Iron-sulfur protein maturation in Helicobacter pylori: identifying a Nfu-type cluster carrier protein and its iron-sulfur protein targets.

Helicobacter pylori is anomalous among non nitrogen-fixing bacteria in containing an incomplete NIF system for Fe-S cluster assembly comprising two essential proteins, NifS (cysteine desulfurase) and NifU (scaffold protein). Although nifU deletion strains cannot be obtained via the conventional gene replacement, a NifU-depleted strain was constructed and shown to be more sensitive to oxidative stress compared to wild-type (WT) strains. The hp1492 gene, encoding a putative Nfu-type Fe-S cluster carrier protein, was disrupted in three different H. pylori strains, indicating that it is not essential. However, Δnfu strains have growth deficiency, are more sensitive to oxidative stress and are unable to colonize mouse stomachs. Moreover, Δnfu strains have lower aconitase activity but higher hydrogenase activity than the WT. Recombinant Nfu was found to bind either one [2Fe-2S] or [4Fe-4S] cluster/dimer, based on analytical, UV-visible absorption/CD and resonance Raman studies. A bacterial two-hybrid system was used to ascertain interactions between Nfu, NifS, NifU and each of 36 putative Fe-S-containing target proteins. Nfu, NifS and NifU were found to interact with 15, 6 and 29 putative Fe-S proteins respectively. The results indicate that Nfu, NifS and NifU play a major role in the biosynthesis and/or delivery of Fe-S clusters in H. pylori.

Noncatalytic Antioxidant Role for Helicobacter pylori Urease.

The well-studied catalytic role of urease, the Ni-dependent conversion of urea into carbon dioxide and ammonia, has been shown to protect Helicobacter pylori against the low pH environment of the stomach lumen. We hypothesized that the abundantly expressed urease protein can play another noncatalytic role in combating oxidative stress via Met residue-mediated quenching of harmful oxidants. Three catalytically inactive urease mutant strains were constructed by single substitutions of Ni binding residues. The mutant versions synthesize normal levels of urease, and the altered versions retained all methionine residues. The three site-directed urease mutants were able to better withstand a hypochlorous acid (HOCl) challenge than a ΔureAB deletion strain. The capacity of purified urease to protect whole cells via oxidant quenching was assessed by adding urease enzyme to nongrowing HOCl-exposed cells. No wild-type cells were recovered with oxidant alone, whereas urease addition significantly aided viability. These results suggest that urease can protect H. pylori against oxidative damage and that the protective ability is distinct from the well-characterized catalytic role. To determine the capability of methionine sulfoxide reductase (Msr) to reduce oxidized Met residues in urease, purified H. pylori urease was exposed to HOCl and a previously described Msr peptide repair mixture was added. Of the 25 methionine residues in urease, 11 were subject to both oxidation and to Msr-mediated repair, as identified by mass spectrometry (MS) analysis; therefore, the oxidant-quenchable Met pool comprising urease can be recycled by the Msr repair system. Noncatalytic urease appears to play an important role in oxidant protection.IMPORTANCE Chronic Helicobacter pylori infection can lead to gastric ulcers and gastric cancers. The enzyme urease contributes to the survival of the bacterium in the harsh environment of the stomach by increasing the local pH. In addition to combating acid, H. pylori must survive host-produced reactive oxygen species to persist in the gastric mucosa. We describe a cyclic amino acid-based antioxidant role of urease, whereby oxidized methionine residues can be recycled by methionine sulfoxide reductase to again quench oxidants. This work expands our understanding of the role of an already acknowledged pathogen virulence factor and specifically expands our knowledge of H. pylori survival mechanisms.

Helicobacter Catalase Devoid of Catalytic Activity Protects the Bacterium against Oxidative Stress.

Catalase, a conserved and abundant enzyme found in all domains of life, dissipates the oxidant hydrogen peroxide (H2O2). The gastric pathogen Helicobacter pylori undergoes host-mediated oxidant stress exposure, and its catalase contains oxidizable methionine (Met) residues. We hypothesized catalase may play a large stress-combating role independent of its classical catalytic one, namely quenching harmful oxidants through its recyclable Met residues, resulting in oxidant protection to the bacterium. Two Helicobacter mutant strains (katAH56A and katAY339A) containing catalase without enzyme activity but that retain all Met residues were created. These strains were much more resistant to oxidants than a catalase-deletion mutant strain. The quenching ability of the altered versions was shown, whereby oxidant-stressed (HOCl-exposed) Helicobacter retained viability even upon extracellular addition of the inactive versions of catalase, in contrast to cells receiving HOCl alone. The importance of the methionine-mediated quenching to the pathogen residing in the oxidant-rich gastric mucus was studied. In contrast to a catalase-null strain, both site-change mutants proficiently colonized the murine gastric mucosa, suggesting that the amino acid composition-dependent oxidant-quenching role of catalase is more important than the well described H2O2-dissipating catalytic role. Over 100 years after the discovery of catalase, these findings reveal a new non-enzymatic protective mechanism of action for the ubiquitous enzyme.

Carbon Fixation Driven by Molecular Hydrogen Results in Chemolithoautotrophically Enhanced Growth of Helicobacter pylori.

UNLABELLED: A molecular hydrogen (H2)-stimulated, chemolithoautotrophic growth mode for the gastric pathogen Helicobacter pylori is reported. In a culture medium containing peptides and amino acids, H2-supplied cells consistently achieved 40 to 60% greater growth yield in 16 h and accumulated 3-fold more carbon from [(14)C]bicarbonate (on a per cell basis) in a 10-h period than cells without H2 Global proteomic comparisons of cells supplied with different atmospheric conditions revealed that addition of H2 led to increased amounts of hydrogenase and the biotin carboxylase subunit of acetyl coenzyme A (acetyl-CoA) carboxylase (ACC), as well as other proteins involved in various cellular functions, including amino acid metabolism, heme synthesis, or protein degradation. In agreement with this result, H2-supplied cells contained 3-fold more ACC activity than cells without H2 Other possible carbon dioxide (CO2) fixation enzymes were not up-expressed under the H2-containing atmosphere. As the gastric mucus is limited in carbon and energy sources and the bacterium lacks mucinase, this new growth mode may contribute to the persistence of the pathogen in vivo This is the first time that chemolithoautotrophic growth is described for a pathogen. IMPORTANCE: Many pathogens must survive within host areas that are poorly supplied with carbon and energy sources, and the gastric pathogen Helicobacter pylori resides almost exclusively in the nutritionally stringent mucus barrier of its host. Although this bacterium is already known to be highly adaptable to gastric niches, a new aspect of its metabolic flexibility, whereby molecular hydrogen use (energy) is coupled to carbon dioxide fixation (carbon acquisition) via a described carbon fixation enzyme, is shown here. This growth mode, which supplements heterotrophy, is termed chemolithoautotrophy and has not been previously reported for a pathogen.

A novel DNA-binding protein plays an important role in Helicobacter pylori stress tolerance and survival in the host.

The gastric pathogen Helicobacter pylori must combat chronic acid and oxidative stress. It does so via many mechanisms, including macromolecule repair and gene regulation. Mitomycin C-sensitive clones from a transposon mutagenesis library were screened. One sensitive strain contained the insertion element at the locus of hp119, a hypothetical gene. No homologous gene exists in any (non-H. pylori) organism. Nevertheless, the predicted protein has some features characteristic of histone-like proteins, and we showed that purified HP119 protein is a DNA-binding protein. A Δhp119 strain was markedly more sensitive (viability loss) to acid or to air exposure, and these phenotypes were restored to wild-type (WT) attributes upon complementation of the mutant with the wild-type version of hp119 at a separate chromosomal locus. The mutant strain was approximately 10-fold more sensitive to macrophage-mediated killing than the parent or the complemented strain. Of 12 mice inoculated with the wild type, all contained H. pylori, whereas 5 of 12 mice contained the mutant strain; the mean colonization numbers were 158-fold less for the mutant strain. A proteomic (two-dimensional PAGE with mass spectrometric analysis) comparison between the Δhp119 mutant and the WT strain under oxidative stress conditions revealed a number of important antioxidant protein differences; SodB, Tpx, TrxR, and NapA, as well as the peptidoglycan deacetylase PgdA, were significantly less expressed in the Δhp119 mutant than in the WT strain. This study identified HP119 as a putative histone-like DNA-binding protein and showed that it plays an important role in Helicobacter pylori stress tolerance and survival in the host.

Aconitase Functions as a Pleiotropic Posttranscriptional Regulator in Helicobacter pylori.

UNLABELLED: Posttranscriptional regulation in bacteria has increasingly become recognized as playing a major role in response to environmental stimuli. Aconitase is a bifunctional protein that not only acts enzymatically but also can be a posttranscriptional regulator. To investigate protein expression regulated by Helicobacter pylori AcnB in response to oxidative stress, a global proteomics study was conducted wherein the ΔacnB strain was compared to the parent strain when both strains were O2 stressed. Many proteins, including some involved in urease activity, in combating oxidative stress, and in motility, were expressed at a significantly lower level in the ΔacnB strain. A bioinformatics prediction tool was used to identify putative targets for aconitase-mediated regulation, and electrophoretic mobility shift assays demonstrated that apo-AcnB is able to bind to RNA transcripts of hpn (encoding a nickel-sequestering protein), ahpC (encoding alkyl hydroperoxide reductase), and flgR (encoding flagellum response regulator). Compared to the wild type (WT), the ΔacnB strain had decreased activities of the nickel-containing enzymes urease and hydrogenase, and this could be correlated with lower total nickel levels within ΔacnB cells. Binding of apo-AcnB to the hpn 5' untranslated region (UTR) may inhibit the expression of Hpn. In agreement with the finding that AcnB regulates the expression of antioxidant proteins such as AhpC, ΔacnB cells displayed oxidative-stress-sensitive phenotypes. The ΔacnB strain has a lesser motility ability than the WT strain, which can likely be explained by the functions of AcnB on the FlgRS-RpoN-FlgE regulatory cascade. Collectively, our results suggest a global role for aconitase as a posttranscriptional regulator in this gastric pathogen. IMPORTANCE: Bacterial survival depends on the ability of the cell to sense and respond to a variety of environmental changes. For Helicobacter pylori, responding to environmental stimuli within the gastric niche is essential for persistence and host colonization. However, there is much to be learned about the regulatory mechanisms that H. pylori employs to orchestrate its response to different stimuli. In this study, we explore the role of aconitase, a bifunctional protein that has been found to act as a posttranscriptional regulator in several other bacteria. Our results shed light on the magnitude of aconitase-mediated regulation in H. pylori, and we propose that aconitase acts as a global regulator of key genes involved in virulence.

Comparative Roles of the Two Helicobacter pylori Thioredoxins in Preventing Macromolecule Damage.

Thioredoxins are highly conserved throughout a wide range of organisms, and they are essential for the isurvival of oxygen-sensitive cells. The gastric pathogen Helicobacter pylori uses the thioredoxin system to maintain its thiol/disulfide balance. There are two thioredoxins present in H. pylori, Trx1 and Trx2 (herein referred to as TrxA and TrxC). TrxA has been shown to be important as an electron donor for some antioxidant enzymes, but the function of TrxC remains unknown (L. M. Baker, A. Raudonikiene, P. S. Hoffman, and L. B. Poole, J Bacteriol 183:1961-1973, 2001; P. Alamuri and R. J. Maier, J Bacteriol 188:5839-5850, 2006). We demonstrate that both TrxA and TrxC are important in protecting H. pylori from oxidative stress. Individual ΔtrxA and ΔtrxC deletion mutant strains each show a greater abundance of lipid peroxides and suffer more DNA damage and more protein carbonylation than the parent. Both deletion mutants were much more sensitive to O2-mediated viability loss than the parent. Unexpectedly, the oxidative DNA damage and protein carbonylation was more severe in the ΔtrxC mutant than in the ΔtrxA mutant; it had 20-fold- and 4-fold-more carbonylated protein content than the wild type and the ΔtrxA strain, respectively, after 4 h of atmospheric O2 stress. trx transcript abundance was altered by the deletion of the heterologous trx gene. The ΔtrxC mutant lacked mouse colonization ability, while the ability to colonize mouse stomachs was significantly reduced in the ΔtrxA mutant.

Host hydrogen rather than that produced by the pathogen is important for Salmonella enterica serovar Typhimurium virulence.

Salmonella enterica serovar Typhimurium utilizes molecular hydrogen as a substrate in various respiratory pathways, via H2-uptake enzymes termed Hya, Hyb, and Hyd. A different hydrogenase, the hydrogen-evolving Hyc enzyme, removes excess reductant during fermentative growth. Virulence phenotypes conferred by mutations in hyc genes, either alone or in combination with mutations in the H2-uptake enzyme genes, are addressed. Anaerobically grown ΔhycB or ΔhycC single-deletion strains were more sensitive to acid than the wild-type strain, but the Δhyc strains were like the virulent parent strain with respect to both mouse morbidity and mortality and in organ burden numbers. Even fecal-recovery numbers for both mutant strains at several time points prior to the animals succumbing to salmonellosis were like those seen with the parent. Neither hydrogen uptake nor evolution of the gas was detected in a hydrogenase quadruple-mutant strain containing deletions in the hya, hyb, hyd, and hyc genes. As previously described, a strain lacking all H2-uptake ability was severely attenuated in its virulence characteristics, and the quadruple-mutant strain had the same (greatly attenuated) phenotype. While H2 levels were greatly reduced in ceca of mice treated with antibiotics, both the ΔhycB and ΔhycC strains were still like the parent in their ability to cause typhoid salmonellosis. It seems that the level of H2 produced by the pathogen (through formate hydrogen lyase [FHL] and Hyc) is insignificant in terms of providing respiratory reductant to facilitate either organ colonization or contributions to gut growth leading to pathogenesis.

Bacterial histone-like proteins: roles in stress resistance.

Histone-like proteins (HLPs) are small and basic bacterial proteins that are associated with a nucleoid and play roles in maintaining DNA architecture and regulating DNA transactions such as replication, recombination/repair and transcription. The studies on HLPs from a variety of bacterial species in recent years are summarized in this mini-review. A recent study reported a novel DNA-binding protein (HP119) in Helicobacter pylori that shows some HLP features. It plays a large role in aiding bacterial stress resistance. We provide herein additional evidence that HP119 is a nucleoid-associated protein, and present some perspectives for future study.

Ammonium metabolism enzymes aid Helicobacter pylori acid resistance.

The gastric pathogen Helicobacter pylori possesses a highly active urease to support acid tolerance. Urea hydrolysis occurs inside the cytoplasm, resulting in the production of NH3 that is immediately protonated to form NH4 (+). This ammonium must be metabolized or effluxed because its presence within the cell is counterproductive to the goal of raising pH while maintaining a viable proton motive force (PMF). Two compatible hypotheses for mitigating intracellular ammonium toxicity include (i) the exit of protonated ammonium outward via the UreI permease, which was shown to facilitate diffusion of both urea and ammonium, and/or (ii) the assimilation of this ammonium, which is supported by evidence that H. pylori assimilates urea nitrogen into its amino acid pools. We investigated the second hypothesis by constructing strains with altered expression of the ammonium-assimilating enzymes glutamine synthetase (GS) and glutamate dehydrogenase (GDH) and the ammonium-evolving periplasmic enzymes glutaminase (Ggt) and asparaginase (AsnB). H. pylori strains expressing elevated levels of either GS or GDH are more acid tolerant than the wild type, exhibit enhanced ammonium production, and are able to alkalize the medium faster than the wild type. Strains lacking the genes for either Ggt or AsnB are acid sensitive, have 8-fold-lower urea-dependent ammonium production, and are more acid sensitive than the parent. Additionally, we found that purified H. pylori GS produces glutamine in the presence of Mg(2+) at a rate similar to that of unadenylated Escherichia coli GS. These data reveal that all four enzymes contribute to whole-cell acid resistance in H. pylori and are likely important for assimilation and/or efflux of urea-derived ammonium.

Random and site-specific mutagenesis of the Helicobacter pylori ferric uptake regulator provides insight into Fur structure-function relationships.

The ferric uptake regulator (Fur) of Helicobacter pylori is a global regulator that is important for colonization and survival within the gastric mucosa. H. pylori Fur is unique in its ability to activate and repress gene expression in both the iron-bound (Fe-Fur) and apo forms (apo-Fur). In the current study we combined random and site-specific mutagenesis to identify amino acid residues important for both Fe-Fur and apo-Fur function. We identified 25 mutations that affected Fe-Fur repression and 23 mutations that affected apo-Fur repression, as determined by transcriptional analyses of the Fe-Fur target gene amiE, and the apo-Fur target gene, pfr. In addition, eight of these mutations also significantly affected levels of Fur in the cell. Based on regulatory phenotypes, we selected several representative mutations to characterize further. Of those selected, we purified the wild-type (HpFurWT) and three mutant Fur proteins (HpFurE5A, HpFurA92T and HpFurH134Y), which represent mutations in the N-terminal extension, the regulatory metal binding site (S2) and the structural metal binding site (S3) respectively. Purified proteins were evaluated for secondary structure by circular dichroism spectroscopy, iron-binding by atomic absorption spectrophotometry, oligomerization in manganese-substituted and apo conditions by in vitro cross-linking assays, and DNA binding to Fe-Fur and apo-Fur target sequences by fluorescence anisotropy. The results showed that the N-terminal, S2 and S3 regions play distinct roles in terms of Fur structure-function relationships. Overall, these studies provide novel information regarding the role of these residues in Fur function, and provide mechanistic insight into how H. pylori Fur regulates gene expression in both the iron-bound and apo forms of the protein.

Role of Helicobacter pylori methionine sulfoxide reductase in urease maturation.

The persistence of the gastric pathogen Helicobacter pylori is due in part to urease and Msr (methionine sulfoxide reductase). Upon exposure to relatively mild (21% partial pressure of O2) oxidative stress, a Δmsr mutant showed both decreased urease specific activity in cell-free extracts and decreased nickel associated with the partially purified urease fraction as compared with the parent strain, yet urease apoprotein levels were the same for the Δmsr and wild-type extracts. Urease activity of the Δmsr mutant was not significantly different from the wild-type upon non-stress microaerobic incubation of strains. Urease maturation occurs through nickel mobilization via a suite of known accessory proteins, one being the GTPase UreG. Treatment of UreG with H2O2 resulted in oxidation of MS-identified methionine residues and loss of up to 70% of its GTPase activity. Incubation of pure H2O2-treated UreG with Msr led to reductive repair of nine methionine residues and recovery of up to full enzyme activity. Binding of Msr to both oxidized and non-oxidized UreG was observed by cross-linking. Therefore we conclude Msr aids the survival of H. pylori in part by ensuring continual UreG-mediated urease maturation under stress conditions.

D-aspartate, an amino-acid important for human health, supports anaerobic respiration in several Campylobacter species.

Despite being classified as microaerophilic microorganisms, most Campylobacter species can grow anaerobically, using formate or molecular hydrogen (H2) as electron donors, and various nitrogenous and sulfurous compounds as electron acceptors. Herein, we showed that both L-asparagine (L-Asn) and L-aspartic acid (L-Asp) bolster H2-driven anaerobic growth in several Campylobacter species, whereas the D-enantiomer form of both asparagine (D-Asn) and aspartic acid (D-Asp) only increased anaerobic growth in C. concisus strain 13826 and C. ureolyticus strain NCTC10941. A gene annotated as racD encoding for a putative D/L-Asp racemase was identified in the genome of both strains. Disruption of racD in Cc13826 resulted in the inability of the mutant strain to use either D-enantiomer during anaerobic growth. Hence, our results suggest that the racD gene is required for campylobacters to use either D-Asp or D-Asn. The use of D-Asp by various human opportunistic bacterial pathogens, including C. concisus, C. ureolyticus, and also possibly select strains of C. gracilis, C. rectus and C. showae, is significant, because D-Asp is an important signal molecule for both human nervous and neuroendocrine systems. To our knowledge, this is the first report of pathogens scavenging a D-amino acid essential for human health.

Aconitase-mediated posttranscriptional regulation of Helicobacter pylori peptidoglycan deacetylase.

Some bacterial aconitases are bifunctional proteins that function in the citric acid cycle and act as posttranscriptional regulators in response to iron levels and oxidative stress. We explore the role of aconitase (AcnB) in Helicobacter pylori as a posttranscriptional regulator of the cell wall-modifying enzyme peptidoglycan deacetylase, PgdA. Under oxidative stress, PgdA is highly expressed and confers resistance to lysozyme in wild-type cells. PgdA protein expression as well as transcript abundance is significantly decreased in an acnB mutant. In the wild type, pgdA mRNA half-life was 13 min, whereas the half-life for the acnB strain was 7 min. Based on electrophoretic mobility shift assays and RNA footprinting, the H. pylori apo-AcnB binds to the 3'-untranslated region of the pgdA RNA transcript. Some of the protected bases (from footprinting) were localized in proposed stem-loop structures. AcnB-pgdA transcript binding was abolished by the addition of iron. The acnB strain is more susceptible to lysozyme-mediated killing and was attenuated in its ability to colonize mice. The results support a model whereby apo-AcnB directly interacts with the pgdA transcript to enhance stability and increase deacetylase enzyme expression, which impacts in vivo survival.

Identification of a new class of adenosine deaminase from Helicobacter pylori with homologs among diverse taxa.

Early studies of Helicobacter pylori's nutritional requirements alluded to a complete purine salvage network in this organism. Recently, this hypothesis was confirmed in two strains of H. pylori, whose purine requirements were satisfied by any single purine base or nucleoside. Most of the purine conversion enzymes in H. pylori have been studied using mutant analysis; however, the gene encoding adenosine deaminase (ADD) in H. pylori remained unidentified. Through stepwise protein purification followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF), we discovered that H. pylori ADD is encoded by hp0267, an apparently essential gene. Hp0267 shares no sequence homology with previously characterized ADDs, yet both are members of the amidohydrolase superfamily. Hp0267 is grouped within cog0402, while other ADDs studied to date are found in cog1816. The hp0267 locus was previously misannotated as encoding a chlorohydrolase. Using purified recombinant Hp0267, we determined the enzyme's pH optimum, temperature optimum, substrate specificity, and estimated kinetic constants. In contrast to other known ADDs, Hp0267 contains Fe(II) as the relevant metal ligand. Furthermore, Hp0267 exhibits very low deaminase activity on 2'-deoxyadenosine, a substrate that is readily hydrolyzed by cog1816 ADDs. Our preliminary comparative genomic analysis suggests that Hp0267 represents a second enzyme class of adenosine deaminase whose phyletic distribution among prokaryotes is broad.

Alkyl hydroperoxide reductase repair by Helicobacter pylori methionine sulfoxide reductase.

Protein exposure to oxidants such as HOCl leads to formation of methionine sulfoxide (MetSO) residues, which can be repaired by methionine sulfoxide reductase (Msr). A Helicobacter pylori msr strain was more sensitive to HOCl-mediated killing than the parent. Because of its abundance in H. pylori and its high methionine content, alkyl hydroperoxide reductase C (AhpC) was hypothesized to be prone to methionine oxidation. AhpC was expressed as a recombinant protein in Escherichia coli. AhpC activity was abolished by HOCl, while all six methionine residues of the enzyme were fully to partially oxidized. Upon incubation with a Msr repair mixture, AhpC activity was restored to nonoxidized levels and the MetSO residues were repaired to methionine, albeit to different degrees. The two most highly oxidized and then Msr-repaired methionine residues in AhpC, Met101 and Met133, were replaced with isoleucine residues by site-directed mutagenesis, either individually or together. E. coli cells expressing variant versions were more sensitive to t-butyl hydroperoxide than cells expressing native protein, and purified AhpC variant proteins had 5% to 39% of the native enzyme activity. Variant proteins were still able to oligomerize like the native version, and circular dichroism (CD) spectra of variant proteins revealed no significant change in AhpC conformation, indicating that the loss of activity in these variants was not related to major structural alterations. Our results suggest that both Met101 and Met133 residues are important for AhpC catalytic activity and that their integrity relies on the presence of a functional Msr.

Helicobacter pylori stores nickel to aid its host colonization.

The transition metal nickel (Ni) is critical for the pathogenicity of Helicobacter pylori. Indeed the element is a required component of two enzymes, hydrogenase and urease, that have been shown to be important for in vivo colonization of the host gastric mucosa. Urease accounts for up to 10% of the total cellular H. pylori protein content, and therefore the bacterial Ni demand is very high. H. pylori possess two small and abundant histidine-rich, Ni-binding proteins, Hpn and Hpn-like, whose physiological role in the host have not been investigated. In this study, special husbandry conditions were used to control Ni levels in the host (mouse), including the use of Ni-free versus Ni-supplemented food. The efficacy of each diet was confirmed by measuring the Ni concentrations in sera of mice fed with either diet. Colonization levels (based on rank tests) of the Δhpn Δhpn-like double mutants isolated from the mice provided Ni-deficient chow were statistically lower than those for mice given Ni in their diet. In contrast, H. pylori wild-type colonization levels were similar in both host groups (e.g., regardless of Ni levels). Our results indicate that the gastric pathogen H. pylori can utilize stored Ni via defined histidine-rich proteins to aid colonization of the host.

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

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 8µM and 14µM 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.

Helicobacter hepaticus NikR controls urease and hydrogenase activities via the NikABDE and HH0418 putative nickel import proteins.

Helicobacter hepaticus open reading frame HH0352 was identified as a nickel-responsive regulator NikR. The gene was disrupted by insertion of an erythromycin resistance cassette. The H. hepaticus nikR mutant had five- to sixfold higher urease activity and at least twofold greater hydrogenase activity than the wild-type strain. However, the urease apo-protein levels were similar in both the wild-type and the mutant, suggesting the increase in urease activity in the mutant was due to enhanced Ni-maturation of the urease. Compared with the wild-type strain, the nikR strain had increased cytoplasmic nickel levels. Transcription of nikABDE (putative inner membrane Ni transport system) and hh0418 (putative outer membrane Ni transporter) was nickel- and NikR-repressed. Electrophoretic mobility shift assays (EMSAs) revealed that purified HhNikR could bind to the nikABDE promoter (P(nikA)), but not to the urease or the hydrogenase promoter; NikR-P(nikA) binding was enhanced in the presence of nickel. Also, qRT-PCR and EMSAs indicated that neither nikR nor the exbB-exbD-tonB were under the control of the NikR regulator, in contrast with their Helicobacter pylori homologues. Taken together, our results suggest that HhNikR modulates urease and hydrogenase activities by repressing the nickel transport/nickel internalization systems in H. hepaticus, without direct regulation of the Ni-enzyme genes (the latter is the case for H. pylori). Finally, the nikR strain had a two- to threefold lower growth yield than the parent, suggesting that the regulatory protein might play additional roles in the mouse liver pathogen.

The Campylobacter concisus BisA protein plays a dual role: oxide-dependent anaerobic respiration and periplasmic methionine sulfoxide repair.

IMPORTANCE: Campylobacter concisus is an excellent model organism to study respiration diversity, including anaerobic respiration of physiologically relevant N-/S-oxides compounds, such as biotin sulfoxide, dimethyl sulfoxide, methionine sulfoxide (MetO), nicotinamide N-oxide, and trimethylamine N-oxide. All C. concisus strains harbor at least two, often three, and up to five genes encoding for putative periplasmic Mo/W-bisPGD-containing N-/S-oxide reductases. The respective role (substrate specificity) of each enzyme was studied using a mutagenesis approach. One of the N/SOR enzymes, annotated as "BisA", was found to be essential for anaerobic respiration of both N- and S-oxides. Additional phenotypes associated with disruption of the bisA gene included increased sensitivity toward oxidative stress and elongated cell morphology. Furthermore, a biochemical approach confirmed that BisA can repair protein-bound MetO residues. Hence, we propose that BisA plays a role as a periplasmic methionine sulfoxide reductase. This is the first report of a Mo/W-bisPGD-enzyme supporting both N- or S-oxide respiration and protein-bound MetO repair in a pathogen.