The Helicobacter pylori MutS protein confers protection from oxidative DNA damage

The human gastric pathogenic bacterium Helicobacter pylori lacks a MutSLH-like DNA mismatch repair system. Here, we have investigated the functional roles of a mutS homologue found in H. pylori, and show that it plays an important physiological role in repairing oxidative DNA damage. H. pylori mutS mutants are more sensitive than wild-type cells to oxidative stress induced by agents such as H2O2, paraquat or oxygen. Exposure of mutS cells to oxidative stress results in a significant ( approximately 10-fold) elevation of mutagenesis. Strikingly, most mutations in mutS cells under oxidative stress condition are G:C to T:A transversions, a signature of 8-oxoguanine (8-oxoG). Purified H. pylori MutS protein binds with a high specific affinity to double-stranded DNA (dsDNA) containing 8-oxoG as well as to DNA Holliday junction structures, but only weakly to dsDNA containing a G:A mismatch. Under oxidative stress conditions, mutS cells accumulate higher levels (approximately threefold) of 8-oxoG DNA lesions than wild-type cells. Finally, we observe that mutS mutant cells have reduced colonization capacity in comparison to wild-type cells in a mouse infection model.     

A histone-like protein of mycobacteria possesses ferritin superfamily protein-like activity and protects against DNA damage by Fenton reaction

Iron is an essential metal for living organisms but its level must be strictly controlled in cells, because ferrous ion induces toxicity by generating highly active reactive oxygen, hydroxyl radicals, through the Fenton reaction. In addition, ferric ion shows low solubility under physiological conditions. To overcome these obstacles living organisms possess Ferritin superfamily proteins that are distributed in all three domains of life: bacteria, archaea, and eukaryotes. These proteins minimize hydroxyl radical formation by ferroxidase activity that converts Fe(2+) into Fe(3+) and sequesters iron by storing it as a mineral inside a protein cage. In this study, we discovered that mycobacterial DNA-binding protein 1 (MDP1), a histone-like protein, has similar activity to ferritin superfamily proteins. MDP1 prevented the Fenton reaction and protects DNA by the ferroxidase activity. The K(m) values of the ferroxidase activity by MDP1 of Mycobacterium bovis bacillus Calmette-Guérin (BCG-3007c), Mycobacterium tuberculosis (Rv2986c), and Mycobacterium leprae (ML1683; ML-LBP) were 0.292, 0.252, and 0.129 mM, respectively. Furthermore, one MDP1 molecule directly captured 81.4±19.1 iron atoms, suggesting the role of this protein in iron storage. This study describes for the first time a ferroxidase-iron storage protein outside of the ferritin superfamily proteins and the protective role of this bacterial protein from DNA damage.     

Adherence protects the binding sites of Helicobacter pylori urease from acid-induced damage

Colonization by Helicobacter pylori partly depends on acid-dependent adherence by urease to gastric mucin. To further verify the relevance of urease adherence to colonization, the influence of acidity on the binding sites of H. pylori urease was investigated. When enzyme-based in vitro ligand capture assays were used, the effect of acidity on the binding site of H. pylori urease was determined against a backdrop medium consisting of acidic buffers simulating the luminal side of gastric mucus. A high degree of stability was exhibited by adherent urease, suggesting a pivotal role by the denatured enzyme in the persistence of the bacterium within the acidified compartment of gastric mucus.     

Oxidative stress defense mechanisms to counter iron-promoted DNA damage in Helicobacter pylori

Iron, a key element in Fenton chemistry, causes oxygen-related toxicity to cells of most living organisms. Helicobacter pylori is a microaerophilic bacterium that infects human gastric mucosa and causes a series of gastric diseases. Exposure of H. pylori cells to air for 2 h elevated the level of free iron by about 4-fold as measured by electron paramagnetic resonance spectroscopy. H. pylori cells accumulated more free iron as they approached stationary phase growth, and they concomitantly suffered more DNA damage as indicated by DNA fragmentation analysis. Relationships between the intracellular free iron level, specific oxidative stress enzymes, and DNA damage were identified, and new roles for three oxidative stress-combating enzymes in H. pylori are proposed. Mutant cells defective in either catalase (KatA), in superoxide dismutase (SodB) or in alkyl hydroperoxide reductase (AhpC) were more sensitive to oxidative stress conditions; and they accumulated more free (toxic) iron; and they suffered more DNA fragmentation compared to wild type cells. A significant proportion of cells of sodB, ahpC, or katA mutant strains developed into the stress-induced coccoid form or lysed; they also contained significantly higher amounts of 8-oxo-guanine associated with their DNA, compared to wild type cells.     

Werner protein protects nonproliferating cells from oxidative DNA damage

Werner syndrome, caused by mutations of the WRN gene, mimics many changes of normal aging. Although roles for WRN protein in DNA replication, recombination, and telomere maintenance have been suggested, the pathology of rapidly dividing cells is not a feature of Werner syndrome. To identify cellular events that are specifically vulnerable to WRN deficiency, we used RNA interference (RNAi) to knockdown WRN or BLM (the RecQ helicase mutated in Bloom syndrome) expression in primary human fibroblasts. Withdrawal of WRN or BLM produced accelerated cellular senescence phenotype and DNA damage response in normal fibroblasts, as evidenced by induction of gammaH2AX and 53BP1 nuclear foci. After WRN depletion, the induction of these foci was seen most prominently in nondividing cells. Growth in physiological (3%) oxygen or in the presence of an antioxidant prevented the development of the DNA damage foci in WRN-depleted cells, whereas acute oxidative stress led to inefficient repair of the lesions. Furthermore, WRN RNAi-induced DNA damage was suppressed by overexpression of the telomere-binding protein TRF2. These conditions, however, did not prevent the DNA damage response in BLM-ablated cells, suggesting a distinct role for WRN in DNA homeostasis in vivo. Thus, manifestations of Werner syndrome may reflect an impaired ability of slowly dividing cells to limit oxidative DNA damage.     

A novel apoptosis-inducing protein from Helicobacter pylori

Helicobacter pylori infection induces apoptosis in gastric epithelial cells. Here, we report a novel apoptosis-inducing protein that functions as a leading factor in H. pylori-mediated apoptosis induction. We purified the protein from H. pylori by separating fractions that showed apoptosis-inducing activity. This protein induced apoptosis of AGS cells in a dose-dependent manner. The purified protein consisted of two protein fragments with molecular masses of about 40 and 22 kDa, which combined to constitute a single complex in their natural form. N-terminal sequencing indicated that both these protein fragments were encoded by the HP1118 gene. The purified protein exhibited gamma-glutamyl transpeptidase activity, the inhibition of which by 6-diazo-5-oxo-l-norleucine resulted in a complete loss of apoptosis-inducing activity. To the best of our knowledge, the apoptosis-inducing function is a newly identified physiological role for bacterial gamma-glutamyl transpeptidase. The apoptosis-inducing activity of the isogenic mutant gamma-glutamyl transpeptidase-deficient strain was significantly lower compared with that of the parent strain, demonstrating that gamma-glutamyl transpeptidase plays a significant role in H. pylori-mediated apoptosis. Our findings provide new insights into H. pylori pathogenicity and reveal a novel aspect of the bacterial gamma-glutamyl transpeptidase function.     

Nucleosomal histone protein protects DNA from iron-mediated damage.

Iron promotes DNA damage by catalyzing hydroxyl radical formation. We examined the effect of chromatin structure on DNA susceptibility to oxidant damage. Oxygen radicals generated by H2O2, ascorbate and iron-ADP (1:2 ratio of Fe2+:ADP) extensively and randomly fragmented protein-free DNA, with double-strand breaks demonstrable even at 1 microM iron. In contrast, polynucleosomes from chicken erythrocytes were converted to nucleosome-sized fragments by iron-ADP even up to 250 microM iron. Cleavage occurred only in bare areas where DNA is unassociated with histone. In confirmation, reassembly of nucleosomes from calf thymus DNA and chicken erythrocyte histone also yielded nucleosomes resistant to fragmentation. Protection of DNA by histone was dependent on nucleosome assembly and did not simply reflect presence of scavenging protein. In contrast to this specific cleavage of internucleosomal linker DNA by iron-ADP, iron-EDTA cleaved polynucleosomes indiscriminately at all sites. The hydroxyl radical scavenger thiourea completely inhibited the random cleavage of polynucleosomes by iron-EDTA but inhibited the nonrandom cleavage of polynucleosomes by iron-ADP less completely, suggesting the possibility that the lower affinity iron-ADP chelate may allow association of free iron with DNA. Thus, oxygen radicals generated by iron-ADP indiscriminately cleaved naked DNA but cleaved chromatin preferentially at internucleosomal bare linker sites, perhaps because of nonrandom iron binding by DNA. These findings suggest that the DNA-damaging effects of iron may be nonrandom, site-directed and modified by histone protein.     

Pathogenesis and host response of Helicobacter pylori

The 5th International Workshop on Pathogenesis and Host Response in Helicobacter Infections was held in Elsinore, Denmark, 4–7 July, 2002.     

Cellular prion protein protects against reactive-oxygen-species-induced DNA damage

Although the cellular form of the prion protein (PrPC) is critical for the development of prion disease through its conformational conversion into the infectious form (PrPSc), the physiological role of PrPC is less clear. Using alkaline single-cell gel electrophoresis (the Comet assay), we show that expression of PrPC protects human neuroblastoma SH-SY5Y cells against DNA damage under basal conditions and following exposure to reactive oxygen species, either hydroxyl radicals following exposure to Cu2+ or Fe2+ or singlet oxygen following exposure to the photosensitizer methylene blue and white light. Cells expressing either PrPDeltaoct which lacks the octapeptide repeats or the prion-disease-associated mutants A116V or PG14 had increased levels of DNA damage compared to cells expressing PrPC. In PrPSc-infected mouse ScN2a cells there was a significant increase in DNA damage over noninfected N2a cells (median tail DNA 2.87 and 7.33%, respectively). Together, these data indicate that PrPC has a critical role to play in protecting cells against reactive-oxygen-species-mediated DNA damage; a function which requires the octapeptide repeats in the protein, is lost in disease-associated mutants of the protein or upon conversion to PrPSc, and thus provide further support for the neuroprotective role for PrPC.     

DNA binding specificity of the replication initiator protein, DnaA from Helicobacter pylori

The key protein in the initiation of Helicobacter pylori chromosome replication, DnaA, has been characterized. The amount of the DnaA protein was estimated to be approximately 3000 molecules per single cell; a large part of the protein was found in the inner membrane. The H.pylori DnaA protein has been analysed using in vitro (gel retardation assay and surface plasmon resonance (SPR)) as well as in silico (comparative computer modeling) studies. DnaA binds a single DnaA box as a monomer, while binding to the fragment containing several DnaA box motifs, the oriC region, leads to the formation of high molecular mass nucleoprotein complexes. In comparison with the Escherichia coli DnaA, the H.pylori DnaA protein exhibits lower DNA-binding specificity; however, it prefers oriC over non-box DNA fragments. As determined by gel retardation techniques, the H.pylori DnaA binds with a moderate level of affinity to its origin of replication (4nM). Comparative computer modelling showed that there are nine residues within the binding domain which are possible determinants of the reduced H.pylori DnaA specificity. Of these, the most interesting is probably the triad PTL; all three residues show significant divergence from the consensus, and Thr398 is the most divergent residue of all.     

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.     

The cysteine-rich protein A from Helicobacter pylori is a beta-lactamase

Among the large number of hypothetical proteins within the genomes of Helicobacter pylori, there is a family of unique and highly disulfide-bridged proteins, designated family 12, for which no function could originally be assigned. Sequence analysis revealed that members of this family possess a modular architecture of alpha/beta-units and a stringent pattern of cysteine residues. The H. pylori cysteine-rich protein A (HcpA), which is a member of this family, was expressed and refolded from inclusion bodies. Six pairs of cysteine residues, which are separated by exactly seven residues, form disulfide bridges. HcpA is a beta-lactamase. It slowly hydrolyzes 6-aminopenicillinic acid and 7-aminocephalosporanic acid (ACA) derivatives. The turnover for 6-aminopenicillinic acid derivatives is 2-3 times greater than for ACA derivatives. The enzyme is efficiently inhibited by cloxacillin and oxacillin but not by ACA derivatives or metal chelators. We suggest that all family 12 members possess similar activities and might be involved in the synthesis of the cell wall peptidoglycan. They might also be responsible for amoxicillin resistance of certain H. pylori strains.     

Role of a MutY DNA glycosylase in combating oxidative DNA damage in Helicobacter pylori

MutY is an adenine glycosylase that has the ability to efficiently remove adenines from adenine/7,8-dihydro-8-oxoguanine (8-oxo-G) or adenine/guanine mismatches, and plays an important role in oxidative DNA damage repair. The human gastric pathogen Helicobacter pylori has a homolog of the MutY enzyme. To investigate the physiological roles of MutY in H. pylori, we constructed and characterized a mutY mutant. H. pylori mutY mutants incubated at 5% O2 have a 325-fold higher spontaneous mutation rate than its parent. The mutation rate is further increased by exposing the mutant to atmospheric levels of oxygen, an effect that is not seen in an E. coli mutY mutant. Most of the mutations that occurred in H. pylori mutY mutants, as examined by rpoB sequence changes that confer rifampicin resistance, are GC to TA transversions. The H. pylori enzyme has the ability to complement an E. coli mutY mutant, restoring its mutation frequency to the wild-type level. Pure H. pylori MutY has the ability to remove adenines from A/8-oxo-G mismatches, but strikingly no ability to cleave A/G mismatches. This is surprising because E. coli MutY can more rapidly turnover A/G than A/8-oxo-G. Thus, H. pylori MutY is an adenine glycosylase involved in the repair of oxidative DNA damage with a specificity for detecting 8-oxo-G. In addition, H. pylori mutY mutants are only 30% as efficient as wild-type in colonizing the stomach of mice, indicating that H. pylori MutY plays a significant role in oxidative DNA damage repair in vivo.     

8-Oxoguanine induces intramolecular DNA damage but free 8-oxoguanine protects intermolecular DNA from oxidative stress

7,8-Dihydro-8-oxoguanine (8-oxoguanine; 8-oxo-G), one of the major oxidative DNA adducts, is highly susceptible to further oxidation by radicals. We confirmed the higher reactivity of 8-oxo-G toward reactive oxygen (singlet oxygen and hydroxyl radical) or nitrogen (peroxynitrite) species as compared to unmodified base. In this study, we raised the question about the effect of this high reactivity toward radicals on intramolecular and intermolecular DNA damage. We found that the amount of intact nucleoside in oligodeoxynucleotide containing 8-oxo-G decreased more by various radicals at higher levels of 8-oxo-G incorporation, and that the oligodeoxynucleotide damage and plasmid cleavage by hydroxyl radical were inhibited in the presence of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG). We conclude that 8-oxo-G within DNA induces intramolecular DNA base damage, but that free 8-oxo-G protects intermolecular DNA from oxidative stress. These results suggest that 8-oxo-G within DNA must be rapidly released to protect DNA from overall oxidative damage.     

Structure of the neutrophil-activating protein from Helicobacter pylori

Helicobacter pylori is a major human pathogen associated with severe gastroduodenal diseases, including ulcers and cancers. An H.pylori protein that is highly immunogenic in humans and mice has been identified recently. This protein has been termed HP-NAP, due to its ability of activating neutrophils. In order to achieve a molecular understanding of its unique immunogenic and pro-inflammatory properties, we have determined its three-dimensional structure. Its quaternary structure is similar to that of the dodecameric bacterial ferritins (Dps-like family), but it has a different surface potential charge distribution. This is due to the presence of a large number of positively charged residues, which could well account for its unique ability in activating human leukocytes.     

Carbohydrate-dependent inhibition of Helicobacter pylori colonization using porcine milk

Breast milk has a well-known anti-microbial effect, which is in part due to the many different carbohydrate structures expressed. This renders it a position as a potential therapeutic for treatment of infection by different pathogens, thus avoiding the drawbacks of many antibiotics. In a previous study, we showed that pigs express the Helicobacter pylori receptors, sialyl Lewis x (Le x) and Le b, on various milk proteins. Here, we investigate the pig breed- and individual-specific expression of these epitopes, as well as the inhibitory capacity of porcine milk on H. pylori binding and colonization. Milk proteins from three different pig breeds were analysed by western blotting using antibodies with known carbohydrate specificity. An adhesion assay was used to investigate the capacity of pig milk to inhibit H. pylori binding to neoglycoproteins carrying Le b and sialyl-di-Le x. alpha1,3/4-fucosyltransferase transgenic FVB/N mice, known to express Le b and sialyl Le x in their gastric epithelium, were colonized by H. pylori and were subsequently treated with Le b- and sialyl Le x-expressing or nonexpressing porcine milk, or water (control) only. The degree of H. pylori colonization in the different treatment groups was quantified. The expression of the Le b and sialyl Le x carbohydrate epitopes on pig milk proteins was breed- and individual specific and correlated to the ability of porcine milk to inhibit H. pylori adhesion in vitro and H. pylori colonization in vivo. Milk from certain pig breeds may have a therapeutic and/or prophylactic effect on H. pylori infection.     

Purification and characterization of protein methylase II from Helicobacter pylori

Protein methylase II (AdoMet:protein-carboxyl O-methyltransferase, EC 2.1.1.24) was identified and purified 115-fold from Helicobacter pylori through Q-Sepharose ion exchange column, AdoHcy-Sepharose 4B column, and Superdex 200 HR column chromatography using FPLC. The purified preparation showed two protein bands of about 78 kDa and 29 kDa molecular mass on SDS-PAGE. On non-denaturing gel electrophoresis, the enzyme migrated as a single band with a molecular mass of 410 kDa. In addition, MALDI-TOF-MS analysis and Superdex 200 HR column chromatography of the purified enzyme showed a major mass signal with molecular mass values of 425 kDa and 430 kDa, respectively. Therefore, the above results led us to suggest that protein methylase II purified from H. pylori is composed of four heterodimers with 425 kDa (4x(78+29)=428 kDa). This magnitude of molecular mass is unusual for protein methylases II so far reported. The enzyme has an optimal pH of 6.0, a K(m) value of 5.0x10(-6) M for S-adenosyl-L-methionine and a V(max) of 205 pmol methyl-(14)C transferred min(-1) mg(-1) protein.     

DNA-binding activity of TNF-alpha inducing protein from Helicobacter pylori

Tumor necrosis factor-alpha (TNF-alpha) inducing protein (Tipalpha) is a carcinogenic factor secreted from Helicobacter pylori (H. pylori), mediated through both enhanced expression of TNF-alpha and chemokine genes and activation of nuclear factor-kappaB. Since Tipalpha enters gastric cancer cells, the Tipalpha binding molecules in the cells should be investigated. The direct DNA-binding activity of Tipalpha was observed by pull down assay using single- and double-stranded genomic DNA cellulose. The surface plasmon resonance assay, indicating an association between Tipalpha and DNA, revealed that the affinity of Tipalpha for (dGdC)10 is 2400 times stronger than that of del-Tipalpha, an inactive Tipalpha. This suggests a strong correlation between DNA-binding activity and carcinogenic activity of Tipalpha. And the DNA-binding activity of Tipalpha was first demonstrated with a molecule secreted from H. pylori.