Robert J. Maier

Requirement of nickel metabolism proteins HypA and HypB for full activity of both hydrogenase and urease in Helicobacter pylori

The nickel‐containing enzymes hydrogenase and urease require accessory proteins in order to incorporate properly the nickel atom(s) into the active sites. The Helicobacter pylori genome contains the full complement of both urease and hydrogenase accessory proteins. Two of these, the hydrogenase accessory proteins HypA (encoded by hypA) and HypB (encoded by hypB), are required for the full activity of both the hydrogenase and the urease enzymes in H. pylori. Under normal growth conditions, hydrogenase activity is abolished in strains in which either hypA (HypA:kan) or hypB (HypB:kan) have been interrupted by a kanamycin resistance cassette. Urease activity in these strains is 40 (HypA:kan)‐ and 200 (HypB:kan)‐fold lower than for the wild‐type (wt) strain 43504. Nickel supplementation in the growth media restored urease activity to almost wt levels. Hydrogenase activity was restored to a lesser extent, as has been observed for hyp mutants in other (H2‐oxidizing) bacteria. Expression levels of UreB (the urease large subunit) were not affected by inactivation of either hypA or hypB, as determined by immunoblotting. Urease activity was not affected by lesions in the genes for either the hydrogenase accessory proteins HypD or HypF or the hydrogenase large subunit structural gene, indicating that the urease deficiency was not caused by lack of hydrogenase activity. When crude extracts of wt, HypA:kan and HypB:kan were separated by anion exchange chromatography, the urease‐containing fractions of the mutant strains contained about four (HypA:kan)‐ and five (HypB:kan)‐fold less nickel than did the urease from wt, indicating that the lack of urease activity in these strains results from a nickel deficiency in the urease enzyme.

The diverse antioxidant systems of Helicobacter pylori

The gastric pathogen Helicobacter pylori induces a strong inflammatory host response, yet the bacterium maintains long‐term persistence in the host. H. pylori combats oxidative stress via a battery of diverse activities, some of which are unique or newly described. In addition to using the well‐studied bacterial oxidative stress resistance enzymes superoxide dismutase and catalase, H. pylori depends on a family of peroxiredoxins (alkylhydroperoxide reductase, bacterioferritin co‐migratory protein and a thiol‐peroxidase) that function to detoxify organic peroxides. Newly described antioxidant proteins include a soluble NADPH quinone reductase (MdaB) and an iron sequestering protein (NapA) that has dual roles – host inflammation stimulation and minimizing reactive oxygen species production within H. pylori. An H. pylori arginase attenuates host inflammation, a thioredoxin required as a reductant for many oxidative stress enzymes is also a chaperon, and some novel properties of KatA and AhpC were discovered. To repair oxidative DNA damage, H. pylori uses an endonuclease (Nth), DNA recombination pathways and a newly described type of bacterial MutS2 that specifically recognizes 8‐oxoguanine. A methionine sulphoxide reductase (Msr) plays a role in reducing the overall oxidized protein content of the cell, although it specifically targets oxidized Met residues. H. pylori possess few stress regulator proteins, but the key roles of a ferric uptake regulator (Fur) and a post‐transcriptional regulator CsrA in antioxidant protein expression are described. The roles of all of these antioxidant systems have been addressed by a targeted mutant analysis approach and almost all are shown to be important in host colonization. The described antioxidant systems in H. pylori are expected to be relevant to many bacterial‐associated diseases, as genes for most of the enzymes carrying out the newly described roles are present in a number of pathogenic bacteria.

Superoxide dismutase-deficient mutants of Helicobacter pylori are hypersensitive to oxidative stress and defective in host colonization.

ABSTRACT Superoxide dismutase (SOD) is a nearly ubiquitous enzyme among organisms that are exposed to oxic environments. The single SOD ofHelicobacter pylori, encoded by the sodB gene, has been suspected to be a virulence factor for this pathogenic microaerophile, but mutations in this gene have not been reported previously. We have isolated mutants with interruptions in thesodB gene and have characterized them with respect to their response to oxidative stress and ability to colonize the mouse stomach. The sodB mutants are devoid of SOD activity, based on activity staining in nondenaturing gels and quantitative assays of cell extracts. Though wild-type H. pylori is microaerophilic, the mutants are even more sensitive to O2 for both growth and viability. While the wild-type strain is routinely grown at 12% O2, growth of the mutant strains is severely inhibited at above 5 to 6% O2. The effect of O2 on viability was determined by subjecting nongrowing cells to atmospheric levels of O2 and plating for survivors at 2-h time intervals. Wild-type cell viability dropped by about 1 order of magnitude after 6 h, while viability of the sodBmutant decreased by more than 6 orders of magnitude at the same time point. The mutants are also more sensitive to H2O2, and this sensitivity is exacerbated by increased O2 concentrations. Since oxidative stress has been correlated with DNA damage, the frequency of spontaneous mutation to rifampin resistance was studied. The frequency of mutagenesis of ansodB mutant strain is about 15-fold greater than that of the wild-type strain. In the mouse colonization model, only 1 out of 23 mice inoculated with an SOD-deficient mutant of a mouse-adapted strain became H. pylori positive, while 15 out of 17 mice inoculated with the wild-type strain were shown to harbor the organism. Therefore, SOD is a virulence factor which affects the ability of this organism to colonize the mouse stomach and is important for the growth and survival of H. pylori under conditions of oxidative stress.

Characterization of Helicobacter pylori Nickel Metabolism Accessory Proteins Needed for Maturation of both Urease and Hydrogenase

Previous studies demonstrated that two accessory proteins, HypA and HypB, play a role in nickel-dependent maturation of both hydrogenase and urease in Helicobacter pylori. Here, the two proteins were purified and characterized. HypA bound two Ni(2+) ions per dimer with positive cooperativity (Hill coefficient, approximately 2.0). The dissociation constants K(1) and K(2) for Ni(2+) were 58 and 1.3 microM, respectively. Studies on purified site-directed mutant proteins in each of the five histidine residues within HypA, revealed that only one histidine residue (His2) is vital for nickel binding. Nuclear magnetic resonance analysis showed that this purified mutant version (H2A) was similar in structure to that of the wild-type HypA protein. A chromosomal site-directed mutant of hypA (in the codon for His2) lacked hydrogenase activity and possessed only 2% of the wild-type urease activity. Purified HypB had a GTPase activity of 5 nmol of GTP hydrolyzed per nmol of HypB per min. Site-directed mutagenesis within the lysine residue in the conserved GTP-binding motif of HypB (Lys59) nearly abolished the GTPase activity of the mutant protein (K59A). In native solution, both HypA and HypB exist as homodimers with molecular masses of 25.8 and 52.4 kDa, respectively. However, a 1:1 molar mixture of HypA plus HypB gave rise to a 43.6-kDa species composed of both proteins. A 43-kDa heterodimeric HypA-HypB complex was also detected by cross-linking. The cross-linked adduct was still observed in the presence of 0.5 mM GTP or 1 microM nickel or when the mutant version of HypA (altered in His2) and HypB (altered in Lys59) were tested. Individually, HypA and HypB formed homodimeric cross-linked adducts. An interaction between HypA and the Hp0868 protein (encoded by the gene downstream of hypA) could not be detected via cross-linking, although such an interaction was predicted by yeast two-hybrid studies. In addition, the phenotype of an insertional mutation within the Hp0868 gene indicated that its presence is not critical for either the urease or the hydrogenase activity.

An NADPH Quinone Reductase of Helicobacter pylori Plays an Important Role in Oxidative Stress Resistance and Host Colonization

ABSTRACT Oxidative stress resistance is one of the key properties that enable pathogenic bacteria to survive the toxic reactive oxygen species released by the host. In a previous study characterizing oxidative stress resistance mutants of Helicobacter pylori, a novel potential antioxidant protein (MdaB) was identified by the observation that the expression of this protein was significantly upregulated to compensate for the loss of other major antioxidant components. In this study, we characterized an H. pylori mdaB mutant and the MdaB protein. While the wild-type strain can tolerate 10% oxygen for growth, the growth of the mdaB mutant was significantly inhibited by this oxygen condition. The mdaB mutant is also more sensitive to H2O2, organic hydroperoxides, and the superoxide-generating agent paraquat. Although the wild-type strain can survive more than 10 h of air exposure, exposure of the mutant strain to air for 8 h resulted in recovery of no viable cells. The oxidative stress sensitivity of the mdaB mutant resulted in a deficiency in the ability to colonize mouse stomachs. H. pylori was recovered from 10 of 11 mouse stomachs inoculated with the wild-type strain, with about 5,000 to 45,000 CFU/g of stomach. However, only 3 of 12 mice that were inoculated with the mdaB mutant strain were found to harbor any H. pylori, and these 3 contained less than 2,000 CFU/g of stomach. A His-tagged MdaB protein was purified and characterized. It was shown to be a flavoprotein that catalyzes two-electron transfer from NAD(P)H to quinones. It reduces both ubiquinones and menaquinones with similar efficiencies and preferably uses NADPH as an electron donor. We propose that the physiological function of the H. pylori MdaB protein is that of an NADPH quinone reductase that plays an important role in managing oxidative stress and contributes to successful colonization of the host.

Oxidative-Stress Resistance Mutants of Helicobacter pylori

Within a large family of peroxidases, one member that catalyzes the reduction of organic peroxides to alcohols is known as alkyl hydroperoxide reductase, or AhpC. Gene disruption mutations in the gene encoding AhpC of Helicobacter pylori (ahpC) were generated by screening transformants under low-oxygen conditions. Two classes of mutants were obtained. Both types lack AhpC protein, but the major class (type I) isolated was found to synthesize increased levels (five times more than the wild type) of another proposed antioxidant protein, an iron-binding, neutrophil-activating protein (NapA). The other class of mutants, the minor class (type II), produced wild-type levels of NapA. The two types of AhpC mutants differed in their frequencies of spontaneous mutation to rifampin resistance and in their sensitivities to oxidative-stress chemicals, with the type I mutants exhibiting less sensitivity to organic hydroperoxides as well as having a lower mutation frequency. The napA promoter regions of the two types of AhpC mutants were identical, and primer extension analysis revealed their transcription start site to be the same as for the wild type. Gene disruption mutations were obtained in napA alone, and a double mutant strain (ahpC napA) was also created. All four of the oxidative-stress resistance mutants could be distinguished from the wild type in oxygen sensitivity or in some other oxidative-stress resistance phenotype (i.e., in sensitivity to stress-related chemicals and spontaneous mutation frequency). For example, growth of the NapA mutant was more sensitive to oxygen than that of the wild-type strain and both of the AhpC-type mutants were highly sensitive to paraquat and to cumene hydroperoxide. Of the four types of mutants, the double mutant was the most sensitive to growth inhibition by oxygen and by organic peroxides and it had the highest spontaneous mutation frequency. Notably, two-dimensional gel electrophoresis combined with protein sequence analysis identified another possible oxidative-stress resistance protein (HP0630) that was up-regulated in the double mutant. However, the transcription start site of the HP0630 gene was the same for the double mutant as for the wild type. It appears that H. pylori can readily modulate the expression of other resistance factors as a compensatory response to loss of a major oxidative-stress resistance component.

Contribution of the Helicobacter pylori Thiol Peroxidase Bacterioferritin Comigratory Protein to Oxidative Stress Resistance and Host Colonization

ABSTRACT Peroxiredoxins, the enzymes that catalyze the reduction of hydrogen peroxide and organic hydroperoxides, are ubiquitous proteins that protect organisms from damage by reactive oxygen species. Helicobacter pylori contains three members of the peroxiredoxin family: AhpC (alkyl hydroperoxide reductase), Tpx (thiol-specific peroxidase), and bacterioferritin comigratory protein (BCP). In this study, we characterized H. pylori bcp mutant strains and wild-type BCP. Compared to the parent strain and the ahpC mutant strain, the bcp mutant showed moderate sensitivity to the superoxide-generating agent paraquat and to organic hydroperoxides. Upon exposure of 108 cells to air for 10 h, 106 wild-type cells survived but none of the 108bcp mutant cells were recovered. Introduction of an intact bcp gene at an unrelated locus in the bcp strain restored the wild-type-like oxidative stress resistance phenotype. Purified BCP was shown to be a thiol peroxidase that depends on the reducing activity of thioredoxin and thioredoxin reductase. Among a series of peroxides tested, linoleic acid hydroperoxide was the preferred substrate of BCP. By examining the profiles of protein expression within H. pylori cells, we confirmed that AhpC is much more abundant than BCP. The overlapping functions and activities of BCP and AhpC probably explain why the bcp mutant displayed a relatively weak oxidative stress resistance phenotype. The bcp mutant strain could colonize mouse stomachs, although colonization by the wild-type strain was slightly better than that by the mutant strain at 1 week after host inoculation. However, at 3 weeks after inoculation, the colonization ability of the wild type was significantly greater than that of the bcp mutant; for example, H. pylori was recovered from 10 of 11 mouse stomachs inoculated with the wild-type strain but from only 4 of 12 mice that were inoculated with the bcp mutant strain. This indicates that H. pylori BCP plays a significant role in efficient host colonization.

Respiratory Hydrogen Use by Salmonella enterica Serovar Typhimurium Is Essential for Virulence

ABSTRACT Based on available annotated gene sequence information, the enteric pathogen salmonella, like other enteric bacteria, contains three putative membrane-associated H2-using hydrogenase enzymes. These enzymes split molecular H2, releasing low-potential electrons that are used to reduce quinone or heme-containing components of the respiratory chain. Here we show that each of the three distinct membrane-associated hydrogenases of Salmonella enterica serovar Typhimurium is coupled to a respiratory pathway that uses oxygen as the terminal electron acceptor. Cells grown in a blood-based medium expressed four times the amount of hydrogenase (H2 oxidation) activity that cells grown on Luria Bertani medium did. Cells suspended in phosphate-buffered saline consumed 2 mol of H2 per mol of O2 used in the H2-O2 respiratory pathway, and the activity was inhibited by the respiration inhibitor cyanide. Molecular hydrogen levels averaging over 40 μM were measured in organs (i.e., livers and spleens) of live mice, and levels within the intestinal tract (the presumed origin of the gas) were four times greater than this. The half-saturation affinity of S. enterica serovar Typhimurium for H2 is only 2.1 μM, so it is expected that H2-utilizing hydrogenase enzymes are saturated with the reducing substrate in vivo. All three hydrogenase enzymes contribute to the virulence of the bacterium in a typhoid fever-mouse model, based on results from strains with mutations in each of the three hydrogenase genes. The introduced mutations are nonpolar, and growth of the mutant strains was like that of the parent strain. The combined removal of all three hydrogenases resulted in a strain that is avirulent and (in contrast to the parent strain) one that is unable to invade liver or spleen tissue. The introduction of one of the hydrogenase genes into the triple mutant strain on a low-copy-number plasmid resulted in a strain that was able to both oxidize H2 and cause morbidity in mice within 11 days of inoculation; therefore, the avirulent phenotype of the triple mutant is not due to an unknown spurious mutation. We conclude that H2 utilization in a respiratory fashion is required for energy production to permit salmonella growth and subsequent virulence during infection.

Dual roles of Bradyrhizobium japonicum nickelin protein in nickel storage and GTP-dependent Ni mobilization.

The hydrogenase accessory protein HypB, or nickelin, has two functions in the N(2)-fixing, H(2)-oxidizing bacterium Bradyrhizobium japonicum. One function of HypB involves the mobilization of nickel into hydrogenase. HypB also carries out a nickel storage/sequestering function in B. japonicum, binding nine nickel ions per monomer. Here we report that the two roles (nickel mobilization and storage) of HypB can be separated in vitro and in vivo using molecular and biochemical approaches. The role of HypB in hydrogenase maturation is completely dependent on its intrinsic GTPase activity; strains which produce a HypB protein that is severely deficient in GTPase activity but that fully retains nickel-sequestering ability cannot produce active hydrogenase even upon prolonged nickel supplementation. A HypB protein that lacks the nickel-binding polyhistidine region near the N terminus lacks only the nickel storage capacity function; it is still able to bind a single nickel ion and also retains complete GTPase activity.

Association of Helicobacter pylori Antioxidant Activities with Host Colonization Proficiency

ABSTRACT To assess the importance of two separate antioxidant activities in Helicobacter pylori, we tested the abilities of strains with mutations in either tpx (encoding thiolperoxidase) or ahpC (encoding alkyl hydroperoxide reductase [AhpC]) to colonize the stomachs of mice. The tpx strain was clearly more sensitive than the parent strain to both oxygen and cumene hydroperoxide. The strain colonized only 5% of the inoculated mice. Two different classes of oxygen-sensitive ahpC mutants in the type strain (ATCC 43504) were recently described (A. A. Olczak, J. W. Olson, and R. J. Maier, J. Bacteriol. 184:3186-3193, 2002). The same two classes of mutants were recovered upon ahpC mutagenesis of the mouse-adapted strain, SS1. Neither of these mutants was able to colonize mouse stomachs, whereas 78% of the mice inoculated with the parent strain became H. pylori positive.