Regulation of ferritin-mediated cytoplasmic iron storage by the ferric uptake regulator homolog (Fur) of Helicobacter pylori

Homologs of the ferric uptake regulator Fur and the iron storage protein ferritin play a central role in maintaining iron homeostasis in bacteria. The gastric pathogen Helicobacter pylori contains an iron-induced prokaryotic ferritin (Pfr) which has been shown to be involved in protection against metal toxicity and a Fur homolog which has not been functionally characterized in H. pylori. Analysis of an isogenic fur-negative mutant revealed that H. pylori Fur is required for metal-dependent regulation of ferritin. Iron starvation, as well as medium supplementation with nickel, zinc, copper, and manganese at nontoxic concentrations, repressed synthesis of ferritin in the wild-type strain but not in the H. pylori fur mutant. Fur-mediated regulation of ferritin synthesis occurs at the mRNA level. With respect to the regulation of ferritin expression, Fur behaves like a global metal-dependent repressor which is activated under iron-restricted conditions but also responds to different metals. Downregulation of ferritin expression by Fur might secure the availability of free iron in the cytoplasm, especially if iron is scarce or titrated out by other metals.     

Structural and functional analysis of the riboflavin synthesis genes encoding GTP cyclohydrolase II (ribA), DHBP synthase (ribBA), riboflavin synthase (ribC), and riboflavin deaminase/reductase (ribD) from Helicobacter pylori strain P1

The functions of the riboflavin synthesis gene homologues ribA, ribBA, ribC, and ribD from Helicobacter pylori strain P1 were confirmed by complementation of defined Escherichia coli mutant strains. The H. pylori ribBA gene, which is similar to bifunctional ribBA genes of Gram-positive bacteria, fully complemented the ribB mutation and partially restored growth in a ribC mutant. However, ribBA did not complement the ribA mutation in E. coli, thus explaining the presence of the additional separate copy of the ribA gene in the H. pylori chromosome. In E. coli exclusively ribA conferred hemolytic activity and gave rise to production of molecules with fluorescence characteristics similar to flavins, as observed earlier. The E. coli hemolysin ClyA was not involved in causing the hemolytic phenotype. No riboflavin synthesis genes on plasmids conferred iron uptake functions to a siderophore-deficient mutant of E. coli. Marker exchange mutagenesis of the genes in H. pylori was not successful indicating that riboflavin synthesis is essential for basic metabolic functions of the gastric pathogen.     

Contribution of dppA to urease activity in Helicobacter pylori 26695

BACKGROUND: The gastric pathogen Helicobacter pylori produces urease in amounts up to 10% of its cell protein. This enzyme, which catalyzes the hydrolysis of urea to ammonia and carbon dioxide, protects the bacterium from gastric acid. Urease, a nickel metalloenzyme, requires active uptake of nickel ions from the environment to maintain its activity. NixA is a nickel transport protein that resides in the cytoplasmic membrane. Mutation of nixA significantly reduces but does not abolish urease activity, strongly suggesting the presence of a second transporter. We postulated that the dipeptide permease (dpp) genes that are homologous to the nik operon of Escherichia coli could be a second nickel transporter. The predicted Dpp polypeptides DppA, DppC, and DppD of H. pylori share approximately 40%, 53%, and 56% amino acid sequence identity with their respective E. coli homologs. METHODS: A mutation in dppA, constructed by insertional inactivation with a chloramphenicol resistance cassette, was introduced by allelic exchange into H. pylori strain 26695. RESULTS: When compared to the parental strain, urease activity was not decreased in a dppA mutant. CONCLUSIONS: DppA does not contribute to the synthesis of catalytically active urease in H. pylori 26695 and is likely not a nickel importer in H. pylori.     

Crystal structure of Helicobacter pylori neutrophil-activating protein with a di-nuclear ferroxidase center in a zinc or cadmium-bound form

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a Dps-like iron storage protein forming a dodecameric shell, and promotes adhesion of neutrophils to endothelial cells. The crystal structure of HP-NAP in a Zn(2+)- or Cd(2+)-bound form reveals the binding of two zinc or two cadmium ions and their bridged water molecule at the ferroxidase center (FOC). The two zinc ions are coordinated in a tetrahedral manner to the conserved residues among HP-NAP and Dps proteins. The two cadmium ions are coordinated in a trigonal-bipyramidal and distorted octahedral manner. In both structures, the second ion is more weakly coordinated than the first. Another zinc ion is found inside of the negatively-charged threefold-related pore, which is suitable for metal ions to pass through.     

Purification and characterization of Helicobacter pylori arginase, RocF: unique features among the arginase superfamily.

The urea cycle enzyme arginase (EC 3.5.3.1) hydrolyzes l-arginine to l-ornithine and urea. Mammalian arginases require manganese, have a highly alkaline pH optimum and are resistant to reducing agents. The gastric human pathogen, Helicobacter pylori, also has a complete urea cycle and contains the rocF gene encoding arginase (RocF), which is involved in the pathogenesis of H. pylori infection. Its arginase is specifically involved in acid resistance and inhibits host nitric oxide production. The rocF gene was found to confer arginase activity to Escherichia coli; disruption of plasmid-borne rocF abolished arginase activity. A translationally fused His(6)-RocF was purified from E. coli under nondenaturing conditions and had catalytic activity. Remarkably, the purified enzyme had an acidic pH optimum of 6.1. Both purified arginase and arginase-containing H. pylori extracts exhibited optimal catalytic activity with cobalt as a metal cofactor; manganese and nickel were significantly less efficient in catalyzing the hydrolysis of arginine. Viable H. pylori or E. coli containing rocF had significantly more arginase activity when grown with cobalt in the culture medium than when grown with manganese or no divalent metal. His(6)-RocF arginase activity was inhibited by low concentrations of reducing agents. Antibodies raised to purified His(6)-RocF reacted with both H. pylori and E. coli extracts containing arginase, but not with extracts from rocF mutants of H. pylori or E. coli lacking the rocF gene. The results indicate that H. pylori RocF is necessary and sufficient for arginase activity and has unparalleled features among the arginase superfamily, which may reflect the unique gastric ecological niche of this organism.     

大肠杆菌拓扑异构酶I结合重金属的特性及功能影响

【背景】大肠杆菌拓扑异构酶Ⅰ(Escherichia coli topoisomerase I,E.coli TopA)在DNA复制、转录、重组和基因表达调控等过程发挥关键作用。研究表明E.coli TopA只有结合锌离子才具有活性,然而E.coli TopA能否结合其他金属离子尤其是重金属离子,以及结合其他金属后是否具有活性,目前仍不清楚。【目的】探究大肠杆菌拓扑异构酶Ⅰ是否结合环境中常见重金属离子,研究重金属离子结合E.coli TopA蛋白后对其活性的影响。【方法】在分别添加有锌、钴、镍、镉、铁、汞、砷、铬、铅、铜离子的M9基础培养中表达、纯化出E.coli TopA蛋白,并对纯化得到的蛋白用电感耦合等离子体质谱仪进行相应金属离子含量的测定;利用表达E.coli TopA锌指结构的突变体蛋白鉴定重金属离子的结合位点;通过体外超螺旋DNA松弛实验测定不同金属结合E.coli TopA的拓扑异构酶活性;通过测定蛋白内源性荧光推测不同金属结合E.coli TopA的空间构象差异。【结果】E.coli TopA在体内除了能结合锌和铁之外,还能够结合钴、镍、镉3种离子,但是不能结合汞、砷、铬、铅、铜离子。钴、镍、镉结合形式的E.coli TopA,每个蛋白分子最多可以结合3个相应的金属离子,他们与TopA蛋白的结合位点也是位于3个锌指结构域,而且每个锌指结构域结合1个金属离子。此外,E.coli TopA结合钴、镍、镉离子后,其DNA拓扑异构酶活性并未受到影响,可能是由于钴、镍、镉离子结合形式的E.coli TopA蛋白,其空间构象与锌结合形式相比并未发生显著变化。【结论】由于DNA拓扑异构酶在维持细胞正常生理功能中发挥关键作用,研究表明E.coli TopA的功能不会受到常见重金属的干扰(不结合或者结合后活性无影响),这也有可能是大肠杆菌在进化过程中产生的对抗环境中重金属离子毒害作用的一种自我保护和耐受机制,具有重要的生理意义。     

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.     

Identification of rcnA (yohM), a nickel and cobalt resistance gene in Escherichia coli

We report here on the isolation and primary characterization of the yohM gene of Escherichia coli. We show that yohM encodes a membrane-bound polypeptide conferring increased nickel and cobalt resistance in E. coli. yohM was specifically induced by nickel or cobalt but not by cadmium, zinc, or copper. Mutation of yohM increased the accumulation of nickel inside the cell, whereas cells harboring yohM in multicopy displayed reduced intracellular nickel content. Our data support the hypothesis that YohM is the first described efflux system for nickel and cobalt in E. coli. We propose rcnA (resistance to cobalt and nickel) as the new denomination of yohM.     

Helicobacter pylori neutrophil-activating protein activates neutrophils by its C-terminal region even without dodecamer formation, which is a prerequisite for DNA protection--novel approaches against Helicobacter pylori inflammation

Helicobacter pylori neutrophil-activating protein (HP-NAP) protects DNA from free radicals as a dodecamer through its ferroxidase activity without, however, directly binding to it. The retardation that was observed at pH 7.5 could be easily attributed to an iron effect, as it was revealed by experiments in the absence of HP-NAP. A total loss of ferroxidase activity, dodecamer formation and DNA protection in environments rich in free radicals was observed after replacement of His25, His37, Asp52 and Lys134, which are located within the ferroxidase site, with Ala. Molecular dynamics simulations revealed that dimer formation is highly unlikely following mutation of the above amino acids, as the Fe(2+) is no longer attracted with equal strength by both subunits. These findings probably indicate that iron plays an important role in the conformation of HP-NAP by initiating the formation of stable dimers that are indispensable for the ensuing dodecamer structure. Very surprisingly, neutrophil activation appeared to be stimulated by structural elements that are localized within the C-terminal region of both mutant HP-NAP and wild-type dodecamer HP-NAP. In particular, the dodecamer conformation does not seem to be necessary for activation, and helices H3 (Leu69-Leu75) and H4 (Lys89-Leu114) or the linking coils (His63-Thr68 and Thr76-Ser88) are probably critical in stimulating neutrophil activation.     

The sialic acid binding SabA adhesin of Helicobacter pylori is essential for nonopsonic activation of human neutrophils

Infiltration of neutrophils and monocytes into the gastric mucosa is a hallmark of chronic gastritis caused by Helicobacter pylori. Certain H. pylori strains nonopsonized stimulate neutrophils to production of reactive oxygen species causing oxidative damage of the gastric epithelium. Here, the contribution of some H. pylori virulence factors, the blood group antigen-binding adhesin BabA, the sialic acid-binding adhesin SabA, the neutrophil-activating protein HP-NAP, and the vacuolating cytotoxin VacA, to the activation of human neutrophils in terms of adherence, phagocytosis, and oxidative burst was investigated. Neutrophils were challenged with wild type bacteria and isogenic mutants lacking BabA, SabA, HP-NAP, or VacA. Mutant and wild type strains lacking SabA had no neutrophil-activating capacity, demonstrating that binding of H. pylori to sialylated neutrophil receptors plays a pivotal initial role in the adherence and phagocytosis of the bacteria and the induction of the oxidative burst. The link between receptor binding and oxidative burst involves a G-protein-linked signaling pathway and downstream activation of phosphatidylinositol 3-kinase as shown by experiments using signal transduction inhibitors. Collectively our data suggest that the sialic acid-binding SabA adhesin is a prerequisite for the nonopsonic activation of human neutrophils and, thus, is a virulence factor important for the pathogenesis of H. pylori infection.     

The neutrophil-activating protein of Helicobacter pylori (HP-NAP) as an immune modulating agent

During evolution microorganisms have developed several immune modulating strategies. The Helicobacter pylori neutrophil-activating protein (HP-NAP) is a virulence factor that attracts and activates neutrophils, and promotes their endothelial adhesion and the production of oxygen radicals and chemokines, including CXCL8, CCL3 and CCL4. HP-NAP, a TLR2 agonist, is an immune modulator able to induce the expression of interleukin-12 (IL-12) and IL-23 by human neutrophils and monocytes. In fact, HP-NAP has the potential to shift antigen-specific T-cell responses from a predominant Th2 to a polarized Th1 cytotoxic phenotype, characterized by high levels of interferon-gamma and tumor necrosis factor-alpha production. Thus, HP-NAP is a key factor driving Th1 inflammation in H. pylori infection and may be a new tool for future therapeutic strategies aimed at redirecting Th2 into Th1 responses, for example in atopy, vaccinology and cancer immunotherapy.     

Helicobacter pylori induce neutrophil transendothelial migration: role of the bacterial HP-NAP

Continuous recruitment of neutrophils into the inflamed gastric mucosal tissue is a hallmark of Helicobacter pylori infection in humans. In this study, we examined the ability of H. pylori to induce transendothelial migration of neutrophils using a transwell system consisting of a cultured monolayer of human endothelial cells as barrier between two chambers. We showed for the first time that live H. pylori, but not formalin-killed bacteria, induced a significantly increased transendothelial migration of neutrophils. H. pylori conditioned culture medium also induced significantly increased transendothelial migration, whereas heat-inactivated culture filtrates had no effect, suggesting that the chemotactic factor was proteinaceous. Depletion of H. pylori-neutrophil activating protein (HP-NAP) from the culture filtrates resulted in significant reduction of the transmigration. Culture filtrates from isogenic HP-NAP deficient mutant bacteria also induced significantly less neutrophil migration than culture filtrates obtained from wild-type bacteria. HP-NAP did not induce endothelial cell activation, suggesting that HP-NAP acts directly on the neutrophils. In conclusion, our results demonstrate that secreted HP-NAP is one of the factors resulting in H. pylori induced neutrophil transendothelial migration. We propose that HP-NAP contributes to the continuous recruitment of neutrophils to the gastric mucosa of H. pylori infected individuals.     

Helicobacter pylori cadA encodes an essential Cd(II)–Zn(II)–Co(II) resistance factor influencing urease activity

Inactivation of Helicobacter pylori cadA, encoding a putative transition metal ATPase, was only possible in one of four natural competent H. pylori strains, designated 69A. All tested cadA mutants showed increased growth sensitivity to Cd(II) and Zn(II). In addition, some of them showed both reduced 63Ni accumulation during growth and no or impaired urease activity, which was not due to lack of urease enzyme subunits. Gene complementation experiments with plasmid (pY178)-derived H. pylori cadA failed to correct the deficiencies, whereas resistance to Cd(II) and Zn(II) was restored. Moreover, pY178 conferred increased Co(II) resistance to both the cadA mutants and the wild-type strain 69A. Heterologous expression of H. pylori cadA in an Escherichia coli zntA mutant resulted in an elevated resistance to Cd(II) and Zn(II). Expression of cadA in E. coli SE5000 harbouring H. pylori nixA, which encodes a divalent cation importer along with the H. pylori urease gene cluster, led to about a threefold increase in urease activity compared with E. coli control cells lacking the H. pylori cadA gene. These results suggest that H. pylori CadA is an essential resistance pump with ion specificity towards Cd(II), Zn(II) and Co(II). They also point to a possible role of H. pylori CadA in high-level activity of H. pylori urease, an enzyme sensitive to a variety of metal ions.     

HybF, a zinc-containing protein involved in NiFe hydrogenase maturation

HypA and HypB are maturation proteins required for incorporation of nickel into the hydrogenase large subunit. To examine the functions of these proteins in nickel insertion, the hybF gene, which is a homolog of hypA essential for maturation of hydrogenases 1 and 2 from Escherichia coli, was overexpressed, and the product was purified. This protein behaves like a monomer in gel filtration and contains stoichiometric amounts of zinc but insignificant or undetectable amounts of nickel and iron. In filter binding assays radioactively labeled nickel binds to HybF with a K(D) of 1.87 microM and in a stoichiometric ratio. To identify amino acid residues of HybF involved in nickel and/or zinc binding, variants in which conserved residues were replaced were studied. An H2Q replacement eliminated both in vivo activity and in vitro binding of nickel. The purified protein, however, contained zinc at the level characteristic of the wild-type protein. When E3 was replaced by Q, activity was retained, but an E3L exchange was detrimental. Replacement of each of the four conserved cysteine residues of a zinc finger motif reduced the cellular amount of HybF protein without a loss of in vivo activity, indicating that these residues play a purely structural role. A triple mutant deficient in the synthesis or activity of HypA, HybF, and HypB was constructed, and it exhibited the same responsiveness for phenotypic complementation by high nickel as mutants with a single lesion in one of the genes exhibited. The results are interpreted in terms of a concerted action of HypB and HybF in nickel insertion in which HybF (as well as its homolog, HypA) functions as a metallochaperone and HypB functions as a regulator that controls the interaction of HybF with the target protein.     

Metal complexes of salicylhydroxamic acid (H2Sha), anthranilic hydroxamic acid and benzohydroxamic acid. Crystal and molecular structure of [Cu(phen)2(Cl)]Cl x H2Sha, a model for a peroxidase-inhibitor complex

Stability constants of iron(III), copper(II), nickel(II) and zinc(II) complexes of salicylhydroxamic acid (H2Sha), anthranilic hydroxamic acid (HAha) and benzohydroxamic acid (HBha) have been determined at 25.0 degrees C, I=0.2 mol dm(-3) KCl in aqueous solution. The complex stability order, iron(III) > copper(II) > nickel(II) approximately = zinc(II) was observed whilst complexes of H2Sha were found to be more stable than those of the other two ligands. In the preparation of ternary metal ion complexes of these ligands and 1,10-phenanthroline (phen) the crystalline complex [Cu(phen)2(Cl)]Cl x H2Sha was obtained and its crystal structure determined. This complex is a model for hydroxamate-peroxidase inhibitor interactions.     

The Helicobacter pylori neutrophil-activating protein is an iron-binding protein with dodecameric structure

The neutrophil-activating protein (HP-NAP) of Helicobacter pylori is a major 17 kDa antigen of the immune response of infected individuals. Amino acid sequence comparison indicated a high similarity between HP-NAP and both bacterial DNA-protecting proteins (Dps) and ferritins. The structure prediction and spectroscopic analysis presented here indicate a close similarity between HP-NAP and Dps. Electron microscopy revealed that HP-NAP forms hexagonal rings of 9-10 nm diameter with a hollow central core as seen in Dps proteins, clearly different from the 12 nm icositetrameric (24 subunits) ferritins. However, HP-NAP is resistant to thermal and chemical denaturation similar to the ferritin family of proteins. In addition, HP-NAP binds up to 40 atoms of iron per monomer and does not bind DNA. We therefore conclude that HP-NAP is an unusual, small, ferritin that folds into a four-helix bundle that oligomerizes into dodecamers with a central hole capable of binding up to 500 iron atoms per oligomer.     

Helicobacter pylori utilises urea for amino acid synthesis

Abstract Helicobacter pylori has one of the highest urease activities of all known bacteria. Its enzymatic production of ammonia protects the organism from acid damage by gastric juice. The possibility that the urease activity allows the bacterium to utilise urea as a nitrogen source for the synthesis of amino acids was investigated. H. pylori (NCTC 11638) was incubated with 50 mM urea, enriched to 5 atom% excess ¹⁵N, that is the excess enrichment of ¹⁵N above the normal background, in the presence of either NaCl pH 6.0, or 0.2M citrate pH 6.0. E. coli (NCTC 9001) was used as a urease-negative control. ¹⁵N enrichment was detected by isotope ratio mass spectrometry. H. pylori showed intracellular incorporation of ¹⁵N in the presence of citrate buffer pH 6.0 but there was no significant incorporation of ¹⁵N in unbuffered saline or by E. coli in either pH 6.0 citrate buffer or unbuffered saline. The intracellular fate of the urea-nitrogen was determined by means of gas chromatography/mass spectrometry following incubation with ¹⁵N enriched 5 mM urea in the presence of either 0.2 M citrate buffer pH 6.0 or 0.2 M acetate buffer pH 6.0. After 5 min incubation in either buffer the ¹⁵n label appeared in glutamate, glutamine, phenylalanine, aspartate and alanine. It appears, therefore, that at pH and urea concentrations typical of the gastric mucosal surface, H. pylori utilises exogenous urea as a nitrogen source for amino acid synthesis. The ammonia produced by H. pylori urease activity thus facilitates the organism's nitrogen metabolism at neutral pH as well as protecting it from acid damage at low pH.