<i>Helicobacter pylori</i> urease activates blood platelets through a lipoxygenase‐mediated pathway

Wassermann, German Enrique; Olivera‐Severo, Deiber; Uberti, Augusto F.; Carlini, Célia R.

doi:10.1111/j.1582-4934.2009.00901.x

Cited by 41 publications

(60 citation statements)

References 55 publications

(100 reference statements)

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“…As a consequence of urease-induced ADP secretion from dense granules, platelets from different species, including humans, undergo aggregation upon exposure to nanomolar concentrations of plant (jack bean and soybean) or bacterial (Bacillus pasteurii and H. pylori) [12] ureases. The platelet-activating properties of these ureases were shown to be independent of urea hydrolysis [12][13][14]. Ureases also show potent proinflammatory activity.…”

Section: Introductionmentioning

confidence: 99%

Cryptococcus gattii urease as a virulence factor and the relevance of enzymatic activity in cryptococcosis pathogenesis

et al. 2015

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Ureases (EC 3.5.1.5) are Ni(2+) -dependent metalloenzymes produced by plants, fungi and bacteria that hydrolyze urea to produce ammonia and CO2 . The insertion of nickel atoms into the apo-urease is better characterized in bacteria, and requires at least three accessory proteins: UreD, UreF, and UreG. Our group has demonstrated that ureases possess ureolytic activity-independent biological properties that could contribute to the pathogenicity of urease-producing microorganisms. The presence of urease in pathogenic bacteria strongly correlates with pathogenesis in some human diseases. Some medically important fungi also produce urease, including Cryptococcus neoformans and Cryptococcus gattii. C. gattii is an etiological agent of cryptococcosis, most often affecting immunocompetent individuals. The cryptococcal urease might play an important role in pathogenesis. It has been proposed that ammonia produced via urease action might damage the host endothelium, which would enable yeast transmigration towards the central nervous system. To analyze the role of urease as a virulence factor in C. gattii, we constructed knockout mutants for the structural urease-coding gene URE1 and for genes that code the accessory proteins Ure4 and Ure6. All knockout mutants showed reduced multiplication within macrophages. In intranasally infected mice, the ure1Δ (lacking urease protein) and ure4Δ (enzymatically inactive apo-urease) mutants caused reduced blood burdens and a delayed time of death, whereas the ure6Δ (enzymatically inactive apo-urease) mutant showed time and dose dependency with regard to fungal burden. Our results suggest that C. gattii urease plays an important role in virulence, in part possibly through enzyme activity-independent mechanism(s).

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Section: Introductionmentioning

confidence: 99%

Cryptococcus gattii urease as a virulence factor and the relevance of enzymatic activity in cryptococcosis pathogenesis

et al. 2015

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“…To address this, single lipid bilayer nanotubes (LNT) rich in phosphatidylserine (PS), capable of binding FVIII with high affinity 8,9 and resembling the activated platelet surface have been developed 10 . Consecutive helical organization of FVIII bound to LNT has been proven to be effective for structure determination of FVIII membrane-bound state by Cryo-EM 5 .…”

Section: Introductionmentioning

confidence: 99%

Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes

Miller

Dalm

Koyfman

et al. 2014

JoVE

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Cryo-electron microscopy (Cryo-EM)1 is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and membrane environment2. Coagulation Factor VIII (FVIII)3 is a multi-domain blood plasma glycoprotein. Defect or deficiency of FVIII is the cause for Hemophilia type A - a severe bleeding disorder. Upon proteolytic activation, FVIII binds to the serine protease Factor IXa on the negatively charged platelet membrane, which is critical for normal blood clotting4. Despite the pivotal role FVIII plays in coagulation, structural information for its membrane-bound state is incomplete5. Recombinant FVIII concentrate is the most effective drug against Hemophilia type A and commercially available FVIII can be expressed as human or porcine, both forming functional complexes with human Factor IXa6,7. In this study we present a combination of Cryo-electron microscopy (Cryo-EM), lipid nanotechnology and structure analysis applied to resolve the membrane-bound structure of two highly homologous FVIII forms: human and porcine. The methodology developed in our laboratory to helically organize the two functional recombinant FVIII forms on negatively charged lipid nanotubes (LNT) is described. The representative results demonstrate that our approach is sufficiently sensitive to define the differences in the helical organization between the two highly homologous in sequence (86% sequence identity) proteins. Detailed protocols for the helical organization, Cryo-EM and electron tomography (ET) data acquisition are given. The two-dimensional (2D) and three-dimensional (3D) structure analysis applied to obtain the 3D reconstructions of human and porcine FVIII-LNT is discussed. The presented human and porcine FVIII-LNT structures show the potential of the proposed methodology to calculate the functional, membrane-bound organization of blood coagulation Factor VIII at high resolution.

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“…Nevertheless, recent evidences have highlighted pathophysiologic properties of HPU unrelated to its ureolytic activity. We have previously reported that non‐catalytic HPU has pro‐inflammatory activity in mice and activates host cells, inducing blood platelet aggregation, chemotaxis, and ROS production by human neutrophils . More recently, it was demonstrated that HPU was internalized by epithelial gastric cells, triggering the expression of pro‐angiogenic mediators, and induced angiogenesis in vivo in the chicken chorioallantoic membrane model .…”

Section: Discussionmentioning

confidence: 99%

“…HPU also induced in vitro platelet aggregation, human neutrophil chemotaxis, and production of reactive oxygen species (ROS) . Platelets activated by HPU display a pro‐inflammatory phenotype with modifications in the pre‐mRNA processing of pro‐inflammatory proteins, and increased levels of mRNAs encoding IL‐1β and CD14 . These results suggest that the inflammatory response triggered by HPU may contribute to amplify local inflammation, worsening mucosal damage during bacterial infection.…”

Section: Introductionmentioning

confidence: 94%

Helicobacter pylori urease induces pro‐inflammatory effects and differentiation of human endothelial cells: Cellular and molecular mechanism

et al. 2019

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Background Helicobacter pylori urease (HPU) is a key virulence factor that enables bacteria to colonize and survive in the stomach. We early demonstrated that HPU, independent of its catalytic activity, induced inflammatory and angiogenic responses in vivo and directly activated human neutrophils to produce reactive oxygen species (ROS). We have investigated the effects of HPU on endothelial cells, focusing on the signaling mechanism involved. Methods Monolayers of human microvascular endothelial cells (HMEC‐1) were stimulated with HPU (up to 10 nmol/L): Paracellular permeability was accessed through dextran‐FITC passage. NO and ROS production was evaluated using intracellular probes. Proteins or mRNA expressions were detected by Western blotting and fluorescence microscopy or qPCR assays, respectively. Results Treatment with HPU enhanced paracellular permeability of HMEC‐1, preceded by VE‐cadherin phosphorylation and its dissociation from cell‐cell junctions. This caused profound alterations in actin cytoskeleton dynamics and focal adhesion kinase (FAK) phosphorylation. HPU triggered ROS and nitric oxide (NO) production by endothelial cells. Increased intracellular ROS resulted in nuclear factor kappa B (NF‐κB) activation and upregulated expression of cyclooxygenase‐2 (COX‐2), hemeoxygenase‐1 (HO‐1), interleukin‐1β (IL‐1β), and intercellular adhesion molecule‐1 (ICAM‐1). Higher ICAM‐1 and E‐selectin expression was associated with increased neutrophil adhesion on HPU‐stimulated HMEC monolayers. The effects of HPU on endothelial cells were dependent on ROS production and lipoxygenase pathway activation, being inhibited by esculetin. Additionally, HPU improved vascular endothelial growth factor receptor 2 (VEGFR‐2) expression. Conclusion The data suggest that the pro‐inflammatory properties of HPU drive endothelial cell to a ROS‐dependent program of differentiation that contributes to the progression of H pylori infection.

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Helicobacter pylori urease activates blood platelets through a lipoxygenase‐mediated pathway

Cited by 41 publications

References 55 publications

Cryptococcus gattii urease as a virulence factor and the relevance of enzymatic activity in cryptococcosis pathogenesis

Cryptococcus gattii urease as a virulence factor and the relevance of enzymatic activity in cryptococcosis pathogenesis

Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes

Helicobacter pylori urease induces pro‐inflammatory effects and differentiation of human endothelial cells: Cellular and molecular mechanism

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