scholarly journals Characterization of a Helicobacter hepaticus putA Mutant Strain in Host Colonization and Oxidative Stress

2008 ◽  
Vol 76 (7) ◽  
pp. 3037-3044 ◽  
Author(s):  
Navasona Krishnan ◽  
Alan R. Doster ◽  
Gerald E. Duhamel ◽  
Donald F. Becker
Keyword(s):  
Oxidative Stress ◽  
Mutant Strain ◽  
Helicobacter Hepaticus ◽  
Proline Metabolism ◽  
Wild Type ◽  
Knockout Mutant ◽  
Antioxidant Genes ◽  
Host Infection ◽  
H Pylori

ABSTRACT Helicobacter hepaticus is a gram-negative, spiral-shaped microaerophilic bacterium associated with chronic intestinal infection leading to hepatitis and colonic and hepatic carcinomas in susceptible strains of mice. In the closely related human pathogen Helicobacter pylori, l-proline is a preferred respiratory substrate and is found at significantly high levels in the gastric juice of infected patients. A previous study of the proline catabolic PutA flavoenzymes from H. pylori and H. hepaticus revealed that Helicobacter PutA generates reactive oxygen species during proline oxidation by transferring electrons from reduced flavin to molecular oxygen. We further explored the preference for proline as a respiratory substrate and the potential impact of proline metabolism on the redox environment in Helicobacter species during host infection by disrupting the putA gene in H. hepaticus. The resulting putA knockout mutant strain was characterized by oxidative stress analysis and mouse infection studies. The putA mutant strain of H. hepaticus exhibited increased proline levels and resistance to oxidative stress relative to that of the wild-type strain, consistent with proline's role as an antioxidant. The significant increase in stress resistance was attributed to higher proline content, as no upregulation of antioxidant genes was observed for the putA mutant strain. The wild-type and putA mutant H. hepaticus strains displayed similar levels of infection in mice, but in mice challenged with the putA mutant strain, significantly reduced inflammation was observed, suggesting a role for proline metabolism in H. hepaticus pathogenicity in vivo.

scholarly journals A Helicobacter hepaticus catalase mutant is hypersensitive to oxidative stress and suffers increased DNA damage

2007 ◽  
Vol 56 (4) ◽  
pp. 557-562 ◽  
Author(s):  
Yang Hong ◽  
Ge Wang ◽  
Robert J. Maier
Keyword(s):  
Oxidative Stress ◽  
Dna Damage ◽  
Catalase Activity ◽  
Insertional Mutagenesis ◽  
Specific Activity ◽  
Bacterial Species ◽  
Helicobacter Hepaticus ◽  
Wild Type ◽  
Crude Cell Extract ◽  
H Pylori

Catalase (KatA) is known to play an important role in oxidative stress resistance in many bacterial species and a homologue exists in Helicobacter hepaticus, a member of the enterohepatic Helicobacter species. Here, a katA mutant was constructed by insertional mutagenesis and its oxidative stress phenotype was investigated. Catalase activity was readily detected [196 units (mg protein crude cell extract)−1] in the wild-type, whereas the mutant strain was deficient in, but not devoid of, activity. In contrast, Helicobacter pylori katA strains lack detectable catalase activity and wild-type H. pylori generally contains higher specific activity than H. hepaticus. Wild-type H. hepaticus cells tolerated 6 % O2 for growth, whilst the katA mutant could not survive at this oxygen level. Even at the optimal O2 level, the growth of the H. hepaticus katA strain was severely inhibited, which is also in contrast to H. pylori katA strains. Wild-type H. hepaticus cells withstood exposure to 100 mM H2O2 but the katA mutant cells were killed by the same treatment. Wild-type cells suffered no significant DNA damage by H2O2 treatment (100 mM for 6 min), whilst the same treatment resulted in severe DNA fragmentation in the katA mutant. Thus H. hepaticus KatA plays an important role as an antioxidant protein.

scholarly journals Proline Metabolism IncreaseskatGExpression and Oxidative Stress Resistance in Escherichia coli

2014 ◽  
Vol 197 (3) ◽  
pp. 431-440 ◽  
Author(s):  
Lu Zhang ◽  
James R. Alfano ◽  
Donald F. Becker
Keyword(s):  
Oxidative Stress ◽  
Escherichia Coli ◽  
Hydrogen Peroxide ◽  
Mutant Strain ◽  
Stress Resistance ◽  
Proline Metabolism ◽  
Wild Type ◽  
Content Type ◽  
Oxidative Stress Resistance ◽  
Proline Utilization

The oxidation ofl-proline to glutamate in Gram-negative bacteria is catalyzed by the proline utilization A (PutA) flavoenzyme, which contains proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate (P5C) dehydrogenase domains in a single polypeptide. Previous studies have suggested that aside from providing energy, proline metabolism influences oxidative stress resistance in different organisms. To explore this potential role and the mechanism, we characterized the oxidative stress resistance of wild-type andputAmutant strains ofEscherichia coli. Initial stress assays revealed that theputAmutant strain was significantly more sensitive to oxidative stress than the parental wild-type strain. Expression of PutA in theputAmutant strain restored oxidative stress resistance, confirming that depletion of PutA was responsible for the oxidative stress phenotype. Treatment of wild-type cells with proline significantly increased hydroperoxidase I (encoded bykatG) expression and activity. Furthermore, the ΔkatGstrain failed to respond to proline, indicating a critical role for hydroperoxidase I in the mechanism of proline protection. The global regulator OxyR activates the expression ofkatGalong with several other genes involved in oxidative stress defense. In addition tokatG, proline increased the expression ofgrxA(glutaredoxin 1) andtrxC(thioredoxin 2) of the OxyR regulon, implicating OxyR in proline protection. Proline oxidative metabolism was shown to generate hydrogen peroxide, indicating that proline increases oxidative stress tolerance inE. colivia a preadaptive effect involving endogenous hydrogen peroxide production and enhanced catalase-peroxidase activity.

scholarly journals Dual Roles of Helicobacter pylori NapA in Inducing and Combating Oxidative Stress

2006 ◽  
Vol 74 (12) ◽  
pp. 6839-6846 ◽  
Author(s):  
Ge Wang ◽  
Yang Hong ◽  
Adriana Olczak ◽  
Susan E. Maier ◽  
Robert J. Maier
Keyword(s):  
Oxidative Stress ◽  
Dna Damage ◽  
Oxidative Dna Damage ◽  
Wild Type Mouse ◽  
Wild Type ◽  
Oxygen Intermediates ◽  
Physiological Analysis ◽  
H Pylori

ABSTRACT Neutrophil-activating protein (NapA) has been well documented to play roles in human neutrophil recruitment and in stimulating host cell production of reactive oxygen intermediates (ROI). A separate role for NapA in combating oxidative stress within H. pylori was implied by studies of various H. pylori mutant strains. Here, physiological analysis of a napA strain was the approach used to assess the iron-sequestering and stress resistance roles of NapA, its role in preventing oxidative DNA damage, and its importance to mouse colonization. The napA strain was more sensitive to oxidative stress reagents and to oxygen, and it contained fourfold more intracellular free iron and more damaged DNA than the parent strain. Pure, iron-loaded NapA bound to DNA, but native NapA did not, presumably linking iron levels sensed by NapA to DNA damage protection. Despite its in vitro phenotype of sensitivity to oxidative stress, the napA strain showed normal (like that of the wild type) mouse colonization efficiency in the conventional in vivo assay. By use of a modified mouse inoculation protocol whereby nonviable H. pylori is first inoculated into mice, followed by (live) bacterial strain administration, an in vivo role for NapA in colonization efficiency could be demonstrated. NapA is the critical component responsible for inducing host-mediated ROI production, thus inhibiting colonization by the napA strain. An animal colonization experiment with a mixed-strain infection protocol further demonstrated that the napA strain has significantly decreased ability to survive when competing with the wild type. H. pylori NapA has unique and separate roles in gastric pathogenesis.

scholarly journals Helicobacter pylori and Complex Gangliosides

2004 ◽  
Vol 72 (3) ◽  
pp. 1519-1529 ◽  
Author(s):  
Niamh Roche ◽  
Jonas Ångström ◽  
Marina Hurtig ◽  
Thomas Larsson ◽  
Thomas Borén ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Sialic Acid ◽  
Mutant Strain ◽  
Carbohydrate Chain ◽  
Proton Nuclear Magnetic Resonance ◽  
Bacterial Cells ◽  
Wild Type ◽  
Knockout Mutant ◽  
Factors Affecting ◽  
H Pylori

ABSTRACT Recognition of sialic acid-containing glycoconjugates by the human gastric pathogen Helicobacter pylori has been repeatedly demonstrated. To investigate the structural requirements for H. pylori binding to complex gangliosides, a large number of gangliosides were isolated and characterized by mass spectrometry and proton nuclear magnetic resonance. Ganglioside binding of sialic acid-recognizing H. pylori strains (strains J99 and CCUG 17874) and knockout mutant strains with the sialic acid binding adhesin SabA or the NeuAcα3Galβ4GlcNAcβ3Galβ4GlcNAcβ-binding neutrophil-activating protein HPNAP deleted was investigated using the thin-layer chromatogram binding assay. The wild-type bacteria bound to N-acetyllactosamine-based gangliosides with terminal α3-linked NeuAc, while gangliosides with terminal NeuGcα3, NeuAcα6, or NeuAcα8NeuAcα3 were not recognized. The factors affecting binding affinity were identified as (i) the length of the N-acetyllactosamine carbohydrate chain, (ii) the branches of the carbohydrate chain, and (iii) fucose substitution of the N-acetyllactosamine core chain. While the J99/NAP− mutant strain displayed a ganglioside binding pattern identical to that of the parent J99 wild-type strain, no ganglioside binding was obtained with the J99/SabA− mutant strain, demonstrating that the SabA adhesin is the sole factor responsible for the binding of H. pylori bacterial cells to gangliosides.

scholarly journals The in vivo gene expression signature of oxidative stress

2008 ◽  
Vol 34 (1) ◽  
pp. 112-126 ◽  
Author(s):  
Eun-Soo Han ◽  
Florian L. Muller ◽  
Viviana I. Pérez ◽  
Wenbo Qi ◽  
Huiyun Liang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Oxidative Damage ◽  
Target Genes ◽  
Wild Type ◽  
Antioxidant Genes ◽  
Genetic Ablation ◽  
Expression Response ◽  
P53 Target Genes

How higher organisms respond to elevated oxidative stress in vivo is poorly understood. Therefore, we measured oxidative stress parameters and gene expression alterations (Affymetrix arrays) in the liver caused by elevated reactive oxygen species induced in vivo by diquat or by genetic ablation of the major antioxidant enzymes CuZn-superoxide dismutase ( Sod1) and glutathione peroxidase-1 ( Gpx1). Diquat (50 mg/kg) treatment resulted in a significant increase in oxidative damage within 3–6 h in wild-type mice without any lethality. In contrast, treatment of Sod1−/− or Gpx1−/− mice with a similar concentration of diquat resulted in a significant increase in oxidative damage within an hour of treatment and was lethal, i.e., these mice are extremely sensitive to the oxidative stress generated by diquat. The expression response to elevated oxidative stress in vivo does not involve an upregulation of classic antioxidant genes, although long-term oxidative stress in Sod1−/− mice leads to a significant upregulation of thiol antioxidants (e.g., Mt1, Srxn1, Gclc, Txnrd1), which appears to be mediated by the redox-sensitive transcription factor Nrf2. The main finding of our study is that the common response to elevated oxidative stress with diquat treatment in wild-type, Gpx1−/−, and Sod1−/− mice and in untreated Sod1−/− mice is an upregulation of p53 target genes ( p21, Gdf15, Plk3, Atf3, Trp53inp1, Ddit4, Gadd45a, Btg2, Ndrg1). A retrospective comparison with previous studies shows that induction of these p53 target genes is a conserved expression response to oxidative stress, in vivo and in vitro, in different species and different cells/organs.

scholarly journals A Novel DNA-Binding Protein Plays an Important Role in Helicobacter pylori Stress Tolerance and Survival in the Host

2014 ◽  
Vol 197 (5) ◽  
pp. 973-982 ◽  
Author(s):  
Ge Wang ◽  
Robert J. Maier
Keyword(s):  
Oxidative Stress ◽  
Helicobacter Pylori ◽  
Dna Binding ◽  
Stress Tolerance ◽  
Mutant Strain ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Wild Type ◽  
Content Type ◽  
H Pylori

The gastric pathogenHelicobacter pylorimust 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 ofhp119, 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 Δhp119strain 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 ofhp119at a separate chromosomal locus. The mutant strain was approximately10-fold more sensitive to macrophage-mediated killing than the parent or the complemented strain. Of 12 mice inoculated with the wild type, all containedH. 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 Δhp119mutant 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 Δhp119mutant 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 inHelicobacter pyloristress tolerance and survival in the host.

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

2005 ◽  
Vol 73 (1) ◽  
pp. 378-384 ◽  
Author(s):  
Ge Wang ◽  
Adriana A. Olczak ◽  
James P. Walton ◽  
Robert J. Maier
Keyword(s):  
Oxidative Stress ◽  
Mutant Strain ◽  
Stress Resistance ◽  
Type Strain ◽  
Wild Type Strain ◽  
Wild Type ◽  
Oxidative Stress Resistance ◽  
Organic Hydroperoxides ◽  
H Pylori ◽  
Thiol Peroxidase

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 108 bcp 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.

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

2004 ◽  
Vol 72 (3) ◽  
pp. 1391-1396 ◽  
Author(s):  
Ge Wang ◽  
Robert J. Maier
Keyword(s):  
Oxidative Stress ◽  
Helicobacter Pylori ◽  
Mutant Strain ◽  
Stress Resistance ◽  
Type Strain ◽  
Wild Type Strain ◽  
Quinone Reductase ◽  
Wild Type ◽  
Oxidative Stress Resistance ◽  
H Pylori

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.

scholarly journals Systems Modeling of the Role of Interleukin-21 in the Maintenance of Effector CD4+ T Cell Responses during Chronic Helicobacter pylori Infection

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Adria Carbo ◽  
Danyvid Olivares-Villagómez ◽  
Raquel Hontecillas ◽  
Josep Bassaganya-Riera ◽  
Rupesh Chaturvedi ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Helicobacter Pylori ◽  
T Cell ◽  
Wild Type ◽  
Content Type ◽  
Interleukin 21 ◽  
H Pylori ◽  
Deficient Mice

ABSTRACTThe development of gastritis duringHelicobacter pyloriinfection is dependent on an activated adaptive immune response orchestrated by T helper (Th) cells. However, the relative contributions of the Th1 and Th17 subsets to gastritis and control of infection are still under investigation. To investigate the role of interleukin-21 (IL-21) in the gastric mucosa duringH. pyloriinfection, we combined mathematical modeling of CD4+T cell differentiation within vivomechanistic studies. We infected IL-21-deficient and wild-type mice withH. pyloristrain SS1 and assessed colonization, gastric inflammation, cellular infiltration, and cytokine profiles. ChronicallyH. pylori-infected IL-21-deficient mice had higherH. pyloricolonization, significantly less gastritis, and reduced expression of proinflammatory cytokines and chemokines compared to these parameters in infected wild-type littermates. Thesein vivodata were used to calibrate anH. pyloriinfection-dependent, CD4+T cell-specific computational model, which then described the mechanism by which IL-21 activates the production of interferon gamma (IFN-γ) and IL-17 during chronicH. pyloriinfection. The model predicted activated expression of T-bet and RORγt and the phosphorylation of STAT3 and STAT1 and suggested a potential role of IL-21 in the modulation of IL-10. Driven by our modeling-derived predictions, we found reduced levels of CD4+splenocyte-specifictbx21androrcexpression, reduced phosphorylation of STAT1 and STAT3, and an increase in CD4+T cell-specific IL-10 expression inH. pylori-infected IL-21-deficient mice. Our results indicate that IL-21 regulates Th1 and Th17 effector responses during chronicH. pyloriinfection in a STAT1- and STAT3-dependent manner, therefore playing a major role controllingH. pyloriinfection and gastritis.IMPORTANCEHelicobacter pyloriis the dominant member of the gastric microbiota in more than 50% of the world’s population.H. pyloricolonization has been implicated in gastritis and gastric cancer, as infection withH. pyloriis the single most common risk factor for gastric cancer. Current data suggest that, in addition to bacterial virulence factors, the magnitude and types of immune responses influence the outcome of colonization and chronic infection. This study uses a combined computational and experimental approach to investigate how IL-21, a proinflammatory T cell-derived cytokine, maintains the chronic proinflammatory T cell immune response driving chronic gastritis duringH. pyloriinfection. This research will also provide insight into a myriad of other infectious and immune disorders in which IL-21 is increasingly recognized to play a central role. The use of IL-21-related therapies may provide treatment options for individuals chronically colonized withH. pylorias an alternative to aggressive antibiotics.

scholarly journals Oxidative stress promotes liver cancer metastasis via a PKA-activated RNF25/ECAD/YAP circuit

2022 ◽  
Author(s):  
Zhao Huang ◽  
Li Zhou ◽  
Jiufei Duan ◽  
Siyuan Qin ◽  
Yu Wang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cancer Metastasis ◽  
Anoikis Resistance ◽  
Antioxidant Genes ◽  
Redox Sensor ◽  
Stress Signals ◽  
Hcc Cells

Abstract Loss of E-cadherin (ECAD), often caused by epigenetic inactivation, is closely associated with tumor metastasis. However, how ECAD is regulated in response to oxidative stress during tumorigenesis is largely unknown. Here we identify RNF25 as a new E3 ligase of ECAD, whose activation by oxidative stress leads to ECAD protein degradation in hepatocellular carcinoma (HCC). Loss of ECAD activates YAP, which in turn promotes the transcription of RNF25, thus forming a positive feedback loop to sustain the ECAD downregulation. YAP activation mitigates oxidative stress in detached HCC cells by upregulating antioxidant genes, protecting detached HCC cells from ferroptosis, resulting in anoikis resistance. Mechanistically, we found that protein kinase A (PKA) senses oxidative stress by redox modification in its β catalytic subunit (PRKACB) at Cys200 and Cys344, which increases its kinase activity towards RNF25 phosphorylation at Ser450, facilitating RNF25-mediated degradation of ECAD. Moreover, RNF25 expression is associated with HCC metastasis and depletion of RNF25 is sufficient to diminish HCC invasion and metastasis in vitro and in vivo. Together, these results identify a dual role of RNF25 as a critical regulator of ECAD protein turnover, promoting both anoikis resistance and metastasis, and PKA is a necessary redox sensor to enable this process. Our study provides mechanistic insight into how tumor cells sense oxidative stress signals to spread while escaping cell death.

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