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Helicobacter pylori hydrogenase accessory protein HypA and urease accessory protein UreG compete with each other for UreE recognition.

ABSTRACT: BACKGROUND:The gastric pathogen Helicobacter pylori relies on nickel-containing urease and hydrogenase enzymes in order to colonize the host. Incorporation of Ni(2+) into urease is essential for the function of the enzyme and requires the action of several accessory proteins, including the hydrogenase accessory proteins HypA and HypB and the urease accessory proteins UreE, UreF, UreG and UreH. METHODS:Optical biosensing methods (biolayer interferometry and plasmon surface resonance) were used to screen for interactions between HypA, HypB, UreE and UreG. RESULTS:Using both methods, affinity constants were found to be 5nM and 13nM for HypA-UreE and 8?M and 14?M for UreG-UreE. Neither Zn(2+) nor Ni(2+) had an effect on the kinetics or stability of the HypA-UreE complex. By contrast, addition of Zn(2+), but not Ni(2+), altered the kinetics and greatly increased the stability of the UreE-UreG complex, likely due in part to Zn(2+)-mediated oligomerization of UreE. Finally our results unambiguously show that HypA, UreE and UreG cannot form a heterotrimeric protein complex in vitro; instead, HypA and UreG compete with each other for UreE recognition. GENERAL SIGNIFICANCE:Factors influencing the pathogen's nickel budget are important to understand pathogenesis and for future drug design.

SUBMITTER: Benoit SL

PROVIDER: S-EPMC4017372 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Helicobacter pylori hydrogenase accessory protein HypA and urease accessory protein UreG compete with each other for UreE recognition.

Benoit Stéphane L SL McMurry Jonathan L JL Hill Stephanie A SA Maier Robert J RJ

Biochimica et biophysica acta 20120612 10

<h4>Background</h4>The gastric pathogen Helicobacter pylori relies on nickel-containing urease and hydrogenase enzymes in order to colonize the host. Incorporation of Ni(2+) into urease is essential for the function of the enzyme and requires the action of several accessory proteins, including the hydrogenase accessory proteins HypA and HypB and the urease accessory proteins UreE, UreF, UreG and UreH.<h4>Methods</h4>Optical biosensing methods (biolayer interferometry and plasmon surface resonanc ...[more]

PMID: 22698670

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Project description:BACKGROUND: Helicobacter pylori is a human pathogen and during the process of infection, antigens from the bacterium elicit strong host humoral immune responses. In our previous report, native H. pylori UreG protein showed good reactivity with sera from H. pylori patients. This study was aimed at producing the recombinant form of the protein (rUreG) and determining its seroreactivities. METHODS: The coding sequence of H. pylori UreG was cloned and the recombinant protein expressed and purified by affinity chromatography using nickel nitrilotriacetic acid (Ni-NTA) resin. The antigenicity of rUreG to detect H. pylori specific antibodies was determined by western blot, using HRP-conjugated anti-human IgG and IgA antibodies as probes. A total of 70 sera, comprising 30 positive and 40 control serum samples, were used. The positive sera were from culture-positive H. pylori-infected patients with duodenal ulcers, gastric ulcers, or gastritis. The control sera comprised three types of samples without detectable H. pylori antibodies, i.e. healthy individuals (with no history of gastric disorders) (n = 10); patients who attended an endoscopy clinic (because of gastrointestinal complaints) but were H. pylori culture negative (n = 20); and people with other diseases (n = 10). Additionally, hyperimmune mice serum against rUreG was raised and tested with the native and recombinant UreG protein. RESULTS: The ureG gene fragment was successfully cloned and expressed in both soluble and insoluble forms. Western blots on rUreG protein showed 70% (21/30) and 60% (18/30) reactivity with patients' sera when probed with HRP-conjugated anti-human IgG and IgA antibodies, respectively; and the combination of the IgG and IgA western blots showed reactivity of 83.3% (25/30). By comparison, 97.5% and 92.5% of the control sera showed no reactivity when probed with HRP-conjugated anti-human IgG and IgA antibodies, respectively. Both the H. pylori lysate antigen and rUreG protein displayed a distinctive band at the expected molecular weight when probed with the hyperimmune mice serum. CONCLUSION: The rUreG protein was successfully cloned and expressed and showed good reactivity with H. pylori culture-positive patients' sera and no reactivity with most control sera. Thus, the diagnostic potential of this recombinant protein merits further investigation.

Dataset Information

Helicobacter pylori hydrogenase accessory protein HypA and urease accessory protein UreG compete with each other for UreE recognition.

Publications

Helicobacter pylori hydrogenase accessory protein HypA and urease accessory protein UreG compete with each other for UreE recognition.

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