Nickel-binding and accessory proteins facilitating Ni-enzyme maturation in Helicobacter pylori

Helicobacter pylori colonizes the human gastric mucosa and this can lead to chronic gastritis, peptic and duodenal ulcers, and even gastric cancers. The bacterium colonizes over one-half of the worlds population. Nickel plays a major role in the bacteriums colonization and persistence attributes as two nickel enzyme sinks obligately contain the metal. Urease accounts for up to 10% of the total cellular protein made and is required for initial colonization processes, and the hydrogen oxidizing hydrogenase provides the bacterium a high-energy substrate yielding low potential electrons for energy generation. A battery of accessory proteins are needed for maturation or activation of each of the apoenzymes. These include Ni-chaperones and GTPases, some of which are unique to each Ni-enzyme and others that are individually required for maturation of both the Ni-enzymes. H. pylori’s need for some conventional hydrogenase maturation proteins playing roles in urease maturation may have to do with the poor nickel-sequestering ability of the UreE urease maturation protein compared to other systems. H. pylori also possesses a NixA nickel specific permease, a nickel dependent regulator (NikR), a recently identified nickel efflux system (CznABC), and a histidine-rich heat shock protein, HspA. Based on mutant analysis approaches all these proteins have roles in nickel homeostasis, in urease expression, and in host colonization. The His-rich putative nickel storage proteins Hpn and Hpn-like play roles in nickel detoxification and may influence the levels of Ni-activated urease that can be achieved.     

幽门螺杆菌在胃内定植的相关影响因素

胃内定植是引起幽门螺杆菌(Helicobacter pylori,H.pylori)感染的先决条件。H.pylori可穿过胃黏液层并与胃上皮细胞相互作用。这个定植过程主要受到H.pylori动力和尿素酶的影响。同时H.pylori形态、胃内pH、外膜蛋白及益生菌等也在其中扮演重要角色。该研究主要对H.pylori胃内定植过程中的相关影响因素进行综述。     

Novel nickel transport mechanism across the bacterial outer membrane energized by the TonB/ExbB/ExbD machinery

Nickel is a cofactor for various microbial enzymes, yet as a trace element, its scavenging is challenging. In the case of the pathogen Helicobacter pylori, nickel is essential for the survival in the human stomach, because it is the cofactor of the important virulence factor urease. While nickel transport across the cytoplasmic membrane is accomplished by the nickel permease NixA, the mechanism by which nickel traverses the outer membrane (OM) of this Gram-negative bacterium is unknown. Import of iron-siderophores and cobalamin through the bacterial OM is carried out by specific receptors energized by the TonB/ExbB/ExbD machinery. In this study, we show for the first time that H. pylori utilizes TonB/ExbB/ExbD for nickel uptake in addition to iron acquisition. We have identified the nickel-regulated protein FrpB4, homologous to TonB-dependent proteins, as an OM receptor involved in nickel uptake. We demonstrate that ExbB/ExbD/TonB and FrpB4 deficient bacteria are unable to efficiently scavenge nickel at low pH. This condition mimics those encountered by H. pylori during stomach colonization, under which nickel supply and full urease activity are essential to combat acidity. We anticipate that this nickel scavenging system is not restricted to H. pylori, but will be represented more largely among Gram-negative bacteria.     

In vivo interactome of Helicobacter pylori urease revealed by tandem affinity purification

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. 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 Ni(2+) 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 Ni(2+) 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.     

幽门螺杆菌表面抗原免疫保护作用的体外与活体研究

目的：调查幽门螺杆菌（Hp)几种表面蛋白体外对T细胞增殖的影响和在小鼠体内的免疫保护作用。方法：评价Hp全菌抗原、尿原酶（Urease)、黏附素（hpaA)、外膜蛋白25（Hop25)和38（Hop38)对人外周血T细胞及小鼠CD4^ T细胞增殖的影响;与佐剂合用,评价上述重组蛋白对小鼠Hp感染的免疫预防作用。结果：Urease和Hop25可刺激人及鼠T细胞增殖,hapA只能刺激Hp^ PBL增殖,而Hop38则有毒性作用;Hop25和Hop38均可产生60％的完全保护,hpaA可产生100％的部分保护即降低细菌定植密度,而Urease只能产生40％的部分保护。结论：外膜蛋白可能是一组高效的Hp疫苗免疫原;其长期免疫效果及对T细胞功能的活体调节作用尚需进一步评价。国际上尚未见相关报道。     

Activities of Urease and Nickel Uptake of Helicobacter pylori Proteins are Media- and Host-Dependent

Background:  Nickel-dependent urease activity and nickel supply are essential for successful colonization of Helicobacter pylori in the acidic environment of the human stomach. A comparison of media effects on these two activities have never been carried out. Additionally to H. pylori we cultivated an Escherichia coli strain expressing the urease and the nickel transporter NixA of H. pylori on the same four media and measured in all cases urease and nickel uptake activity.
Aim:  To compare nickel uptake and urease activity on an inter- and intraspecies level.
Results:  In H. pylori nickel uptake (four to 200 times) and urease activities (400 to 30,000 times) were found to be much higher in comparison to the tested E. coli strain after growth on all media. These differences could not be explained by reduced protein amounts in the heterologous host E. coli . On which media the two bacteria extracted most of the nickel were organism-dependent: E. coli on Brucella Broth, H. pylori on Trypticase Soy Broth, and Minimal Media.
Conclusion:  H. pylori took nickel much more efficiently up than E. coli . The observed differences in urease activity are most likely due to additional protein components absent in the recombinant E. coli strain. The observed variety in nickel uptake and urease activities on the different media in the same organism depended on the intrinsic nickel content and chelating capacities of media components. Different culture conditions may lead to varying results; generalizations should be concluded only after excluding their media dependence.     

Helicobacter pylori AddAB helicase-nuclease and RecA promote recombination-related DNA repair and survival during stomach colonization

Helicobacter pylori colonization of the human stomach is characterized by profound disease-causing inflammation. Bacterial proteins that detoxify reactive oxygen species or recognize damaged DNA adducts promote infection, suggesting that H. pylori requires DNA damage repair for successful in vivo colonization. The molecular mechanisms of repair remain unknown. We identified homologues of the AddAB class of helicase-nuclease enzymes, related to the Escherichia coli RecBCD enzyme, which, with RecA, is required for repair of DNA breaks and homologous recombination. H. pylori mutants lacking addA or addB genes lack detectable ATP-dependent nuclease activity, and the cloned H. pylori addAB genes restore both nuclease and helicase activities to an E. coli recBCD deletion mutant. H. pylori addAB and recA mutants have a reduced capacity for stomach colonization. These mutants are sensitive to DNA damaging agents and have reduced frequencies of apparent gene conversion between homologous genes encoding outer membrane proteins. Our results reveal requirements for double-strand break repair and recombination during both acute and chronic phases of H. pylori stomach infection.     

Therapeutic effects of molecularly designed antigen UREB138 for mice infected with Helicobacter pylori

Helicobacter pylori (H. pylori) is a bacteria that is well known as the principal cause of chronic gastritis and peptic ulcer disease in humans. Because no effective vaccine has yet been established, we designed a new biomolecule as a vaccination antigen capable of preventing the infection of H. pylori. The designed biomolecule involves a 138 stretch (aa 201-aa 338 of beta-subunit of H. pylori urease), which is the functionally important region for urease activity. The region was expressed as a recombinant protein, called UREB138. The therapeutic vaccination was performed using UREB138 in mice persistently infected with H. pylori. The subcutaneous administration of UREB138 remarkably reduced the number of bacteria in the mice stomach compared with the control. Immunization with UREB138 enhanced the urease-specific IgA and IgG1 in the serum. Immunohistochemical staining for IgA in gastric mucosa showed that the number of mice positively stained with IgA was significantly higher in UREB138-immunized mice than in control mice. Furthermore, the expression of interferon-gamma mRNA in the gastric tissues with eradicated bacteria was higher than in the non-eradicated group. The combination of Th1- and Th2-mediated immunity plays a role in reducing the colonization of bacterial numbers of H. pylori.     

Recombinant antigen from Helicobacter pylori urease as vaccine against H. pylori-associated disease

It is well documented that the enzymatic active site of Helicobacter pylori urease is present in the beta-subunit. An important sequence of 135 amino acids of the beta-subunit was determined from the structure of H. pylori urease and by a homology-based study of the urease of other bacteria and plants. The sequence (UreB) was expressed in Escherichia coli as a recombinant fusion protein with glutathione-S-transferase (GST). Seventeen monoclonal antibodies, UA-1-17, were produced using the UreB-GST as the immunogen. The obtained monoclonal antibodies showed a high specificity to UreB, and some of the MAbs cross-reacted with Jack bean urease. About 70% of the established MAbs displayed an inhibitory effect on the enzymatic activity of the urease. Among them, UA-15 MAb could reduce the activity by 53% and it immunologically binds to the bacterium infecting the human stomach mucosa. The antiserum induced by immunization with a recombinant UreB-GST into rabbits displayed a specific binding to mucosal surfaces of the human stomach infected with the pathogen H. pylori. Moreover, the antiserum suppressed the enzymatic activity of H. pylori urease, while the purified H. pylori urease could not induce such an antiserum.