Helicobacter pylori Genes Involved in Avoidance of Mutations Induced by 8-Oxoguanine

ABSTRACT Chromosomal rearrangements and base substitutions contribute to the large intraspecies genetic diversity of Helicobacter pylori. Here we explored the base excision repair pathway for the highly mutagenic 8-oxo-7,8-dihydroguanine (8-oxoG), a ubiquitous form of oxidized guanine. In most organisms, 8-oxoG is removed by a specific DNA glycosylase (Fpg in bacteria or OGG1 in eukaryotes). In the case where replication of the lesion yields an A/8-oxoG base pair, a second DNA glycosylase (MutY) can excise the adenine and thus avoid the fixation of the mutation in the next round of replication. In a genetic screen for H. pylori genes complementing the hypermutator phenotype of an Escherichia coli fpg mutY strain, open reading frame HP0142, a putative MutY coding gene, was isolated. Besides its capacity to complement E. coli mutY strains, HP0142 expression resulted in a strong adenine DNA glycosylase activity in E. coli mutY extracts. Consistently, the purified protein also exhibited such an activity. Inactivation of HP0142 in H. pylori resulted in an increase in spontaneous mutation frequencies. An Mg-dependent AP (abasic site) endonuclease activity, potentially allowing the processing of the abasic site resulting from H. pylori MutY activity, was detected in H. pylori cell extracts. Disruption of HP1526, a putative xth homolog, confirmed that this gene is responsible for the AP endonuclease activity. The lack of evidence for an Fpg/OGG1 functional homolog is also discussed.

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Antimutator Role of the DNA Glycosylase mutY Gene in Helicobacter pylori

Journal of Bacteriology ◽

10.1128/jb.00477-06 ◽

2006 ◽

Vol 188 (17) ◽

pp. 6224-6234 ◽

Cited By ~ 29

Author(s):

Shuyan Huang ◽

Josephine Kang ◽

Martin J. Blaser

Keyword(s):

Helicobacter Pylori ◽

Base Excision Repair ◽

Excision Repair ◽

Spontaneous Mutation ◽

Phase Variable ◽

Wild Type ◽

Recombination Rates ◽

Base Excision ◽

H Pylori

ABSTRACT Helicobacter pylori has a highly variable genome with ongoing diversification via inter- and intragenomic recombination and spontaneous mutation. DNA repair genes modulating mutation and recombination rates that influence diversification have not been well characterized for H. pylori. To examine the role of putative base excision repair ung and mutY glycosylase and xthA apurinic/apyrimidinic endonuclease genes in H. pylori, mutants of each were constructed in strain JP26 by allelic exchange. Spontaneous mutation frequencies of JP26 mutY mutants, assessed by rifampin resistance, were consistently higher (26-fold) than that of the wild type, whereas the ung and xthA mutants showed smaller increases. In trans complementation of the JP26 mutY mutant restored spontaneous mutation frequencies to wild-type levels. In cross-species studies, H. pylori mutY complemented an Escherichia coli mutY mutant and vice versa. In contrast, the ung and mutY mutants did not show higher frequencies of intergenomic recombination or greater sensitivity to UV-induced DNA damage than the wild type. The H. pylori mutY open reading frame contains an eight-adenine homonucleotide tract; we provide evidence that this is subject to slipped-strand mispairing, leading to frameshifts that eliminate gene function. Our findings indicate that H. pylori possesses phase-variable base excision repair, consistent with a tension between repair and mutation.

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Stimulation of DNA Glycosylase Activities by XPC Protein Complex: Roles of Protein-Protein Interactions

Journal of Nucleic Acids ◽

10.4061/2010/805698 ◽

2010 ◽

Vol 2010 ◽

pp. 1-12 ◽

Cited By ~ 14

Author(s):

Yuichiro Shimizu ◽

Yasuhiro Uchimura ◽

Naoshi Dohmae ◽

Hisato Saitoh ◽

Fumio Hanaoka ◽

...

Keyword(s):

Protein Interactions ◽

Molecular Mechanisms ◽

Excision Repair ◽

Dna Glycosylase ◽

Abasic Site ◽

Dna Glycosylases ◽

Protein Protein Interactions ◽

E Coli ◽

Base Excision ◽

Stimulation Of

We showed that XPC complex, which is a DNA damage detector for nucleotide excision repair, stimulates activity of thymine DNA glycosylase (TDG) that initiates base excision repair. XPC appeared to facilitate the enzymatic turnover of TDG by promoting displacement from its own product abasic site, although the precise mechanism underlying this stimulation has not been clarified. Here we show that XPC has only marginal effects on the activity ofE. coliTDG homolog (EcMUG), which remains bound to the abasic site like human TDG but does not significantly interacts with XPC. On the contrary, XPC significantly stimulates the activities of sumoylated TDG and SMUG1, both of which exhibit quite different enzymatic kinetics from unmodified TDG but interact with XPC. These results point to importance of physical interactions for stimulation of DNA glycosylases by XPC and have implications in the molecular mechanisms underlying mutagenesis and carcinogenesis in XP-C patients.

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Antimutator Role of DNA Glycosylase MutY in Pathogenic Neisseria Species

Journal of Bacteriology ◽

10.1128/jb.187.8.2801-2809.2005 ◽

2005 ◽

Vol 187 (8) ◽

pp. 2801-2809 ◽

Cited By ~ 30

Author(s):

T. Davidsen ◽

M. Bjørås ◽

E. C. Seeberg ◽

T. Tønjum

Keyword(s):

Excision Repair ◽

Spontaneous Mutation ◽

Dna Glycosylase ◽

Wild Type ◽

Spontaneous Mutation Rate ◽

Neisseria Species ◽

E Coli ◽

Base Excision

ABSTRACT Genome alterations due to horizontal gene transfer and stress constantly generate strain on the gene pool of Neisseria meningitidis, the causative agent of meningococcal (MC) disease. The DNA glycosylase MutY of the base excision repair pathway is involved in the protection against oxidative stress. MC MutY expressed in Escherichia coli exhibited base excision activity towards DNA substrates containing A:7,8-dihydro-8-oxo-2′-deoxyguanosine and A:C mismatches. Expression in E. coli fully suppressed the elevated spontaneous mutation rate found in the E. coli mutY mutant. An assessment of MutY activity in lysates of neisserial wild-type and mutY mutant strains showed that both MC and gonococcal (GC) MutY is expressed and active in vivo. Strikingly, MC and GC mutY mutants exhibited 60- to 140-fold and 20-fold increases in mutation rates, respectively, compared to the wild-type strains. Moreover, the differences in transitions and transversions in rpoB conferring rifampin resistance observed with the wild type and mutants demonstrated that the neisserial MutY enzyme works in preventing GC→AT transversions. These findings are important in the context of models linking mutator phenotypes of disease isolates to microbial fitness.

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Impact of Helicobacter pylori Infection and Its Major Virulence Factor CagA on DNA Damage Repair

Microorganisms ◽

10.3390/microorganisms8122007 ◽

2020 ◽

Vol 8 (12) ◽

pp. 2007

Author(s):

Eleftherios Kontizas ◽

Spyros Tastsoglou ◽

Timokratis Karamitros ◽

Yiannis Karayiannis ◽

Panagoula Kollia ◽

...

Keyword(s):

Dna Damage ◽

Helicobacter Pylori ◽

Virulence Factor ◽

Excision Repair ◽

Dna Damage Repair ◽

Helicobacter Pylori Infection ◽

Dna Glycosylase ◽

Damage Repair ◽

Increased Risk ◽

H Pylori

Helicobacter pylori infection induces a plethora of DNA damages. Gastric epithelial cells, in order to maintain genomic integrity, require an integrous DNA damage repair (DDR) machinery, which, however, is reported to be modulated by the infection. CagA is a major H. pylori virulence factor, associated with increased risk for gastric carcinogenesis. Its pathogenic activity is partly regulated by phosphorylation on EPIYA motifs. Our aim was to identify effects of H. pylori infection and CagA on DDR, investigating the transcriptome of AGS cells, infected with wild-type, ΔCagA and EPIYA-phosphorylation-defective strains. Upon RNA-Seq-based transcriptomic analysis, we observed that a notable number of DDR genes were found deregulated during the infection, potentially resulting to base excision repair and mismatch repair compromise and an intricate deregulation of nucleotide excision repair, homologous recombination and non-homologous end-joining. Transcriptome observations were further investigated on the protein expression level, utilizing infections of AGS and GES-1 cells. We observed that CagA contributed to the downregulation of Nth Like DNA Glycosylase 1 (NTHL1), MutY DNA Glycosylase (MUTYH), Flap Structure-Specific Endonuclease 1 (FEN1), RAD51 Recombinase, DNA Polymerase Delta Catalytic Subunit (POLD1), and DNA Ligase 1 (LIG1) and, contrary to transcriptome results, Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APE1) upregulation. Our study accentuates the role of CagA as a significant contributor of H. pylori infection-mediated DDR modulation, potentially disrupting the balance between DNA damage and repair, thus favoring genomic instability and carcinogenesis.

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Development of an Internal Control for Evaluation and Standardization of a Quantitative PCR Assay for Detection of Helicobacter pylori in Drinking Water

Applied and Environmental Microbiology ◽

10.1128/aem.00687-07 ◽

2007 ◽

Vol 73 (22) ◽

pp. 7380-7387 ◽

Cited By ~ 20

Author(s):

Keya Sen ◽

Nancy A. Schable ◽

Dennis J. Lye

Keyword(s):

Helicobacter Pylori ◽

Drinking Water ◽

Quantitative Pcr ◽

Water Samples ◽

Sodium Thiosulfate ◽

Qpcr Assay ◽

E Coli ◽

H Pylori ◽

Labeled Probe ◽

Treated Drinking Water

ABSTRACT Due to metabolic and morphological changes that can prevent Helicobacter pylori cells in water from growing on conventional media, an H. pylori-specific TaqMan quantitative PCR (qPCR) assay was developed that uses a 6-carboxyfluorescein-labeled probe (A. E. McDaniels, L. Wymer, C. Rankin, and R. Haugland, Water Res. 39:4808-4816, 2005). However, proper internal controls are needed to provide an accurate estimate of low numbers of H. pylori in drinking water. In this study, the 135-bp amplicon described by McDaniels et al. was modified at the probe binding region, using PCR mutagenesis. The fragment was incorporated into a single-copy plasmid to serve as a PCR-positive control and cloned into Escherichia coli to serve as a matrix spike. It was shown to have a detection limit of five copies, using a VIC dye-labeled probe. A DNA extraction kit was optimized that allowed sampling of an entire liter of water. Water samples spiked with the recombinant E. coli cells were shown to behave like H. pylori cells in the qPCR assay. The recombinant E. coli cells were optimized to be used at 10 cells/liter of water, where they were shown not to compete with 5 to 3,000 cells of H. pylori in a duplex qPCR assay. Four treated drinking water samples spiked with H. pylori (100 cells) demonstrated similar cycle threshold values if the chlorine disinfectant was first neutralized by sodium thiosulfate.

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Antibacterial Activity of Geraniol in Combination with Standard Antibiotics against Staphylococcus aureus, Escherichia coli and Helicobacter pylori

Natural Product Communications ◽

10.1177/1934578x1801300701 ◽

2018 ◽

Vol 13 (7) ◽

pp. 1934578X1801300 ◽

Cited By ~ 1

Author(s):

Subrat Kumar Bhattamisra ◽

Chew Hui Kuean ◽

Lee Boon Chieh ◽

Vivian Lee Yean Yan ◽

Chin Koh Lee ◽

...

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Helicobacter Pylori ◽

Antibacterial Activity ◽

Synergistic Effect ◽

Inhibitory Concentration ◽

Therapeutic Potential ◽

E Coli ◽

Checkerboard Assay ◽

H Pylori

The antibacterial activity of geraniol and its effect in combination with ampicillin, amoxicillin and clarithromycin against Staphylococcus aureus, Escherichia coli and Helicobacter pylori was tested. The minimum inhibitory concentrations (MICs) and combinatory effects of geraniol against the bacteria were assessed by using the modified broth microdilution and checkerboard assay, respectively. The combinatory effect is expressed as fractional inhibitory concentration index (FICI). The MIC of geraniol against S. aureus, E. coli and H. pylori was found to be 11200, 5600, and 7325 μg/mL, respectively. A significant synergistic effect was observed with geraniol and ampicillin against S. aureus with FICI in the range 0.19 to 0.32. Geraniol and ampicillin exhibited a partial synergistic effect against E. coli. A similar effect was observed with geraniol and clarithromycin against S. aureus. A partial synergistic effect was observed with clarithromycin and geraniol against H. pylori with the FICI value in the range 0.86 to 0.89. An additive effect was observed with geraniol and amoxicillin combination against H. pylori. However, the amoxicillin and clarithromycin dose was reduced by thirty-two fold when combined with geraniol against H. pylori. The anti- H. pylori effect of geraniol with clarithromycin and amoxicillin could be of potential interest in the treatment of H. pylori infection and associated ulcers in humans. Further, geraniol, in combination with other antibiotics, has substantial therapeutic potential against S. aureus and E.coli infection.

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MSH2–MSH6 stimulates DNA polymerase η, suggesting a role for A:T mutations in antibody genes

Journal of Experimental Medicine ◽

10.1084/jem.20042066 ◽

2005 ◽

Vol 201 (4) ◽

pp. 637-645 ◽

Cited By ~ 136

Author(s):

Teresa M. Wilson ◽

Alexandra Vaisman ◽

Stella A. Martomo ◽

Patsa Sullivan ◽

Li Lan ◽

...

Keyword(s):

Dna Polymerase ◽

Base Excision Repair ◽

Somatic Hypermutation ◽

Excision Repair ◽

Cytidine Deaminase ◽

Abasic Site ◽

Cell Extracts ◽

Activation Induced Cytidine Deaminase ◽

Base Excision

Activation-induced cytidine deaminase deaminates cytosine to uracil (dU) in DNA, which leads to mutations at C:G basepairs in immunoglobulin genes during somatic hypermutation. The mechanism that generates mutations at A:T basepairs, however, remains unclear. It appears to require the MSH2–MSH6 mismatch repair heterodimer and DNA polymerase (pol) η, as mutations of A:T are decreased in mice and humans lacking these proteins. Here, we demonstrate that these proteins interact physically and functionally. First, we show that MSH2–MSH6 binds to a U:G mismatch but not to other DNA intermediates produced during base excision repair of dUs, including an abasic site and a deoxyribose phosphate group. Second, MSH2 binds to pol η in solution, and endogenous MSH2 associates with the pol in cell extracts. Third, MSH2–MSH6 stimulates the catalytic activity of pol η in vitro. These observations suggest that the interaction between MSH2–MSH6 and DNA pol η stimulates synthesis of mutations at bases located downstream of the initial dU lesion, including A:T pairs.

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Protection against Helicobacter pylori infection in mice by intragastric vaccination with H. pylori antigens is achieved using a non-toxic mutant of E. coli heat-labile enterotoxin (LT) as adjuvant

Vaccine ◽

10.1016/s0264-410x(97)00153-9 ◽

1998 ◽

Vol 16 (1) ◽

pp. 33-37 ◽

Cited By ~ 127

Author(s):

Marta Marchetti ◽

Michela Rossi ◽

Valentina Giannelli ◽

Marzia M Giuliani ◽

Mariagrazia Pizza ◽

...

Keyword(s):

Helicobacter Pylori ◽

Helicobacter Pylori Infection ◽

E Coli ◽

Heat Labile ◽

H Pylori

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Bacterial killing in gastric juice – effect of pH and pepsin on Escherichia coli and Helicobacter pylori

Journal of Medical Microbiology ◽

10.1099/jmm.0.46611-0 ◽

2006 ◽

Vol 55 (9) ◽

pp. 1265-1270 ◽

Cited By ~ 59

Author(s):

H. Zhu ◽

C. A. Hart ◽

D. Sales ◽

N. B. Roberts

Keyword(s):

Escherichia Coli ◽

Helicobacter Pylori ◽

Gastric Juice ◽

Bacterial Killing ◽

E Coli ◽

Test Organisms ◽

H Pylori ◽

Ph Range ◽

K 12 ◽

Rough Mutant

The susceptibility of Escherichia coli and Helicobacter pylori to pH and the effect of pepsin-mediated proteolysis were investigated. This was to establish the relative importance of their bacterial killing properties in gastric juice. Solutions in the pH range 1.5–7.4 with or without pig pepsin A were used, together with seven gastric juice samples obtained from patients undergoing routine gastric collection. Escherichia coli C690 (a capsulate strain), E. coli K-12 (a rough mutant) and Helicobacter pylori E5 were selected as the test organisms. Suspensions of bacteria (1×106 E. coli ml−1 and 1×108 H. pylori ml−1) were pre-incubated with test solutions at 37 °C for up to 2 h, and then cultured to establish the effect on subsequent growth. Survival of bacteria was diminished at pHs of less than 3.5, whereas killing required a pH of less than 2.5. Pre-incubation with pig pepsin at 0.5, 1.0 and 2.0 mg ml−1 at pH 3.5 reduced viable counts by 100 % for E. coli 690 and E. coli K-12 after 100 min incubation. With H. pylori, the viable counts decreased to 50 % of the control after 20 min incubation in 1 mg pepsin ml−1 at pH 2.5, 3.0 and 3.5. The gastric juices showed bactericidal activity at pH 3.5, and the rate of killing was juice dependent, with complete death of E. coli 690 occurring between 5 and 40 min post-incubation. Thus, killing of E. coli and H. pylori occurs optimally at pHs of less than 2.5. At pH 3.5, little effect is observed, whereas addition of pepsin alone or in gastric juice causes a marked increase in bacterial susceptibility, suggesting an important role for proteolysis in the killing of bacteria.

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16S rRNA Mutations That Confer Tetracycline Resistance in Helicobacter pylori Decrease Drug Binding in Escherichia coli Ribosomes

Journal of Bacteriology ◽

10.1128/jb.187.11.3708-3712.2005 ◽

2005 ◽

Vol 187 (11) ◽

pp. 3708-3712 ◽

Cited By ~ 27

Author(s):

Lisa Nonaka ◽

Sean R. Connell ◽

Diane E. Taylor

Keyword(s):

Escherichia Coli ◽

Helicobacter Pylori ◽

16S Rrna ◽

Binding Site ◽

Tetracycline Resistance ◽

Drug Binding ◽

Multicopy Plasmid ◽

Wild Type ◽

E Coli ◽

H Pylori

ABSTRACT Tetracycline resistance in clinical isolates of Helicobacter pylori has been associated with nucleotide substitutions at positions 965 to 967 in the 16S rRNA. We constructed mutants which had different sequences at 965 to 967 in the 16S rRNA gene present on a multicopy plasmid in Escherichia coli strain TA527, in which all seven rrn genes were deleted. The MICs for tetracycline of all mutants having single, double, or triple substitutions at the 965 to 967 region that were previously found in highly resistant H. pylori isolates were higher than that of the mutant exhibiting the wild-type sequence of tetracycline-susceptible H. pylori. The MIC of the mutant with the 965TTC967 triple substitution was 32 times higher than that of the E. coli mutant with the 965AGA967 substitution present in wild-type H. pylori. The ribosomes extracted from the tetracycline-resistant E. coli 965TTC967 variant bound less tetracycline than E. coli with the wild-type H. pylori sequence at this region. The concentration of tetracycline bound to the ribosome was 40% that of the wild type. The results of this study suggest that tetracycline binding to the primary binding site (Tet-1) of the ribosome at positions 965 to 967 is influenced by its sequence patterns, which form the primary binding site for tetracycline.

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