scholarly journals Role of Helicobacter pylori methionine sulfoxide reductase in urease maturation

2013 ◽  
Vol 450 (1) ◽  
pp. 141-148 ◽  
Author(s):  
Lisa G. Kuhns ◽  
Manish Mahawar ◽  
Joshua S. Sharp ◽  
Stéphane Benoit ◽  
Robert J. Maier
Keyword(s):  
Helicobacter Pylori ◽  
Parent Strain ◽  
Specific Activity ◽  
Methionine Sulfoxide ◽  
Methionine Sulfoxide Reductase ◽  
Accessory Proteins ◽  
Wild Type ◽  
Methionine Residues ◽  
H Pylori

The persistence of the gastric pathogen Helicobacter pylori is due in part to urease and Msr (methionine sulfoxide reductase). Upon exposure to relatively mild (21% partial pressure of O2) oxidative stress, a Δmsr mutant showed both decreased urease specific activity in cell-free extracts and decreased nickel associated with the partially purified urease fraction as compared with the parent strain, yet urease apoprotein levels were the same for the Δmsr and wild-type extracts. Urease activity of the Δmsr mutant was not significantly different from the wild-type upon non-stress microaerobic incubation of strains. Urease maturation occurs through nickel mobilization via a suite of known accessory proteins, one being the GTPase UreG. Treatment of UreG with H2O2 resulted in oxidation of MS-identified methionine residues and loss of up to 70% of its GTPase activity. Incubation of pure H2O2-treated UreG with Msr led to reductive repair of nine methionine residues and recovery of up to full enzyme activity. Binding of Msr to both oxidized and non-oxidized UreG was observed by cross-linking. Therefore we conclude Msr aids the survival of H. pylori in part by ensuring continual UreG-mediated urease maturation under stress conditions.

scholarly journals Noncatalytic Antioxidant Role forHelicobacter pyloriUrease

2018 ◽  
Vol 200 (17) ◽  
Author(s):  
Alan A. Schmalstig ◽  
Stéphane L. Benoit ◽  
Sandeep K. Misra ◽  
Joshua S. Sharp ◽  
Robert J. Maier
Keyword(s):  
Helicobacter Pylori ◽  
Methionine Sulfoxide ◽  
Methionine Sulfoxide Reductase ◽  
Repair System ◽  
Content Type ◽  
Urease Enzyme ◽  
Catalytic Role ◽  
Methionine Residues ◽  
H Pylori

ABSTRACTThe well-studied catalytic role of urease, the Ni-dependent conversion of urea into carbon dioxide and ammonia, has been shown to protectHelicobacter pyloriagainst the low pH environment of the stomach lumen. We hypothesized that the abundantly expressed urease protein can play another noncatalytic role in combating oxidative stress via Met residue-mediated quenching of harmful oxidants. Three catalytically inactive urease mutant strains were constructed by single substitutions of Ni binding residues. The mutant versions synthesize normal levels of urease, and the altered versions retained all methionine residues. The three site-directed urease mutants were able to better withstand a hypochlorous acid (HOCl) challenge than a ΔureABdeletion strain. The capacity of purified urease to protect whole cells via oxidant quenching was assessed by adding urease enzyme to nongrowing HOCl-exposed cells. No wild-type cells were recovered with oxidant alone, whereas urease addition significantly aided viability. These results suggest that urease can protectH. pyloriagainst oxidative damage and that the protective ability is distinct from the well-characterized catalytic role. To determine the capability of methionine sulfoxide reductase (Msr) to reduce oxidized Met residues in urease, purifiedH. pyloriurease was exposed to HOCl and a previously described Msr peptide repair mixture was added. Of the 25 methionine residues in urease, 11 were subject to both oxidation and to Msr-mediated repair, as identified by mass spectrometry (MS) analysis; therefore, the oxidant-quenchable Met pool comprising urease can be recycled by the Msr repair system. Noncatalytic urease appears to play an important role in oxidant protection.IMPORTANCEChronicHelicobacter pyloriinfection can lead to gastric ulcers and gastric cancers. The enzyme urease contributes to the survival of the bacterium in the harsh environment of the stomach by increasing the local pH. In addition to combating acid,H. pylorimust survive host-produced reactive oxygen species to persist in the gastric mucosa. We describe a cyclic amino acid-based antioxidant role of urease, whereby oxidized methionine residues can be recycled by methionine sulfoxide reductase to again quench oxidants. This work expands our understanding of the role of an already acknowledged pathogen virulence factor and specifically expands our knowledge ofH. pylorisurvival mechanisms.

scholarly journals Methionine Sulfoxide Reductase in Helicobacter pylori: Interaction with Methionine-Rich Proteins and Stress-Induced Expression

2006 ◽  
Vol 188 (16) ◽  
pp. 5839-5850 ◽  
Author(s):  
Praveen Alamuri ◽  
Robert J. Maier
Keyword(s):  
Helicobacter Pylori ◽  
Specific Activity ◽  
Methionine Sulfoxide ◽  
Stress Conditions ◽  
Methionine Sulfoxide Reductase ◽  
Iron Starvation ◽  
Protein Levels ◽  
Cell Extracts ◽  
Methionine Residues ◽  
H Pylori

ABSTRACT The reductive repair of oxidized methionine residues performed by methionine sulfoxide reductase is important for the gastric pathogen Helicobacter pylori to maintain persistent stomach colonization. Methionine-containing proteins that are targeted for repair by Msr were identified from whole-cell extracts (after cells were exposed to O2 stress) by using a coimmunoprecipitation approach. Proteins identified as Msr-interacting included catalase, GroEL, thioredoxin-1 (Trx1), and site-specific recombinase; with one exception (Trx1, the reductant for Msr) all these proteins have approximately twofold higher methionine (Met) content than other proteins. These Met-rich proteins were purified and were shown to individually form a cross-linked adduct with Msr. Catalase-specific activity in an msr strain was one-half that of the parent strain; this difference was only observed under oxidative stress conditions, and the activity was restored to nearly wild-type levels by adding Msr plus dithiothreitol to msr strain extracts. In agreement with the cross-linking study, pure Msr used Trx1 but not Trx2 as a reductant. Comparative structure modeling classified the H. pylori Msr in class II within the MsrB family, like the Neisseria enzymes. Pure H. pylori enzyme reduced only the R isomer of methyl p-tolyl-sulfoxide with an apparent Km of 4.1 mM for the substrate. Stress conditions (peroxide, peroxynitrite, and iron starvation) all caused approximately 3- to 3.5-fold transcriptional up-regulation of msr. Neither the O2 level during growth nor the use of background regulatory mutants had a significant effect on msr transcription. Late log and stationary phase cultures had the highest Msr protein levels and specific activity.

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 Growth Phase Regulation of flaA Expression in Helicobacter pylori Is luxS Dependent

2004 ◽  
Vol 72 (9) ◽  
pp. 5506-5510 ◽  
Author(s):  
John T. Loh ◽  
Mark H. Forsyth ◽  
Timothy L. Cover
Keyword(s):  
Helicobacter Pylori ◽  
Growth Phase ◽  
Wild Type ◽  
Phase Dependence ◽  
Autoinducer 2 ◽  
Extracellular Signaling ◽  
Reporter Strains ◽  
Phase Regulation ◽  
H Pylori

ABSTRACT LuxS plays a role in the synthesis of an extracellular signaling molecule, autoinducer 2 (AI-2). To analyze a possible role of AI-2 in regulating Helicobacter pylori gene expression, we constructed a panel of transcriptional reporter strains. We show that the expression of H. pylori flaA is growth phase dependent and that flaA transcription increases in association with increased culture density. Mutating the luxS gene eliminates growth-phase-dependent control of flaA, and this growth phase dependence is restored when the luxS mutant strain is complemented with the wild-type luxS gene.

scholarly journals Colonization and Inflammation Deficiencies in Mongolian Gerbils Infected by Helicobacter pylori Chemotaxis Mutants

2005 ◽  
Vol 73 (3) ◽  
pp. 1820-1827 ◽  
Author(s):  
David J. McGee ◽  
Melanie L. Langford ◽  
Emily L. Watson ◽  
J. Elliot Carter ◽  
Yu-Ting Chen ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Immune Cell ◽  
Response Regulator ◽  
Critical Role ◽  
Mongolian Gerbils ◽  
Wild Type ◽  
In The Wild ◽  
H Pylori

ABSTRACT Helicobacter pylori causes disease in the human stomach and in mouse and gerbil stomach models. Previous results have shown that motility is critical for H. pylori to colonize mice, gerbils, and other animal models. The role of chemotaxis, however, in colonization and disease is less well understood. Two genes in the H. pylori chemotaxis pathway, cheY and tlpB, which encode the chemotaxis response regulator and a methyl-accepting chemoreceptor, respectively, were disrupted. The cheY mutation was complemented with a wild-type copy of cheY inserted into the chromosomal rdxA gene. The cheY mutant lost chemotaxis but retained motility, while all other strains were motile and chemotactic in vitro. These strains were inoculated into gerbils either alone or in combination with the wild-type strain, and colonization and inflammation were assessed. While the cheY mutant completely failed to colonize gerbil stomachs, the tlpB mutant colonized at levels similar to those of the wild type. With the tlpB mutant, there was a substantial decrease in inflammation in the gerbil stomach compared to that with the wild type. Furthermore, there were differences in the numbers of each immune cell in the tlpB-mutant-infected stomach: the ratio of lymphocytes to neutrophils was about 8 to 1 in the wild type but only about 1 to 1 in the mutant. These results suggest that the TlpB chemoreceptor plays an important role in the inflammatory response while the CheY chemotaxis regulator plays a critical role in initial colonization. Chemotaxis mutants may provide new insights into the steps involved in H. pylori pathogenesis.

scholarly journals In Helicobacter pylori, LuxS Is a Key Enzyme in Cysteine Provision through a Reverse Transsulfuration Pathway

2010 ◽  
Vol 192 (5) ◽  
pp. 1184-1192 ◽  
Author(s):  
Neil C. Doherty ◽  
Feifei Shen ◽  
Nigel M. Halliday ◽  
David A. Barrett ◽  
Kim R. Hardie ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Wild Type ◽  
Metabolic Role ◽  
Absolute Requirement ◽  
Transsulfuration Pathway ◽  
Cysteine Synthesis ◽  
Key Enzyme ◽  
H Pylori ◽  
Isogenic Strains

ABSTRACT In many bacteria, LuxS functions as a quorum-sensing molecule synthase. However, it also has a second, more central metabolic function in the activated methyl cycle (AMC), which generates the S-adenosylmethionine required by methyltransferases and recycles the product via methionine. Helicobacter pylori lacks an enzyme catalyzing homocysteine-to-methionine conversion, rendering the AMC incomplete and thus making any metabolic role of H. pylori LuxS (LuxSHp) unclear. Interestingly, luxS Hp is located next to genes annotated as cysK Hp and metB Hp, involved in other bacteria in cysteine and methionine metabolism. We showed that isogenic strains carrying mutations in luxS Hp, cysK Hp, and metB Hp could not grow without added cysteine (whereas the wild type could), suggesting roles in cysteine synthesis. Growth of the ΔluxS Hp mutant was restored by homocysteine or cystathionine and growth of the ΔcysK Hp mutant by cystathionine only. The ΔmetB Hp mutant had an absolute requirement for cysteine. Metabolite analyses showed that S-ribosylhomocysteine accumulated in the ΔluxS Hp mutant, homocysteine in the ΔcysK Hp mutant, and cystathionine in the ΔmetB Hp mutant. This suggests that S-ribosylhomocysteine is converted by LuxSHp to homocysteine (as in the classic AMC) and thence by CysKHp to cystathionine and by MetBHp to cysteine. In silico analysis suggested that cysK-metB-luxS were acquired by H. pylori from a Gram-positive source. We conclude that cysK-metB-luxS encode the capacity to generate cysteine from products of the incomplete AMC of H. pylori in a process of reverse transsulfuration. We recommend that the misnamed genes cysK Hp and metB Hp be renamed mccA (methionine-to-cysteine-conversion gene A) and mccB, respectively.

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

2007 ◽  
Vol 76 (1) ◽  
pp. 153-160 ◽  
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.

scholarly journals The Role of a Dipeptide Transporter in the Virulence of Human Pathogen, Helicobacter pylori

2021 ◽  
Vol 12 ◽  
Author(s):  
Xiaohong Xu ◽  
Junwei Chen ◽  
Xiaoxing Huang ◽  
Shunhang Feng ◽  
Xiaoyan Zhang ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Virulence Factors ◽  
Outer Membrane Proteins ◽  
Wild Type ◽  
Ags Cells ◽  
Dipeptide Transporter ◽  
Adhesion Level ◽  
H Pylori ◽  
Substrate Binding Protein

Helicobacter pylori harbors a dipeptide (Dpp) transporter consisting of a substrate-binding protein (DppA), two permeases (DppB and C), and two ATPases (DppD and F). The Dpp transporter is responsible for the transportation of dipeptides and short peptides. We found that its expression is important for the growth of H. pylori. To understand the role of the Dpp transporter in the pathogenesis of H. pylori, the expression of virulence factors and H. pylori-induced IL-8 production were investigated in H. pylori wild-type and isogenic H. pylori Dpp transporter mutants. We found that expression of CagA was downregulated, while expression of type 4 secretion system (T4SS) components was upregulated in Dpp transporter mutants. The DppA mutant strain expressed higher levels of outer membrane proteins (OMPs), including BabA, HopZ, OipA, and SabA, and showed a higher adhesion level to gastric epithelial AGS cells compared with the H. pylori 26695 wild-type strain. After infection of AGS cells, H. pylori ΔdppA induced a higher level of NF-κB activation and IL-8 production compared with wild-type. These results suggested that in addition to supporting the growth of H. pylori, the Dpp transporter causes bacteria to alter the expression of virulence factors and reduces H. pylori-induced NF-κB activation and IL-8 production in gastric epithelial cells.

scholarly journals Identification of a d-glycero-d-manno-Heptosyltransferase Gene from Helicobacter pylori

2005 ◽  
Vol 187 (15) ◽  
pp. 5156-5165 ◽  
Author(s):  
Koji Hiratsuka ◽  
Susan M. Logan ◽  
J. Wayne Conlan ◽  
Vandana Chandan ◽  
Annie Aubry ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Critical Role ◽  
Outer Core ◽  
Core Region ◽  
Wild Type ◽  
Core Oligosaccharide ◽  
Detailed Structural Analysis ◽  
Insertional Inactivation ◽  
H Pylori

ABSTRACT We have identified a Helicobacter pylori d-glycero-d-manno-heptosyltransferase gene, HP0479, which is involved in the biosynthesis of the outer core region of H. pylori lipopolysaccharide (LPS). Insertional inactivation of HP0479 resulted in formation of a truncated LPS molecule lacking an α-1,6-glucan-, dd-heptose-containing outer core region and O-chain polysaccharide. Detailed structural analysis of purified LPS from HP0479 mutants of strains SS1, 26695, O:3, and PJ1 by a combination of chemical and mass spectrometric methods showed that HP0479 likely encodes α-1,2-d-glycero-d-manno-heptosyltransferase, which adds a d-glycero-d-manno-heptose residue (DDHepII) to a distal dd-heptose of the core oligosaccharide backbone of H. pylori LPS. When the wild-type HP0479 gene was reintegrated into the chromosome of strain 26695 by using an “antibiotic cassette swapping” method, the complete LPS structure was restored. Introduction of the HP0479 mutation into the H. pylori mouse-colonizing Sydney (SS1) strain and the clinical isolate PJ1, which expresses dd-heptoglycan, resulted in the loss of colonization in a mouse model. This indicates that H. pylori expressing a deeply truncated LPS is unable to successfully colonize the murine stomach and provides evidence for a critical role of the outer core region of H. pylori LPS in colonization.

scholarly journals Role of the Helicobacter pylori outer-membrane proteins AlpA and AlpB in colonization of the guinea pig stomach

2004 ◽  
Vol 53 (5) ◽  
pp. 375-379 ◽  
Author(s):  
Ramon de Jonge ◽  
Zarmina Durrani ◽  
Sjoerd G. Rijpkema ◽  
Ernst J. Kuipers ◽  
Arnoud H.M. van Vliet ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Membrane Proteins ◽  
Guinea Pig ◽  
Outer Membrane ◽  
Type Strain ◽  
Wild Type Strain ◽  
Outer Membrane Proteins ◽  
Wild Type ◽  
H Pylori

The human gastric pathogen Helicobacter pylori expresses several putative outer-membrane proteins (OMPs), but the role of individual OMPs in colonization of the stomach by H. pylori is still poorly understood. The role of four such OMPs (AlpA, AlpB, OipA and HopZ) in a guinea pig model of H. pylori infection has been investigated. Single alpA, alpB, hopZ and oipA isogenic mutants were constructed in the guinea pig-adapted, wild-type H. pylori strain GP15. Guinea pigs were inoculated intragastrically with the wild-type strain, single mutants or a mixture of the wild-type and a single mutant in a 1 : 1 ratio. Three weeks after infection, H. pylori could be isolated from stomach sections of all animals that were infected with the wild-type, the hopZ mutant or the oipA mutant, but from only five of nine (P = 0.18) and one of seven (P = 0.02) animals that were infected with the alpA or alpB mutants, respectively. The hopZ and oipA mutants colonized the majority of animals that were inoculated with the strain mixture, whereas alpA and alpB mutants could not be isolated from animals that were infected with the strain mixture (P < 0.01). Specific IgG antibody responses were observed in all animals that were infected with either the wild-type or a mutant, but IgG levels were lower in animals that were infected with either the alpA or the alpB mutants, compared to the wild-type strain (P < 0.05). In conclusion, absence of AlpA or AlpB is a serious disadvantage for colonization of the stomach by H. pylori.

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