In Vivo Interactome of Helicobacter pylori Urease Revealed by Tandem Affinity Purification

Abstract

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 Ni 2؉ 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. Protein-protein interactions are operative at the majority of cellular processes; thus, their study gives valuable insight into proteomic and functional associations. Helicobacter pylori is a Gram-negative bacterium that is infecting the stomach of about half of the human population. It is responsible for the development of gastric pathologies such as gastritis, gastroduodenal ulcer, and adenocarcinoma (1). One remarkable feature of H. pylori is its capacity to persist (often during decades) and multiply in the hostile environment of the stomach. In H. pylori, urease is a major virulence factor and is essential for the resistance to acidity because of its capacity to hydrolyze urea into bicarbonate and ammonia (2), which results in pH homeostasis of the bacterium. The protein is an extremely active and highly regulated member of the nickel metalloenzyme family. Activation of urease, i.e. incorporation of nickel into the active site, requires urease-specific accessory proteins whose homologues have been extensively studied genetically and biochemically in Klebsiella pneumoniae (3)(4)(5)(6)(7)(8). It is still enigmatic why vast amounts of urease, up to 10% of the total protein, are produced, whereas only a minor active portion is sufficient for acid resistance (9). Furthermore the observation that urease is even indispensable for the colonization under neutral conditions (10) suggested additional essential functions for this central enzyme that remained unidentified.H. pylori possesses another nickel-containing enzyme, a [NiFe] hydrogenase, allowing this organism to utilize hydrogen as an energy source. Accordingly hydrogenase has been shown to be essential for H. pylori colonization in the mouse model (11). The assembly of the [NiFe] hydrogenase metal center located in the large hydrogenase subunit is partially understood and requires several proteins involved in the sequential delivery of iron before nickel (for a review, see Ref. 12). In addition, the [NiFe] hydrogenase possesses a small subunit containing [Fe-S] clusters. For the assembly of the latter no distinct biosynthetic pathway has been attributed so far, suggesting that the housekeeping [Fe-S] cluster assembly system might be involved (12). H. pylori is unique in that the maturation events of these two nickel-containing essential proteins are interconnected as H. pylori mutants deficient in one of the accessory proteins, HypA or HypB, involved in nickel insertion into hydrogenase, lack significant activation of urease (13).

A Second-generation Protein–Protein Interaction Network of Helicobacter pylori

“…Stingl et al (67) already observed some unexpected proteins in their complexes, for example, cell division factors (MinD, Tig, FtsA, Fig. 6).…”

“…Stingl and colleagues have isolated eight protein complexes associated with urease components (67). Based on the known PPIs we investigated which binary interactions explain the the complexesЈ topology.…”

Crystal Structures of Apo and Metal-Bound Forms of the UreE Protein from Helicobacter pylori: Role of Multiple Metal Binding Sites,

“…For example, an interaction between H. pylori UreE and HypA, the latter protein being a component of the [NiFe] hydrogenase maturation pathway, has been shown to be necessary for urease maturation (9). Similarly, H. pylori UreG can interact with another hydrogenase maturation protein, HypB (6).…”

“…While most bacterial ureases are composed of three different subunits that form a (UreABC) 3 ions into urease requires a series of maturation proteins that have been best-studied in Klebsiella aerogenes, including UreD (UreH in H. pylori), UreE, UreF, and UreG (2). Interactions between the urease apoprotein subunits and maturation proteins, as well as interactions among the maturation proteins themselves, have been identified by various approaches in K. aerogenes (reviewed in ref 1), Proteus mirabilis (3), and H. pylori (4)(5)(6). Although the specific molecular roles of each of the accessory proteins in metal center maturation and the requirements for protein-protein interactions are not clear, the overall conclusion from studies of K. aerogenes is that UreD, UreF, and UreG form a complex with the urease apoprotein while UreE functions in Ni 2þ delivery.…”

Cell-free Determination of Binary Complexes That Comprise Extended Protein-Protein Interaction Networks of Yersinia pestis

“…The basic building blocks of proteomic interaction networks are binary protein-protein interactions (PPi). Proteome-scale PPi studies were described for E. coli (6 -11), and a few gram-negative pathogens (12)(13)(14)(15), although only limited data for Y. pestis and other highly virulent bacteria currently exists.…”