from the literature suggesting that H. pylori urease displays many of the biological activities observed for jack bean ureases and show that bacterial ureases have a secretagogue effect modulated by eicosanoid metabolites through lipoxygenase pathways. These findings could be relevant to the elucidation of the role of urease in the pathogenesis of the gastrointestinal disease caused by H. pylori.

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Ureases (EC 3.5.1.5) are metalloenzymes that hydrolyze urea to produce ammonia and carbon dioxide These enzymes, which are found in fungi, bacteria, and plants, show very similar structures. Despite an abundance of urease in vegetal tissues, the physiological role of this enzyme in plants is still poorly understood. It has been previously described that ureases from the legumes jackbean ( Canavalia ensiformis) and soybean ( Glycine max) have insecticidal activity and antifungal properties. This work presents the physicochemical purification and characterization of a urease from cotton ( Gossypium hirsutum) seeds, the first description of this enzyme in Malvaceae. The urease content varied among different cotton cultivars. Cotton seed urease (98.3 kDa) displayed low ureolytic activity but exhibited potent antifungal properties at sub-micromolar concentrations against different phytopathogenic fungi. As described for other ureases, the antifungal effect of cotton urease persisted after treatment with an irreversible inhibitor of its enzyme activity. The data suggest an important role of these proteins in plant defense.