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Molecular & Cellular Proteomics 7:2429-2441, 2008.
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

In Vivo Interactome of Helicobacter pylori Urease Revealed by Tandem Affinity Purification^*,S

Kerstin Stingl^,,¶, Kristine Schauer^,||,**, Chantal Ecobichon^,, Agnès Labigne, Pascal Lenormand, Jean-Claude Rousselle, Abdelkader Namane and Hilde de Reuse^,||,¶¶

From the Unité de Pathogénie Bactérienne des Muqueuses, ^|| Unité Postulante de Pathogenèse de Helicobacter, G5 Biologie et Génétique de la Paroi Bacterienne, and Plate-forme de Protéomique, Institut Pasteur, 75015 Paris, France and Institut für Allgemeine Zoologie und Genetik, Westfälische Wilhelms-Universität, 48149 Münster, Germany

In the human gastric bacterium Helicobacter pylori, two metalloenzymes, hydrogenase and urease, are essential for in vivo colonization, the latter being a major virulence factor. The UreA and UreB structural subunits of urease and UreG, one of the accessory proteins for Ni²⁺ incorporation into apourease, were taken as baits for tandem affinity purification. The method allows the purification of protein complexes under native conditions and physiological expression levels of the bait protein. Furthermore the tandem affinity purification technology was combined with in vivo cross-link to capture transient interactions. The results revealed different populations of urease complexes: (i) urease captured during activation by Ni²⁺ ions comprising all the accessory proteins and (ii) urease in association with metabolic proteins involved e.g. in ammonium incorporation and the cytoskeleton. Using UreG as a bait protein, we copurified HypB, the accessory protein for Ni²⁺ incorporation into hydrogenase, that is reported to play a role in urease activation. The interactome of HypB partially overlapped with that of urease and revealed interactions with SlyD, which is known to be involved in hydrogenase maturation as well as with proteins implicated in the formation of [Fe-S] clusters present in the small subunit of hydrogenase. In conclusion, this study provides new insight into coupling of ammonium production and assimilation in the gastric pathogen and the intimate link between urease and hydrogenase maturation.

^¶¶ To whom correspondence may be addressed: Inst. Pasteur, Unité Postulante de Pathogenèse de Helicobacter, 28 rue du Docteur Roux, F-75015 Paris Cedex, France. Tel.: 33-1-40-61-36-41; Fax: 33-1-40-61-36-40; E-mail: hdereuse{at}pasteur.fr

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