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

2002 ◽  
Vol 70 (2) ◽  
pp. 685-691 ◽  
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.

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

2003 ◽  
Vol 71 (5) ◽  
pp. 2920-2923 ◽  
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.

scholarly journals Dependence of Helicobacter pylori Urease Activity on the Nickel-Sequestering Ability of the UreE Accessory Protein

2003 ◽  
Vol 185 (16) ◽  
pp. 4787-4795 ◽  
Author(s):  
Stéphane Benoit ◽  
Robert J. Maier
Keyword(s):  
Helicobacter Pylori ◽  
Urease Activity ◽  
Binding Capacity ◽  
Accessory Protein ◽  
Wild Type ◽  
Extra Copy ◽  
Nickel Ion ◽  
Partial Restoration ◽  
H Pylori

ABSTRACT The Helicobacter pylori ureE gene product was previously shown to be required for urease expression, but its characteristics and role have not been determined. The UreE protein has now been overexpressed in Escherichia coli, purified, and characterized, and three altered versions were expressed to address a nickel-sequestering role of UreE. Purified UreE formed a dimer in solution and was capable of binding one nickel ion per dimer. Introduction of an extra copy of ureE into the chromosome of mutants carrying mutations in the Ni maturation proteins HypA and HypB resulted in partial restoration of urease activity (up to 24% of the wild-type levels). Fusion proteins of UreE with increased ability to bind nickel were constructed by adding histidine-rich sequences (His-6 or His-10 to the C terminus and His-10 as a sandwich fusion) to the UreE protein. Each fusion protein was overexpressed in E. coli and purified, and its nickel-binding capacity and affinity were determined. Each construct was also expressed in wild-type H. pylori and in hypA and hypB mutant strains for determining in vivo urease activities. The urease activity was increased by introduction of all the engineered versions, with the greatest Ni-sequestering version (the His-6 version) also conferring the greatest urease activity on both the hypA and hypB mutants. The differences in urease activities were not due to differences in the amounts of urease peptides. Addition of His-6 to another expressed protein (triose phosphate isomerase) did not result in stimulation of urease, so urease activation is not related to the level of nonspecific protein-bound nickel. The results indicate a correlation between H. pylori urease activity and the nickel-sequestering ability of the UreE accessory protein.

scholarly journals In Vivo Complementation of ureB Restores the Ability of Helicobacter pylori To Colonize

2002 ◽  
Vol 70 (2) ◽  
pp. 771-778 ◽  
Author(s):  
Kathryn A. Eaton ◽  
Joanne V. Gilbert ◽  
Elizabeth A. Joyce ◽  
Amy E. Wanken ◽  
Tracy Thevenot ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Urease Activity ◽  
Wild Type ◽  
Colonization Density ◽  
H Pylori ◽  
Epithelial Lesions ◽  
Derivatives Of ◽  
Colonizing Ability

ABSTRACT The objective of this study was to determine (i) if complementation of ureB-negative Helicobacter pylori restores colonization and (ii) if urease is a useful reporter for promoter activity in vivo. Strains used were M6, M6ΔureB, and 10 recombinant derivatives of M6 or M6ΔureB in which urease expression was under the control of different H. pylori promoters. Mice were orally inoculated with either the wild type or one of the mutant strains, and colonization, in vivo urease activity, and extent of gastritis were determined. Of eight M6ΔureB recombinants tested, four colonized mice. Of those, three had the highest in vitro urease activity of any of the recombinants, significantly different from that of the noncolonizing mutants. The fourth colonizing recombinant, with ureB under control of the cag-15 promoter, had in vitro urease activity which did not differ significantly from the noncolonizing strains. In vivo, urease activities of the four colonizing transformants and the wild-type control were indistinguishable. There were no differences in gastritis or epithelial lesions between mice infected with M6 and those infected with the transformants. These results demonstrate that recovery of urease activity can restore colonizing ability to urease-negative H. pylori. They also suggest that cag-15 is upregulated in vivo, as was previously suggested by demonstrating that it is upregulated upon contact with epithelial cells. Finally, our results suggest that total urease activity and colonization density do not contribute to gastritis due to H. pylori.

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.

Enhanced fitness of a Helicobacter pylori babA mutant in a murine model

2021 ◽  
Author(s):  
M. Lorena Harvey ◽  
Aung Soe Lin ◽  
Lili Sun ◽  
Tatsuki Koyama ◽  
Jennifer H. B. Shuman ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Outer Membrane Proteins ◽  
Population Diversity ◽  
Fitness Advantage ◽  
Neutral Locus ◽  
Mutant Strains ◽  
Bacterial Fitness ◽  
H Pylori

Helicobacter pylori genomes encode >60 predicted outer membrane proteins (OMPs). Several OMPs in the Hop family act as adhesins, but the functions of most Hop proteins are unknown. To identify hop mutant strains that exhibit altered fitness in vivo compared to fitness in vitro , we used a genetic barcoding method that allowed us to track changes in the proportional abundance of H. pylori strains within a mixed population. We generated a library of hop mutant strains, each containing a unique nucleotide barcode, as well as a library of control strains, each containing a nucleotide barcode in an intergenic region predicted to be a neutral locus unrelated to bacterial fitness. We orogastrically inoculated each of the libraries into mice and analyzed compositional changes in the populations over time in vivo compared to changes detected in the populations during library passage in vitro . The control library proliferated as a relatively stable community in vitro, but there was a reduction in the population diversity of this library in vivo and marked variation in the dominant strains recovered from individual animals, consistent with the existence of a non-selective bottleneck in vivo . We did not identify any OMP mutants exhibiting fitness defects exclusively in vivo without corresponding fitness defects in vitro . Conversely, a babA mutant exhibited a strong fitness advantage in vivo but not in vitro . These findings, when taken together with results of other studies, suggest that production of BabA may have differential effects on H. pylori fitness depending on the environmental conditions.

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

2000 ◽  
Vol 44 (11) ◽  
pp. 3127-3132 ◽  
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.

scholarly journals Novel Functions for Glycosyltransferases Jhp0562 and GalT in Lewis Antigen Synthesis and Variation in Helicobacter pylori

2012 ◽  
Vol 80 (4) ◽  
pp. 1593-1605 ◽  
Author(s):  
Mary Ann Pohl ◽  
Sabine Kienesberger ◽  
Martin J. Blaser
Keyword(s):  
Helicobacter Pylori ◽  
Wild Type ◽  
Sequence Analyses ◽  
Upstream Gene ◽  
Content Type ◽  
Lewis Antigen ◽  
Mutant Strains ◽  
H Pylori

ABSTRACTLewis (Le) antigens are fucosylated oligosaccharides present in theHelicobacter pylorilipopolysaccharide. Expression of these antigens is believed to be important forH. pyloricolonization, since Le antigens also are expressed on the gastric epithelia in humans. A galactosyltransferase encoded by β-(1,3)galTis essential for production of type 1 (Leaand Leb) antigens. The upstream genejhp0562, which is present in many but not allH. pyloristrains, is homologous to β-(1,3)galTbut is of unknown function. BecauseH. pyloridemonstrates extensive intragenomic recombination, we hypothesized that these two genes could undergo DNA rearrangement. A PCR screen and subsequent sequence analyses revealed that the two genes can recombine at both the 5′ and 3′ ends. Chimeric β-(1,3)galT-like alleles can restore function in a β-(1,3)galTnull mutant, but neither native nor recombinantjhp0562can. Mutagenesis ofjhp0562revealed that it is essential for synthesis of both type 1 and type 2 Le antigens. Transcriptional analyses of both loci showed β-(1,3)galTexpression in all wild-type (WT) and mutant strains tested, whereasjhp0562was not expressed injhp0562null mutants, as expected. Sincejhp0562unexpectedly displayed functions in both type 1 and type 2 Le synthesis, we asked whethergalT, part of the type 2 synthesis pathway, had analogous functions in type 1 synthesis. Mutagenesis and complementation analysis confirmed thatgalTis essential for Lebproduction. In total, these results demonstrate thatgalTandjhp0562have functions that cross the expected Le synthesis pathways and thatjhp0562provides a substrate for intragenomic recombination to generate diverse Le synthesis enzymes.

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

2007 ◽  
Vol 73 (23) ◽  
pp. 7506-7514 ◽  
Author(s):  
Beth M. Carpenter ◽  
Timothy K. McDaniel ◽  
Jeannette M. Whitmire ◽  
Hanan Gancz ◽  
Silvia Guidotti ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Fluorescent Protein ◽  
Current Knowledge ◽  
Kanamycin Resistance ◽  
Multiple Cloning Site ◽  
Wild Type ◽  
Gfp Expression ◽  
Rnase Protection ◽  
Transcriptional Reporter ◽  
H Pylori

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.

scholarly journals Helicobacter pylori Containing Only Cytoplasmic Urease Is Susceptible to Acid

1998 ◽  
Vol 66 (11) ◽  
pp. 5060-5066 ◽  
Author(s):  
Partha Krishnamurthy ◽  
Mary Parlow ◽  
Jason B. Zitzer ◽  
Nimish B. Vakil ◽  
Harry L. T. Mobley ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Urease Activity ◽  
Acid Resistance ◽  
Etiologic Agent ◽  
Stationary Phase Culture ◽  
Colonization Factor ◽  
Acid Environment ◽  
H Pylori

ABSTRACT Helicobacter pylori, an important etiologic agent in a variety of gastroduodenal diseases, produces large amounts of urease as an essential colonization factor. We have demonstrated previously that urease is located within the cytoplasm and on the surface of H. pylori both in vivo and in stationary-phase culture. The purpose of the present study was to assess the relative contributions of cytoplasmic and surface-localized urease to the ability of H. pylori to survive exposure to acid in the presence of urea. Toward this end, we compared the acid resistance in vitro of H. pylori cells which possessed only cytoplasmic urease to that of bacteria which possessed both cytoplasmic and surface-localized or extracellular urease. Bacteria with only cytoplasmic urease activity were generated by using freshly subcultured bacteria or by treating repeatedly subcultured H. pylori with flurofamide (1 μM), a potent, but poorly diffusible urease inhibitor. H. pyloriwith cytoplasmic and surface-located urease activity survived in an acid environment when 5 mM urea was present. In contrast, H. pylori with only cytoplasmic urease shows significantly reduced survival when exposed to acid in the presence of 5 mM urea. Similarly,Escherichia coli SE5000 expressing H. pyloriurease and the Ni2+ transport protein NixA, which expresses cytoplasmic urease activity at levels similar to those in wild-typeH. pylori, survived minimally when exposed to acid in the presence of 5 to 50 mM urea. We conclude that cytoplasmic urease activity alone is not sufficient (although cytoplasmic urease activity is likely to be necessary) to allow survival of H. pyloriin acid; the activity of surface-localized urease is essential for resistance of H. pylori to acid under the assay conditions used. Therefore, the mechanism whereby urease becomes associated with the surface of H. pylori, which involves release of the enzyme from bacteria due to autolysis followed by adsorption of the enzyme to the surface of intact bacteria (“altruistic autolysis”), is essential for survival of H. pylori in an acid environment. The ability of H. pylori to survive exposure to low pH is likely to depend on a combination of both cytoplasmic and surface-associated urease activities.

scholarly journals Effect of Glycine on Helicobacter pylori In Vitro

2004 ◽  
Vol 48 (10) ◽  
pp. 3782-3788 ◽  
Author(s):  
Masaaki Minami ◽  
Takafumi Ando ◽  
Shin-nosuke Hashikawa ◽  
Keizo Torii ◽  
Tadao Hasegawa ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Eradication Therapy ◽  
Resistant Mutant ◽  
Synergistic Activity ◽  
Wild Type ◽  
Mutant Strains ◽  
The Difference ◽  
H Pylori ◽  
Isogenic Mutants

ABSTRACT Glycine is the simplest amino acid and is used as a metabolic product in some bacteria. However, an excess of glycine inhibits the growth of many bacteria, and it is used as a nonspecific antiseptic agent due to its low level of toxicity in animals. The effect of glycine on Helicobacter pylori is not precisely known. The present study was conducted to investigate (i) the effect of glycine on clarithromycin (CLR)-resistant and -susceptible strains of H. pylori, (ii) the effect of glycine in combination with amoxicillin (AMX), and (iii) the postantibiotic effect (PAE). The MIC at which 90% of strains are inhibited for glycine was almost 2.5 mg/ml for 31 strains of H. pylori, including CLR-resistant strains. We constructed isogenic CLR-resistant mutant strains by natural transformation and investigated the difference between clinical wild-type strains and isogenic mutants. There were no differences in the MICs between CLR-resistant and -susceptible strains or between clinical wild-type and mutant strains. The combination of AMX and glycine showed synergistic activity, with the minimum bactericidal concentration of AMX with glycine decreasing to 1/10 that of AMX alone. Glycine showed no PAE against H. pylori. These results suggest that glycine may be a useful antimicrobial agent against H. pylori not only alone but also in combination with antibacterial drugs for the treatment of H. pylori-associated diseases. Glycine may represent a component of a new type of eradication therapy for CLR-resistant H. pylori.

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