A RecB-Like Helicase in Helicobacter pylori Is Important for DNA Repair and Host Colonization

ABSTRACT The human gastric pathogen Helicobacter pylori encounters frequent oxidative and acid stress in its specific niche, and this causes bacterial DNA damage. H. pylori exhibits a very high degree of DNA recombination, which is required for repairing both DNA double-stranded (ds) breaks and blocked replication forks. Nevertheless, few genes encoding components of DNA recombinational repair processes have been identified in H. pylori. An H. pylori mutant defective in a putative helicase gene (HP1553) was constructed and characterized herein. The HP1553 mutant strain was much more sensitive to mitomycin C than the WT strain, indicating that HP1553 is required for repair of DNA ds breaks. Disruption of HP1553 resulted in a significant decrease in the DNA recombination frequency, suggesting that HP1553 is involved in DNA recombination processes, probably functioning as a RecB-like helicase. HP1553 was shown to be important for H. pylori protection against oxidative stress-induced DNA damage, as the exposure of the HP1553 mutant cells to air for 6 h caused significant fragmentation of genomic DNA and led to cell death. In a mouse infection model, the HP1553 mutant strain displayed a greatly reduced ability to colonize the host stomachs, indicating that HP1553 plays a significant role in H. pylori survival/colonization in the host.

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Pyridodiazepine Amines Are Selective Therapeutic Agents for Helicobacter pylori by Suppressing Growth through Inhibition of Glutamate Racemase but Are Predicted To Require Continuous Elevated Levels in Plasma To Achieve Clinical Efficacy

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04410-14 ◽

2015 ◽

Vol 59 (4) ◽

pp. 2337-2342 ◽

Cited By ~ 7

Author(s):

Boudewijn L. M. de Jonge ◽

Amy Kutschke ◽

Joseph V. Newman ◽

Michael T. Rooney ◽

Wei Yang ◽

...

Keyword(s):

Helicobacter Pylori ◽

Therapeutic Agents ◽

Infection Model ◽

Content Type ◽

Peptidoglycan Biosynthesis ◽

Glutamate Racemase ◽

Mouse Infection Model ◽

Exposure Levels ◽

H Pylori ◽

Extended Exposure

ABSTRACTA pyridodiazepine amine inhibitor ofHelicobacter pyloriglutamate racemase (MurI) was characterized. The compound was selectively active againstH. pylori, and growth suppression was shown to be mediated through the inhibition of MurI by several methods. In killing kinetics experiments, the compound showed concentration-independent activity, with about a 2-log loss of viability in 24 h. A demonstration of efficacy in a mouse infection model was attempted but not achieved, and this was attributed to the failure to attain extended exposure levels above the MIC for >95% of the time. This index and magnitude were derived from pharmacokinetic-pharmacodynamic (PK-PD) studies with amoxicillin, another inhibitor of peptidoglycan biosynthesis that showed slow killing kinetics similar to those of the pyridodiazepine amines. These studies indicate that MurI and other enzymes involved in peptidoglycan biosynthesis may be less desirable targets for monotherapy directed againstH. pyloriif once-a-day dosing is required.

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Peptidoglycan Deacetylation in Helicobacter pylori Contributes to Bacterial Survival by Mitigating Host Immune Responses

Infection and Immunity ◽

10.1128/iai.00307-10 ◽

2010 ◽

Vol 78 (11) ◽

pp. 4660-4666 ◽

Cited By ~ 35

Author(s):

Ge Wang ◽

Susan E. Maier ◽

Leja F. Lo ◽

George Maier ◽

Shruti Dosi ◽

...

Keyword(s):

Oxidative Stress ◽

Helicobacter Pylori ◽

Immune Responses ◽

Bacterial Load ◽

Host Cells ◽

Infection Model ◽

Wild Type ◽

Mouse Infection Model ◽

Mouse Infection ◽

H Pylori

ABSTRACT An oxidative stress-induced enzyme, peptidoglycan deacetylase (PgdA), in the human gastric pathogen Helicobacter pylori was previously identified and characterized. In this study, we constructed H. pylori pgdA mutants in two mouse-adapted strains, X47 and B128, to investigate the role of PgdA in vivo (to determine the mutants’ abilities to colonize mice and to induce an immune response). H. pylori pgdA mutant cells showed increased sensitivity to lysozyme compared to the sensitivities of the parent strains. We demonstrated that the expression of PgdA was significantly induced (3.5-fold) when H. pylori cells were in contact with macrophages, similar to the effect observed with oxidative stress as the environmental inducer. Using a mouse infection model, we first examined the mouse colonization ability of an H. pylori pgdA mutant in X47, a strain deficient in the major pathway (cag pathogenicity island [PAI] encoded) for delivery of peptidoglycan into host cells. No animal colonization difference between the wild type and the mutant was observed 3 weeks after inoculation. However, the pgdA mutant showed a significantly attenuated ability to colonize mouse stomachs (9-fold-lower bacterial load) at 9 weeks postinoculation. With the cag PAI-positive strain B128, a significant colonization difference between the wild type and the pgdA mutant was observed at 3 weeks postinoculation (1.32 × 104 versus 1.85 × 103 CFU/gram of stomach). To monitor the immune responses elicited by H. pylori in the mouse infection model, we determined the concentrations of cytokines present in mouse sera. In the mice infected with the pgdA mutant strain, we observed a highly significant increase in the level of MIP-2. In addition, significant increases in interleukin-10 and tumor necrosis factor alpha in the pgdA mutant-infected mice compared to the levels in the wild-type H. pylori-infected mice were also observed. These results indicated that H. pylori peptidoglycan deacetylation is an important mechanism for mitigating host immune detection; this likely contributes to pathogen persistence.

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Identification of Helicobacter pylori Genes That Contribute to Stomach Colonization

Infection and Immunity ◽

10.1128/iai.01176-06 ◽

2006 ◽

Vol 75 (2) ◽

pp. 1005-1016 ◽

Cited By ~ 93

Author(s):

David N. Baldwin ◽

Benjamin Shepherd ◽

Petra Kraemer ◽

Michael K. Hall ◽

Laura K. Sycuro ◽

...

Keyword(s):

Helicobacter Pylori ◽

Pathogenic Bacteria ◽

Null Alleles ◽

Infection Model ◽

Genetic Loci ◽

Pathogenic Potential ◽

Mouse Infection Model ◽

Urease Enzyme ◽

Transposon Mutants ◽

H Pylori

ABSTRACT Chronic infection of the human stomach by Helicobacter pylori leads to a variety of pathological sequelae, including peptic ulcer and gastric cancer, resulting in significant human morbidity and mortality. Several genes have been implicated in disease related to H. pylori infection, including the vacuolating cytotoxin and the cag pathogenicity island. Other factors important for the establishment and maintenance of infection include urease enzyme production, motility, iron uptake, and stress response. We utilized a C57BL/6 mouse infection model to query a collection of 2,400 transposon mutants in two different bacterial strain backgrounds for H. pylori genetic loci contributing to colonization of the stomach. Microarray-based tracking of transposon mutants allowed us to monitor the behavior of transposon insertions in 758 different gene loci. Of the loci measured, 223 (29%) had a predicted colonization defect. These included previously described H. pylori virulence genes, genes implicated in virulence in other pathogenic bacteria, and 81 hypothetical proteins. We have retested 10 previously uncharacterized candidate colonization gene loci by making independent null alleles and have confirmed their colonization phenotypes by using competition experiments and by determining the dose required for 50% infection. Of the genetic loci retested, 60% have strain-specific colonization defects, while 40% have phenotypes in both strain backgrounds for infection, highlighting the profound effect of H. pylori strain variation on the pathogenic potential of this organism.

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Two-Component Systems of Helicobacter pylori Contribute to Virulence in a Mouse Infection Model

Infection and Immunity ◽

10.1128/iai.71.9.5381-5385.2003 ◽

2003 ◽

Vol 71 (9) ◽

pp. 5381-5385 ◽

Cited By ~ 35

Author(s):

Klaus Panthel ◽

Patricia Dietz ◽

Rainer Haas ◽

Dagmar Beier

Keyword(s):

Helicobacter Pylori ◽

Transcriptional Control ◽

Response Regulator ◽

Infection Model ◽

Histidine Kinases ◽

Reading Frame ◽

Mouse Infection Model ◽

Regulatory Systems ◽

Two Component ◽

H Pylori

ABSTRACT Helicobacter pylori encodes three histidine kinases and five response regulators belonging to the family of two-component regulatory systems which are involved in transcriptional control. Here we demonstrate that isogenic mutants of H. pylori P76 with deletions of the response regulator open reading frame (ORF) HP1365 and ORFs HP244, HP165, and HP1364 encoding histidine kinases are unable to colonize the stomachs of BALB/c mice, suggesting an essential role of these systems in the regulation of important virulence properties of H. pylori. Furthermore, we demonstrate that the genes under the control of the PHP1408 and PHP119 promoters which are regulated by the two-component system HP166-HP165 are not essential for single mutant colonization of mice but are required under competitive colonization conditions.

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Basal Body Structures Differentially Affect Transcription of RpoN- and FliA-Dependent Flagellar Genes in Helicobacter pylori

Journal of Bacteriology ◽

10.1128/jb.02533-14 ◽

2015 ◽

Vol 197 (11) ◽

pp. 1921-1930 ◽

Cited By ~ 5

Author(s):

Jennifer Tsang ◽

Timothy R. Hoover

Keyword(s):

Helicobacter Pylori ◽

Basal Body ◽

Protein Interactions ◽

Transcript Levels ◽

Content Type ◽

Genes Encoding ◽

H Pylori ◽

Flagellar Biogenesis ◽

Flagellar Genes

ABSTRACTFlagellar biogenesis inHelicobacter pyloriis regulated by a transcriptional hierarchy governed by three sigma factors, RpoD (σ80), RpoN (σ54), and FliA (σ28), that temporally coordinates gene expression with the assembly of the flagellum. Previous studies showed that loss of flagellar protein export apparatus components inhibits transcription of flagellar genes. The FlgS/FlgR two-component system activates transcription of RpoN-dependent genes though an unknown mechanism. To understand better the extent to which flagellar gene regulation is coupled to flagellar assembly, we disrupted flagellar biogenesis at various points and determined how these mutations affected transcription of RpoN-dependent (flaBandflgE) and FliA-dependent (flaA) genes. The MS ring (encoded byfliF) is one of the earliest flagellar structures assembled. Deletion offliFresulted in the elimination of RpoN-dependent transcripts and an ∼4-fold decrease inflaAtranscript levels. FliH is a cytoplasmic protein that functions with the C ring protein FliN to shuttle substrates to the export apparatus. Deletions offliHand genes encoding C ring components (fliMandfliY) decreased transcript levels offlaBandflgEbut had little or no effect on transcript levels offlaA. Transcript levels offlaBandflgEwere elevated in mutants where genes encoding rod proteins (fliEandflgBC) were deleted, while transcript levels offlaAwas reduced ∼2-fold in both mutants. We propose that FlgS responds to an assembly checkpoint associated with the export apparatus and that FliH and one or more C ring component assist FlgS in engaging this flagellar structure.IMPORTANCEThe mechanisms used by bacteria to couple transcription of flagellar genes with assembly of the flagellum are poorly understood. The results from this study identified components of theH. pyloriflagellar basal body that either positively or negatively affect expression of RpoN-dependent flagellar genes. Some of these basal body proteins may interact directly with regulatory proteins that control transcription of theH. pyloriRpoN regulon, a hypothesis that can be tested by examining protein-protein interactionsin vitro.

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The zinc transporter ZnuABC is critical for the virulence of Chromobacterium violaceum and contributes to diverse zinc-dependent physiological processes

10.1101/2021.06.03.447022 ◽

2021 ◽

Author(s):

Renato E. R. S. Santos ◽

Waldir P. da Silva Júnior ◽

Simone A. Harrison ◽

Eric P Skaar ◽

Walter J. Chazin ◽

...

Keyword(s):

Mutant Strain ◽

Zinc Uptake ◽

Chromobacterium Violaceum ◽

Host Protein ◽

Infection Model ◽

Uptake System ◽

Environmental Bacterium ◽

Mouse Infection Model ◽

Synthetic Metal

Chromobacterium violaceum is a ubiquitous environmental bacterium that causes sporadic life-threatening infections in humans. How C. violaceum acquires zinc to colonize environmental and host niches is unknown. In this work, we demonstrated that C. violaceum employs the zinc uptake system ZnuABC to overcome zinc limitation in the host, ensuring the zinc supply for several physiological demands. Our data indicated that the C. violaceum ZnuABC transporter is encoded in a zur-CV_RS15045-CV_RS15040-znuCBA operon. This operon was repressed by the zinc uptake regulator Zur and derepressed in the presence of the host protein calprotectin (CP) and the synthetic metal chelator EDTA. A ΔznuCBA mutant strain showed impaired growth under these zinc-chelated conditions. Moreover, the deletion of znuCBA provoked a reduction in violacein production, swimming motility, biofilm formation, and bacterial competition. Remarkably, the ΔznuCBA mutant strain was highly attenuated for virulence in an in vivo mouse infection model and showed a low capacity to colonize the liver, grow in the presence of CP, and resist neutrophil killing. Overall, our findings demonstrate that ZnuABC is essential for C. violaceum virulence, contributing to subvert the zinc-based host nutritional immunity.

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Critical Role of RecN in Recombinational DNA Repair and Survival of Helicobacter pylori

Infection and Immunity ◽

10.1128/iai.00791-07 ◽

2007 ◽

Vol 76 (1) ◽

pp. 153-160 ◽

Cited By ~ 32

Author(s):

Ge Wang ◽

Robert J. Maier

Keyword(s):

Helicobacter Pylori ◽

Parent Strain ◽

Dna Recombination ◽

Double Strand Breaks ◽

Recombinational Repair ◽

Wild Type ◽

Dna Double Strand Breaks ◽

Air Exposure ◽

Strand Breaks ◽

H Pylori

ABSTRACT Homologous recombination is one of the key mechanisms responsible for the repair of DNA double-strand breaks. Recombinational repair normally requires a battery of proteins, each with specific DNA recognition, strand transfer, resolution, or other functions. Helicobacter pylori lacks many of the proteins normally involved in the early stage (presynapsis) of recombinational repair, but it has a RecN homologue with an unclear function. A recN mutant strain of H. pylori was shown to be much more sensitive than its parent to mitomycin C, an agent predominantly causing DNA double-strand breaks. The recN strain was unable to survive exposure to either air or acid as well as the parent strain, and air exposure resulted in no viable recN cells recovered after 8 h. In oxidative stress conditions (i.e., air exposure), a recN strain accumulated significantly more damaged (multiply fragmented) DNA than the parent strain. To assess the DNA recombination abilities of strains, their transformation abilities were compared by separately monitoring transformation using H. pylori DNA fragments containing either a site-specific mutation (conferring rifampin resistance) or a large insertion (kanamycin resistance cassette). The transformation frequencies using the two types of DNA donor were 10- and 50-fold lower, respectively, for the recN strain than for the wild type, indicating that RecN plays an important role in facilitating DNA recombination. In two separate mouse colonization experiments, the recN strain colonized most of the stomachs, but the average number of recovered cells was 10-fold less for the mutant than for the parent strain (a statistically significant difference). Complementation of the recN strain by chromosomal insertion of a functional recN gene restored both the recombination frequency and mouse colonization ability to the wild-type levels. Thus, H. pylori RecN, as a component of DNA recombinational repair, plays a significant role in H. pylori survival in vivo.

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Analysis of rdxA and Involvement of Additional Genes Encoding NAD(P)H Flavin Oxidoreductase (FrxA) and Ferredoxin-Like Protein (FdxB) in Metronidazole Resistance of Helicobacter pylori

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.8.2133-2142.2000 ◽

2000 ◽

Vol 44 (8) ◽

pp. 2133-2142 ◽

Cited By ~ 78

Author(s):

Dong-Hyeon Kwon ◽

Fouad A. K. El-Zaatari ◽

Mototsugu Kato ◽

Michael S. Osato ◽

Rita Reddy ◽

...

Keyword(s):

Helicobacter Pylori ◽

Resistant Strain ◽

Resistance Mechanisms ◽

Sensitive Strain ◽

Metronidazole Resistance ◽

Genes Encoding ◽

Resistant Strains ◽

Flavin Oxidoreductase ◽

H Pylori

ABSTRACT Metronidazole (Mtz) is a critical ingredient of modern multidrug therapies for Helicobacter pylori infection. Mtz resistance reduces the effectiveness of these combinations. Although null mutations in a rdxA gene that encodes oxygen-insensitive NAD(P)H nitroreductase was reported in Mtz-resistant H. pylori, an intact rdxA gene has also been reported in Mtz-resistant H. pylori, suggesting that additional Mtz resistance mechanisms exist in H. pylori. We explored the nature of Mtz resistance among 544 clinical H. pyloriisolates to clarify the role of rdxA inactivation in Mtz resistance and to identify another gene(s) responsible for Mtz resistance in H. pylori. Mtz resistance was present in 33% (181 of 544) of the clinical isolates. There was marked heterogeneity of resistance, with Mtz MICs ranging from 8 to ≥256 μg/ml.rdxA inactivation resulted in Mtz MICs of up to 32 μg/ml for 6 Mtz-sensitive H. pylori strains and 128 μg/ml for one Mtz-sensitive strain. Single or dual (with rdxA) inactivation of genes that encode ferredoxin-like protein (designatedfdxB) and NAD(P)H flavin oxidoreductase (frxA) also increased the MICs of Mtz for sensitive and resistant strains with low to moderate levels of Mtz resistance. fdxB inactivation resulted in a lower level of resistance than that from rdxAinactivation, whereas frxA inactivation resulted in MICs similar to those seen with rdxA inactivation. Further evidence for involvement of the frxA gene in Mtz resistance included the finding of a naturally inactivated frxA but an intact rdxA in an Mtz-resistant strain, complementation of Mtz sensitivity from an Mtz-sensitive strain to an Mtz-resistant strain or vice versa by use of naturally inactivated or functionalfrxA genes, respectively, and transformation of an Mtz-resistant Escherichia coli strain to an Mtz sensitive strain by a naturally functional frxA gene but not an inactivated frxA gene. These results are consistent with the hypothesis that null mutations in fdxB,frxA, or rdxA may be involved in Mtz resistance.

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

Infection and Immunity ◽

10.1128/iai.00232-15 ◽

2015 ◽

Vol 83 (7) ◽

pp. 2935-2943 ◽

Cited By ~ 11

Author(s):

Lisa G. Kuhns ◽

Ge Wang ◽

Robert J. Maier

Keyword(s):

Dna Damage ◽

Helicobacter Pylori ◽

Oxidative Dna Damage ◽

Lipid Peroxides ◽

Transcript Abundance ◽

Protein Carbonylation ◽

Content Type ◽

Thioredoxin System ◽

Wide Range ◽

H Pylori

Thioredoxins are highly conserved throughout a wide range of organisms, and they are essential for the isurvival of oxygen-sensitive cells. The gastric pathogenHelicobacter pyloriuses the thioredoxin system to maintain its thiol/disulfide balance. There are two thioredoxins present inH. 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 protectingH. pylorifrom oxidative stress. Individual ΔtrxAand ΔtrxCdeletion 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 ΔtrxCmutant than in the ΔtrxAmutant; it had 20-fold- and 4-fold-more carbonylated protein content than the wild type and the ΔtrxAstrain, respectively, after 4 h of atmospheric O2stress.trxtranscript abundance was altered by the deletion of the heterologoustrxgene. The ΔtrxCmutant lacked mouse colonization ability, while the ability to colonize mouse stomachs was significantly reduced in the ΔtrxAmutant.

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Involvement of CO2 generated by urease in multiplication of Helicobacter pylori

GSC Advanced Research and Reviews ◽

10.30574/gscarr.2021.7.3.0123 ◽

2021 ◽

Vol 7 (3) ◽

pp. 045-053

Author(s):

Masaaki Minami ◽

Shin-nosuke Hashikawa ◽

Takafumi Ando ◽

Hiroshi Kobayashi ◽

Hidemi Goto ◽

...

Keyword(s):

Helicobacter Pylori ◽

Mutant Strain ◽

Type Strain ◽

Wild Type Strain ◽

Neutral Medium ◽

Wild Type ◽

In The Wild ◽

H Pylori ◽

Carbon Dioxide Co2 ◽

The Relationship

Helicobacter pylori (H. pylori) urease generates both ammonia (NH3) and carbon dioxide (CO2) from urea. NH3 helps H. pylori to survive in the stomach in part by neutralizing gastric acid. However, the relationship between CO2 and H. pylori is not completed cleared. We examined the effect of CO2 generated by urease on multiplication of H. pylori by using isogenic ureB mutant and ureB complemented strain from H. pylori strain JP26. Wild-type strain survived in the medium supplement with 1mM urea in room air, however, the urease negative strain did not. To discern whether CO2 was incorporated into H. pylori, 14C in bacillus was counted after 6 hours incubation with 14C urea in both acidic and neutral medium. Significant more 14C uptake was detected in wild-type strain compared to ureB mutant strain and this uptake in the wild-type strain was more under acidic condition compared to under neutral condition, but no difference was identified in the mutant strain. These results suggest that CO2 generated by urease plays a role in multiplication of H. pylori.

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