Expanding the Helicobacter pylori Genetic Toolbox: Modification of an Endogenous Plasmid for Use as a Transcriptional Reporter and Complementation Vector

ABSTRACT Helicobacter pylori is an important human pathogen. However, the study of this organism is often limited by a relative shortage of genetic tools. In an effort to expand the methods available for genetic study, an endogenous H. pylori plasmid was modified for use as a transcriptional reporter and as a complementation vector. This was accomplished by addition of an Escherichia coli origin of replication, a kanamycin resistance cassette, a promoterless gfpmut3 gene, and a functional multiple cloning site to form pTM117. The promoters of amiE and pfr, two well-characterized Fur-regulated promoters, were fused to the promoterless gfpmut3, and green fluorescent protein (GFP) expression of the fusions in wild-type and Δfur strains was analyzed by flow cytometry under iron-replete and iron-depleted conditions. GFP expression was altered as expected based on current knowledge of Fur regulation of these promoters. RNase protection assays were used to determine the ability of this plasmid to serve as a complementation vector by analyzing amiE, pfr, and fur expression in wild-type and Δfur strains carrying a wild-type copy of fur on the plasmid. Proper regulation of these genes was restored in the Δfur background under high- and low-iron conditions, signifying complementation of both iron-bound and apo Fur regulation. These studies show the potential of pTM117 as a molecular tool for genetic analysis of H. pylori.

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Stable Accumulation of σ54 in Helicobacter pylori Requires the Novel Protein HP0958

Journal of Bacteriology ◽

10.1128/jb.187.13.4463-4469.2005 ◽

2005 ◽

Vol 187 (13) ◽

pp. 4463-4469 ◽

Cited By ~ 24

Author(s):

Lara Pereira ◽

Timothy R. Hoover

Keyword(s):

Helicobacter Pylori ◽

Hypothetical Protein ◽

Kanamycin Resistance ◽

Wild Type ◽

Protein Protein Interaction ◽

In The Wild ◽

Interaction Map ◽

H Pylori ◽

A Minor ◽

Flagellar Biogenesis

ABSTRACT Several flagellar genes in Helicobacter pylori are dependent on σ54 (RpoN) for their expression. These genes encode components of the basal body, the hook protein, and a minor flagellin, FlaB. A protein-protein interaction map for H. pylori constructed from a high-throughput screen of a yeast two-hybrid assay (http://pim.hybrigenics.com/pimrider ext/common/) revealed interactions between σ54 and the conserved hypothetical protein HP0958. To see if HP0958 influences σ54 function, the corresponding gene was disrupted with a kanamycin resistance gene (aphA3) in H. pylori ATCC 43504 and the resulting mutant was analyzed. The hp0958:aphA3 mutant was nonmotile and failed to produce flagella. Introduction of a functional copy of hp0958 into the genome of the hp0958:aphA3 mutant restored flagellar biogenesis and motility. The hp0958:aphA3 mutant was deficient in expressing two σ54-dependent reporter genes, flaB′-′xylE and hp1120′-′xylE. Levels of σ54 in the hp0958 mutant were substantially lower than those in the parental strain, suggesting that the failure of the mutant to express the genes in the RpoN regulon and produce flagella was due to reduced σ54 levels. Expressing σ54 at high levels by putting rpoN under the control of the ureA promoter restored flagellar biogenesis and motility in the hp0958:aphA3 mutant. Turnover of σ54 was more rapid in the hp0958:aphA3 mutant than it was in the wild-type strain, suggesting that HP0958 supports wild-type σ54 levels in H. pylori by protecting it from proteolysis.

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The Helicobacter pylori Anti-Sigma Factor FlgM Is Predominantly Cytoplasmic and Cooperates with the Flagellar Basal Body Protein FlhA

Journal of Bacteriology ◽

10.1128/jb.00018-09 ◽

2009 ◽

Vol 191 (15) ◽

pp. 4824-4834 ◽

Cited By ~ 18

Author(s):

Melanie Rust ◽

Sophie Borchert ◽

Eike Niehus ◽

Sarah A. Kuehne ◽

Eugenia Gripp ◽

...

Keyword(s):

Helicobacter Pylori ◽

Basal Body ◽

Sigma Factor ◽

Fluorescent Protein ◽

Regulatory Function ◽

Wild Type ◽

Body Protein ◽

Terminal Domain ◽

Flagellar Proteins ◽

H Pylori

ABSTRACT Helicobacter pylori requires flagellar motility and orientation to persist actively in its habitat. A particular feature of flagella in most Helicobacter species including H. pylori is a membraneous flagellar sheath. The anti-sigma factor FlgM of H. pylori is unusual, since it lacks an N-terminal domain present in other FlgM homologs, e.g., FlgM of Salmonella spp., whose regulatory function is intimately coupled to its secretion through the flagellar type III secretion system. The aim of the present study was to characterize the localization and secretion of the short H. pylori FlgM in the presence of a flagellar sheath and to elucidate its interaction with other flagellar proteins, such as the basal body protein FlhA, which was previously shown to cooperate with FlgM for regulation. H. pylori FlgM was only released into the medium in minor amounts in wild-type bacteria, where the bulk amount of the protein was retained in the cytoplasm. Some FlgM was detected in the flagellar fraction. FlgM was expressed in flhA mutants and was less soluble and differentially localized in bacterial fractions of the flhA mutant in comparison to wild-type bacteria. FlgM-green fluorescent protein and FlgM-V5 translational fusions were generated and expressed in H. pylori. FlgM displayed a predominantly polar distribution and interacted with the C-terminal domain of FlhA (FlhAC). We suggest that, in H. pylori, FlgM secretion may not be paramount for its regulatory function and that protein interactions at the flagellar basal body may determine the turnover and localization of functional FlgM.

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Methylation Motifs in Promoter Sequences May Contribute to the Maintenance of a Conserved m5C Methyltransferase in Helicobacter pylori

Microorganisms ◽

10.3390/microorganisms9122474 ◽

2021 ◽

Vol 9 (12) ◽

pp. 2474

Author(s):

Bowen Meng ◽

Naomi Epp ◽

Winsen Wijaya ◽

Jan Mrázek ◽

Timothy R. Hoover

Keyword(s):

Helicobacter Pylori ◽

Fluorescent Protein ◽

Reporter Genes ◽

Dna Methyltransferases ◽

Wild Type ◽

Promoter Sequences ◽

Expression Of Genes ◽

Gfp Reporter ◽

H Pylori ◽

Green Fluorescent

DNA methylomes of Helicobacter pylori strains are complex due to the large number of DNA methyltransferases (MTases) they possess. H. pylori J99 M.Hpy99III is a 5-methylcytosine (m5C) MTase that converts GCGC motifs to Gm5CGC. Homologs of M.Hpy99III are found in essentially all H. pylori strains. Most of these homologs are orphan MTases that lack a cognate restriction endonuclease, and their retention in H. pylori strains suggest they have roles in gene regulation. To address this hypothesis, green fluorescent protein (GFP) reporter genes were constructed with six putative promoters that had a GCGC motif in the extended −10 region, and the expression of the reporter genes was compared in wild-type H. pylori G27 and a mutant lacking the M.Hpy99III homolog (M.HpyGIII). The expression of three of the GFP reporter genes was decreased significantly in the mutant lacking M.HpyGIII. In addition, the growth rate of the H. pylori G27 mutant lacking M.HpyGIII was reduced markedly compared to that of the wild type. These findings suggest that the methylation of the GCGC motif in many H. pylori GCGC-containing promoters is required for the robust expression of genes controlled by these promoters, which may account for the universal retention of M.Hpy99III homologs in H. pylori strains.

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In Vivo Behavior of a Helicobacter pylori SS1 nixA Mutant with Reduced Urease Activity

Infection and Immunity ◽

10.1128/iai.70.2.685-691.2002 ◽

2002 ◽

Vol 70 (2) ◽

pp. 685-691 ◽

Cited By ~ 55

Author(s):

Kylie J. Nolan ◽

David J. McGee ◽

Hazel M. Mitchell ◽

Tassia Kolesnikow ◽

Janette M. Harro ◽

...

Keyword(s):

Helicobacter Pylori ◽

Urease Activity ◽

Kanamycin Resistance ◽

Wild Type ◽

Bacterial Colony ◽

Nickel Ions ◽

Kanamycin Resistance Cassette ◽

Mutant Strains ◽

H Pylori

ABSTRACT Helicobacter pylori mutants devoid of urease activity fail to colonize the gastric mucosa of mice; however, the effect of decreased levels of urease on colonization has not been examined. The nixA gene, required for full urease activity, encodes a cytoplasmic membrane nickel transporter that imports nickel ions and leads to incorporation of nickel ions into apourease. A nixA mutant of the Sydney strain of H. pylori (SS1) was constructed by disruption of the nixA gene with a kanamycin resistance cassette. This mutant retained only half the urease activity of the wild-type (wild-type) SS1 strain. C57BL/6j (n = 75) and BALB/c (n = 75) mice were inoculated independently with the wild-type or the nixA strain. The level and distribution of colonization were assessed by bacterial colony counts and histological grading at 4, 12, and 24 weeks postinfection. Colonization levels of the nixA strain in BALB/c mice were significantly lower compared with SS1 (P = 0.005), while colonization in C57BL/6j mice was similar for both the wild-type and mutant strains. Subtle differences in colonization of the different regions of the stomach, determined by microscopic grading, were observed between wild-type SS1 and the nixA strain in BALB/c mice. On the contrary, when C57BL/6j (n = 35) and BALB/c (n = 35) mice were coinfected with the wild-type and nixA strains simultaneously, the nixA mutant failed to colonize and was outcompeted by the wild-type SS1 strain, which established normal levels of colonization. These results demonstrate the importance of the nixA gene for increasing the fitness of H. pylori for gastric colonization. Since nixA is required for full urease activity, the decreased fitness of the nixA mutant is likely due to reduced urease activity; however, pleiotropic effects of the mutation cannot be completely ruled out.

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The Helicobacter pylori Autotransporter ImaA (HP0289) Modulates the Immune Response and Contributes to Host Colonization

Infection and Immunity ◽

10.1128/iai.00312-12 ◽

2012 ◽

Vol 80 (7) ◽

pp. 2286-2296 ◽

Cited By ~ 13

Author(s):

William E. Sause ◽

Andrea R. Castillo ◽

Karen M. Ottemann

Keyword(s):

Immune Response ◽

Helicobacter Pylori ◽

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Dependent Manner ◽

Wild Type ◽

Content Type ◽

H Pylori ◽

Murine Infections

ABSTRACTThe human pathogenHelicobacter pyloriemploys a diverse collection of outer membrane proteins to colonize, persist, and drive disease within the acidic gastric environment. In this study, we sought to elucidate the function of the host-induced geneHP0289, which encodes an uncharacterized outer membrane protein. We first generated an isogenicH. pylorimutant that lacksHP0289and found that the mutant has a colonization defect in single-strain infections and is greatly outcompeted in mouse coinfection experiments with wild-typeH. pylori. Furthermore, we used protease assays and biochemical fractionation coupled with an HP0289-targeted peptide antibody to verify that the HP0289 protein resides in the outer membrane. Our previous findings showed that theHP0289promoter is upregulated in the mouse stomach, and here we demonstrate thatHP0289expression is induced under acidic conditions in an ArsRS-dependent manner. Finally, we have shown that theHP0289mutant induces greater expression of the chemokine interleukin-8 (IL-8) and the cytokine tumor necrosis factor alpha (TNF-α) in gastric carcinoma cells (AGS). Similarly, transcription of the IL-8 homolog keratinocyte-derived chemokine (KC) is elevated in murine infections with the HP0289 mutant than in murine infections with wild-typeH. pylori. On the basis of this phenotype, we renamed HP0289 ImaA forimmunomodulatoryautotransporter protein. Our work has revealed that genes inducedin vivoplay an important role inH. pyloripathogenesis. Specifically, the outer membrane protein ImaA modulates a component of the host inflammatory response, and thus may allowH. pylorito fine tune the host immune response based on ImaA expression.

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

mBio ◽

10.1128/mbio.01243-14 ◽

2014 ◽

Vol 5 (4) ◽

Cited By ~ 36

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.

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Mouse-Colonizing Helicobacter pylori SS1 Is Unusually Susceptible to Metronidazole Due to Two Complementary Reductase Activities

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.11.3127-3132.2000 ◽

2000 ◽

Vol 44 (11) ◽

pp. 3127-3132 ◽

Cited By ~ 18

Author(s):

Jin-Yong Jeong ◽

Douglas E. Berg

Keyword(s):

Helicobacter Pylori ◽

Kanamycin Resistance ◽

Phenotypic Traits ◽

Insertion Mutant ◽

Low Concentrations ◽

Mutant Strains ◽

Resistance Markers ◽

Clinically Significant ◽

H Pylori ◽

Derivatives Of

ABSTRACT In most strains of Helicobacter pylori, mutational inactivation of the rdxA (HP0954) gene, which encodes a nitroreductase that converts metronidazole (MTZ) from a harmless prodrug to a mutagenic and bacteriocidal product, is sufficient to make this pathogen resistant to clinically significant levels of MTZ. Here we report that SS1, a strain with the special ability to colonize mice, is unusual in being susceptible to very low concentrations of MTZ (0.5 μg/ml) and in being especially difficult to mutate to MTZ resistance (Mtzr). These phenotypic traits were traced to expression in this strain of the normally quiescent H. pylori frxAgene (HP0642, an rdxA paralog) along with rdxA. Transformation tests using rdxA::camand frxA::kan insertion mutant DNAs, with selection solely for the chloramphenicol and kanamycin resistance markers, and sequence analyses of frxA in spontaneous Mtzr derivatives of rdxA null mutant strains each showed that the development of Mtzr in SS1 required inactivation of both rdxA and frxA. Inactivation of either gene alone left SS1 susceptible to MTZ, although it was readily mutable from an MTZ-susceptible to an Mtzrphenotype. Reverse transcriptase PCR tests showed that frxAmRNA was at least 10-fold more abundant in SS1 than in reference strain 26695. It is proposed that these reductases play primarily nutritional roles during bacterial growth.

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

Infection and Immunity ◽

10.1128/iai.72.9.5506-5510.2004 ◽

2004 ◽

Vol 72 (9) ◽

pp. 5506-5510 ◽

Cited By ~ 27

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.

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The Helicobacter pylori Autotransporter ImaA Tempers the Bacterium's Interaction with α5β1 Integrin

Infection and Immunity ◽

10.1128/iai.00450-16 ◽

2016 ◽

Vol 85 (1) ◽

Cited By ~ 5

Author(s):

William E. Sause ◽

Daniela Keilberg ◽

Soufiane Aboulhouda ◽

Karen M. Ottemann

Keyword(s):

Helicobacter Pylori ◽

Host Cell ◽

Type Iv Secretion ◽

Host Cells ◽

Wild Type ◽

Content Type ◽

Type Iv ◽

H Pylori ◽

Α5β1 Integrin ◽

Cell Interface

ABSTRACT The human pathogen Helicobacter pylori uses the host receptor α5β1 integrin to trigger inflammation in host cells via its cag pathogenicity island (cag PAI) type IV secretion system (T4SS). Here, we report that the H. pylori ImaA protein (HP0289) decreases the action of the cag PAI T4SS via tempering the bacterium's interaction with α5β1 integrin. Previously, imaA-null mutants were found to induce an elevated inflammatory response that was dependent on the cag PAI T4SS; here we extend those findings to show that the elevated response is independent of the CagA effector protein. To understand how ImaA could be affecting cag PAI T4SS activity at the host cell interface, we utilized the Phyre structural threading program and found that ImaA has a region with remote homology to bacterial integrin-binding proteins. This region was required for ImaA function. Unexpectedly, we observed that imaA mutants bound higher levels of α5β1 integrin than wild-type H. pylori, an outcome that required the predicted integrin-binding homology region of ImaA. Lastly, we report that ImaA directly affected the amount of host cell β1 integrin but not other cellular integrins. Our results thus suggest a model in which H. pylori employs ImaA to regulate interactions between integrin and the T4SS and thus alter the host inflammatory strength.

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The Entner-Doudoroff Pathway Has Little Effect on Helicobacter pylori Colonization of Mice

Infection and Immunity ◽

10.1128/iai.71.5.2920-2923.2003 ◽

2003 ◽

Vol 71 (5) ◽

pp. 2920-2923 ◽

Cited By ~ 10

Author(s):

Amy E. Wanken ◽

Tyrrell Conway ◽

Kathryn A. Eaton

Keyword(s):

Helicobacter Pylori ◽

Wild Type ◽

Mutant Strains ◽

H Pylori ◽

Type Strains

ABSTRACT Helicobacter pylori mutants deficient in 6-phosphogluconate dehydratase (6PGD) were constructed. Colonization densities were lower and minimum infectious doses were higher for mutant strains than for wild-type strains. In spite of better colonization, however, wild-type strains did not displace the mutant in cocolonization experiments. Loss of 6PGD diminishes the fitness of H. pylori in vivo, but the pathway is nonessential for colonization.

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