Isolation of Helicobacter pylori Genes That Modulate Urease Activity

ABSTRACT Helicobacter pylori urease, a nickel-requiring metalloenzyme, hydrolyzes urea to NH3 and CO2. We sought to identify H. pylori genes that modulate urease activity by constructing pHP8080, a plasmid which encodes both H. pylori urease and the NixA nickel transporter. Escherichia coli SE5000 and DH5α transformed with pHP8080 resulted in a high-level urease producer and a low-level urease producer, respectively. An H. pylori DNA library was cotransformed into SE5000 (pHP8080) and DH5α (pHP8080) and was screened for cotransformants expressing either lowered or heightened urease activity, respectively. Among the clones carrying urease-enhancing factors, 21 of 23 contained hp0548, a gene that potentially encodes a DNA helicase found within the cag pathogenicity island, and hp0511, a gene that potentially encodes a lipoprotein. Each of these genes, when subcloned, conferred a urease-enhancing activity in E. coli (pHP8080) compared with the vector control. Among clones carrying urease-decreasing factors, 11 of 13 clones contained the flbA (also known asflhA) flagellar biosynthesis/regulatory gene (hp1041), an lcrD homolog. The LcrD protein family is involved in type III secretion and flagellar secretion in pathogenic bacteria. Almost no urease activity was detected in E. coli (pHP8080) containing the subcloned flbA gene. Furthermore, there was significantly reduced synthesis of the urease structural subunits in E. coli (pHP8080) containing the flbA gene, as determined by Western blot analysis with UreA and UreB antiserum. Thus, flagellar biosynthesis and urease activity may be linked in H. pylori. These results suggest that H. pylori genes may modulate urease activity.

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Acid-Responsive Gene Induction of Ammonia-Producing Enzymes in Helicobacter pylori Is Mediated via a Metal-Responsive Repressor Cascade

Infection and Immunity ◽

10.1128/iai.72.2.766-773.2004 ◽

2004 ◽

Vol 72 (2) ◽

pp. 766-773 ◽

Cited By ~ 65

Author(s):

Arnoud H. M. van Vliet ◽

Ernst J. Kuipers ◽

Jeroen Stoof ◽

Sophie W. Poppelaars ◽

Johannes G. Kusters

Keyword(s):

Helicobacter Pylori ◽

Urease Activity ◽

Low Ph ◽

Wild Type ◽

Gene Encoding ◽

Amidase Activity ◽

Adaptive Mechanisms ◽

Acidic Conditions ◽

H Pylori ◽

High Level

ABSTRACT Although the adaptive mechanisms allowing the gastric pathogen Helicobacter pylori to survive acid shocks have been well documented, the mechanisms allowing growth at mildly acidic conditions (pH ∼5.5) are still poorly understood. Here we demonstrate that H. pylori strain 26695 increases the transcription and activity of its urease, amidase, and formamidase enzymes four- to ninefold in response to growth at pH 5.5. Supplementation of growth medium with NiCl2 resulted in a similar induction of urease activity (at low NiCl2 concentration) and amidase activity (at ≥500 μM NiCl2) but did not affect formamidase activity. Mutation of the fur gene, which encodes an iron-responsive repressor of both amidases, resulted in a constitutively high level of amidase and formamidase activity at either pH but did not affect urease activity at pH 7.0 or pH 5.5. In contrast, mutation of the nikR gene, encoding the nickel-responsive activator of urease expression, resulted in a significant reduction of acid-responsive induction of amidase and formamidase activity. Finally, acid-responsive repression of fur transcription was absent in the H. pylori nikR mutant, whereas transcription of the nikR gene itself was increased at pH 5.5 in wild-type H. pylori. We hypothesize that H. pylori uses a repressor cascade to respond to low pH, with NikR initiating the response directly via the urease operon and indirectly via the members of the Fur regulon.

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The Helicobacter pylori flbA flagellar biosynthesis and regulatory gene is required for motility and virulence and modulates urease of H. pylori and Proteus mirabilis

Journal of Medical Microbiology ◽

10.1099/0022-1317-51-11-958 ◽

2002 ◽

Vol 51 (11) ◽

pp. 958-970 ◽

Cited By ~ 26

Author(s):

DAVID J. McGEE ◽

CHRISTOPHER COKER ◽

TRACI L. TESTERMAN ◽

JANETTE M. HARRO ◽

SUSAN V. GIBSON ◽

...

Keyword(s):

Helicobacter Pylori ◽

Proteus Mirabilis ◽

Regulatory Gene ◽

Flagellar Biosynthesis ◽

H Pylori

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Gastric Microenvironment—A Partnership between Innate Immunity and Gastric Microbiota Tricks Helicobacter pylori

Journal of Clinical Medicine ◽

10.3390/jcm10153258 ◽

2021 ◽

Vol 10 (15) ◽

pp. 3258

Author(s):

Cristina Oana Mărginean ◽

Lorena Elena Meliț ◽

Maria Oana Săsăran

Keyword(s):

Helicobacter Pylori ◽

Immune System ◽

Pathogenic Bacteria ◽

T Regulatory Cells ◽

Inflammatory Responses ◽

Mucosal Barrier ◽

Pathogen Recognition ◽

Microenvironmental Factors ◽

H Pylori ◽

Major Factors

Helicobacter pylori (H. pylori) carcinogenicity depends on three major factors: bacterial virulence constituents, environmental factors and host’s genetic susceptibility. The relationship between microenvironmental factors and H. pylori virulence factors are incontestable. H. pylori infection has a major impact on both gastric and colonic microbiota. The presence of non-H. pylori bacteria within the gastric ecosystem is particularly important since they might persistently act as an antigenic stimulus or establish a partnership with H. pylori in order to augment the subsequent inflammatory responses. The gastric ecosystem, i.e., microbiota composition in children with H. pylori infection is dominated by Streptoccocus, Neisseria, Rothia and Staphylococcus. The impairment of this ecosystem enhances growth and invasion of different pathogenic bacteria, further impairing the balance between the immune system and mucosal barrier. Moreover, altered microbiota due to H. pylori infection is involved in increasing the gastric T regulatory cells response in children. Since gastric homeostasis is defined by the partnership between commensal bacteria and host’s immune system, this review is focused on how pathogen recognition through toll-like receptors (TLRs—an essential class of pathogen recognition receptors—PRRs) on the surface of macrophages and dendritic cells impact the immune response in the setting of H. pylori infection. Further studies are required for delineate precise role of bacterial community features and of immune system components.

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Zerumbone Inhibits Helicobacter pylori Urease Activity

Molecules ◽

10.3390/molecules26092663 ◽

2021 ◽

Vol 26 (9) ◽

pp. 2663

Author(s):

Hyun Jun Woo ◽

Ji Yeong Yang ◽

Pyeongjae Lee ◽

Jong-Bae Kim ◽

Sa-Hyun Kim

Keyword(s):

Helicobacter Pylori ◽

Carbon Atom ◽

Natural Compound ◽

Urease Activity ◽

Negative Charge ◽

Positive Charge ◽

Gastric Epithelium ◽

Functional Inhibition ◽

Electrical Gradient ◽

H Pylori

Helicobacter pylori (H. pylori) produces urease in order to improve its settlement and growth in the human gastric epithelium. Urease inhibitors likely represent potentially powerful therapeutics for treating H. pylori; however, their instability and toxicity have proven problematic in human clinical trials. In this study, we investigate the ability of a natural compound extracted from Zingiber zerumbet Smith, zerumbone, to inhibit the urease activity of H. pylori by formation of urease dimers, trimers, or tetramers. As an oxygen atom possesses stronger electronegativity than the first carbon atom bonded to it, in the zerumbone structure, the neighboring second carbon atom shows a relatively negative charge (δ−) and the next carbon atom shows a positive charge (δ+), sequentially. Due to this electrical gradient, it is possible that H. pylori urease with its negative charges (such as thiol radicals) might bind to the β-position carbon of zerumbone. Our results show that zerumbone dimerized, trimerized, or tetramerized with both H. pylori urease A and urease B molecules, and that this formation of complex inhibited H. pylori urease activity. Although zerumbone did not affect either gene transcription or the protein expression of urease A and urease B, our study demonstrated that zerumbone could effectively dimerize with both urease molecules and caused significant functional inhibition of urease activity. In short, our findings suggest that zerumbone may be an effective H. pylori urease inhibitor that may be suitable for therapeutic use in humans.

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Whole-Genome Sequencing and Comparative Genomics of Three Helicobacter pylori Strains Isolated from the Stomach of a Patient with Adenocarcinoma

Pathogens ◽

10.3390/pathogens10030331 ◽

2021 ◽

Vol 10 (3) ◽

pp. 331

Author(s):

Montserrat Palau ◽

Núria Piqué ◽

M. José Ramírez-Lázaro ◽

Sergio Lario ◽

Xavier Calvet ◽

...

Keyword(s):

Helicobacter Pylori ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Virulence Factors ◽

Outer Membrane Proteins ◽

Whole Genome ◽

Flagellar Biosynthesis ◽

H Pylori ◽

Restriction Modification

Helicobacter pylori is a common pathogen associated with several severe digestive diseases. Although multiple virulence factors have been described, it is still unclear the role of virulence factors on H. pylori pathogenesis and disease progression. Whole genome sequencing could help to find genetic markers of virulence strains. In this work, we analyzed three complete genomes from isolates obtained at the same point in time from a stomach of a patient with adenocarcinoma, using multiple available bioinformatics tools. The genome analysis of the strains B508A-S1, B508A-T2A and B508A-T4 revealed that they were cagA, babA and sabB/hopO negative. The differences among the three genomes were mainly related to outer membrane proteins, methylases, restriction modification systems and flagellar biosynthesis proteins. The strain B508A-T2A was the only one presenting the genotype vacA s1, and had the most distinct genome as it exhibited fewer shared genes, higher number of unique genes, and more polymorphisms were found in this genome. With all the accumulated information, no significant differences were found among the isolates regarding virulence and origin of the isolates. Nevertheless, some B508A-T2A genome characteristics could be linked to the pathogenicity of H. pylori.

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E-Test or Agar Dilution for Metronidazole Susceptibility Testing of Helicobacter Pylori: Importance of the Prevalence of Metronidazole Resistance

10.21203/rs.3.rs-677597/v1 ◽

2021 ◽

Author(s):

Jinnan Chen ◽

Yu Huang ◽

Zhaohui Ding ◽

Xiao Liang ◽

Hong Lu

Keyword(s):

Helicobacter Pylori ◽

Susceptibility Testing ◽

Resistance Rate ◽

Test Method ◽

Major Error ◽

Metronidazole Resistance ◽

Agar Dilution ◽

H Pylori ◽

High Level ◽

Level Resistance

Abstract Background: A number of studies have shown that E-test overestimated the presence of Helicobacter pylori (H. pylori) resistance compared to agar dilution.Objective: The purpose of this study was to explore whether E-test could be an alternative for agar dilution to detect the metronidazole susceptibility of H. pylori.Method: E-test and agar dilution were used to assess susceptibility of H. pylori to metronidazole, clarithromycin and levofloxacin in 281 clinical isolates obtained from China where resistance was high. Cohen kappa analysis, McNemar test, essential and categorical agreement analysis were performed for these two methods. Results: Overall, the result of E-test showed similar prevalence of resistance rate to all antibiotics compared with agar dilution. The essential agreement (EA) of E-test method and agar dilution in the evaluation susceptibility of H. pylori to clarithromycin and levofloxacin were moderate, with 89.0% and 79.7% respectively, but only 45.9% for metronidazole. Results showed categorical agreement (CA) between E-test and agar dilution were 100% for both clarithromycin and levofloxacin. As for metronidazole, the CA was 98.7%, no major error was identified, and rate of very major error was 1.8%.Conclusion: E-test can be an alternative method to detect the metronidazole susceptibility of H. pylori in regions where high-level resistance is common.

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Development of an Internal Control for Evaluation and Standardization of a Quantitative PCR Assay for Detection of Helicobacter pylori in Drinking Water

Applied and Environmental Microbiology ◽

10.1128/aem.00687-07 ◽

2007 ◽

Vol 73 (22) ◽

pp. 7380-7387 ◽

Cited By ~ 20

Author(s):

Keya Sen ◽

Nancy A. Schable ◽

Dennis J. Lye

Keyword(s):

Helicobacter Pylori ◽

Drinking Water ◽

Quantitative Pcr ◽

Water Samples ◽

Sodium Thiosulfate ◽

Qpcr Assay ◽

E Coli ◽

H Pylori ◽

Labeled Probe ◽

Treated Drinking Water

ABSTRACT Due to metabolic and morphological changes that can prevent Helicobacter pylori cells in water from growing on conventional media, an H. pylori-specific TaqMan quantitative PCR (qPCR) assay was developed that uses a 6-carboxyfluorescein-labeled probe (A. E. McDaniels, L. Wymer, C. Rankin, and R. Haugland, Water Res. 39:4808-4816, 2005). However, proper internal controls are needed to provide an accurate estimate of low numbers of H. pylori in drinking water. In this study, the 135-bp amplicon described by McDaniels et al. was modified at the probe binding region, using PCR mutagenesis. The fragment was incorporated into a single-copy plasmid to serve as a PCR-positive control and cloned into Escherichia coli to serve as a matrix spike. It was shown to have a detection limit of five copies, using a VIC dye-labeled probe. A DNA extraction kit was optimized that allowed sampling of an entire liter of water. Water samples spiked with the recombinant E. coli cells were shown to behave like H. pylori cells in the qPCR assay. The recombinant E. coli cells were optimized to be used at 10 cells/liter of water, where they were shown not to compete with 5 to 3,000 cells of H. pylori in a duplex qPCR assay. Four treated drinking water samples spiked with H. pylori (100 cells) demonstrated similar cycle threshold values if the chlorine disinfectant was first neutralized by sodium thiosulfate.

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Genetic Diversity of the cagA gene of Helicobacter pylori strains from Sudanese Patients with Different Gastroduodenal Diseases

10.1101/19007435 ◽

2019 ◽

Author(s):

Hadeel Gassim Hassan ◽

Abeer Babiker Idris ◽

Mohamed A. Hassan ◽

Hisham N. Altayb ◽

Kyakonye Yasin ◽

...

Keyword(s):

Helicobacter Pylori ◽

Amino Acid ◽

Novel Mutation ◽

Amino Acid Residues ◽

Specific Amino Acid ◽

Gastrointestinal Malignancy ◽

High Incidence ◽

H Pylori ◽

High Level ◽

Chloride Method

AbstractBackgroundThere is an increase in the prevalence of Helicobacter pylori infection in Sudan, accompanied by a high incidence of upper gastrointestinal malignancy. The cytotoxin-associated gene cagA gene is a marker of a pathogenicity island (PAI) in H. pylori and plays a crucial role in determining the clinical outcome of Helicobacter infections.ObjectiveThis study aimed to determine the frequency and heterogeneity of the cagA gene of H. pylori and correlate the presence of cagA gene with clinical outcomes.Materials and methodsFifty endoscopy biopsies were collected from Fedail and Soba hospitals in Khartoum state. DNA was extracted using the Guanidine chloride method followed by PCR to amplify 16S rRNA and cagA gene of H. pylori using specific primers. DNA amplicons of cagA gene were purified and sequenced. Bioinformatics and statistical analysis were done to characterize and to test the association between cagA gene and gastric complications.ResultsCagA gene was detected in 20/37(54%) of the samples that were found positive for H. pylori. There was no association between endoscopy finding and the presence of the cagA gene (p = 0.225). Specific amino acid variations were found at seven loci related to strains from a patient with duodenitis, gastric ulcer, and gastric atrophy (R448H, T457K, S460L, IT463-464VA, D470E, A482Q, KNV490-491-492TKT) while mutations in cancerous strain were A439P, T457P, and H500Y.ConclusionDisease-specific variations of cagA of H. pylori strains, in the region of amino acid residues 428-510, were evident among Sudanese patients with different gastroduodenal diseases. A novel mutation (K458N) was detected in a patient with duodenitis, which affects the positive electrostatic surface of cagA. Phylogenetic analysis showed a high level of diversity of cagA from Sudanese H. pylori strains.

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Carbonic Anhydrases: New Perspectives on Protein Functional Role and Inhibition in Helicobacter pylori

Frontiers in Microbiology ◽

10.3389/fmicb.2021.629163 ◽

2021 ◽

Vol 12 ◽

Author(s):

Cristina Campestre ◽

Viviana De Luca ◽

Simone Carradori ◽

Rossella Grande ◽

Vincenzo Carginale ◽

...

Keyword(s):

Helicobacter Pylori ◽

Pathogenic Bacteria ◽

Acid Tolerance ◽

Membrane Vesicles ◽

Research Field ◽

Outer Membrane Vesicles ◽

Bacterial Toxin ◽

Carbonic Anhydrases ◽

Ph Homeostasis ◽

H Pylori

Our understanding of the function of bacterial carbonic anhydrases (CAs, EC 4.2.1.1) has increased significantly in the last years. CAs are metalloenzymes able to modulate CO2, HCO3– and H+ concentration through their crucial role in catalysis of reversible CO2 hydration (CO2 + H2O ⇄ HCO3– + H+). In all living organisms, CA activity is linked to physiological processes, such as those related to the transport and supply of CO2 or HCO3–, pH homeostasis, secretion of electrolytes, biosynthetic processes and photosynthesis. These important processes cannot be ensured by the very low rate of the non-catalyzed reaction of CO2 hydration. It has been recently shown that CAs are important biomolecules for many bacteria involved in human infections, such as Vibrio cholerae, Brucella suis, Salmonella enterica, Pseudomonas aeruginosa, and Helicobacter pylori. In these species, CA activity promotes microorganism growth and adaptation in the host, or modulates bacterial toxin production and virulence. In this review, recent literature in this research field and some of the above-mentioned issues are discussed, namely: (i) the implication of CAs from bacterial pathogens in determining the microorganism growth and virulence; (ii) the druggability of these enzymes using classical CA inhibitors (CAIs) of the sulfonamide-type as examples; (iii) the role played by Helicobacter pylori CAs in the acid tolerance/adaptation of the microbe within the human abdomen; (iv) the role of CAs played in the outer membrane vesicles spawned by H. pylori in its planktonic and biofilm phenotypes; (v) the possibility of using H. pylori CAIs in combination with probiotic strains as a novel anti-ulcer treatment approach. The latter approach may represent an innovative and successful strategy to fight gastric infections in the era of increasing resistance of pathogenic bacteria to classical antibiotics.

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The role of acid inhibition in Helicobacter pylori eradication

F1000Research ◽

10.12688/f1000research.8598.1 ◽

2016 ◽

Vol 5 ◽

pp. 1747 ◽

Cited By ~ 10

Author(s):

David R. Scott ◽

George Sachs ◽

Elizabeth A. Marcus

Keyword(s):

Helicobacter Pylori ◽

Urease Activity ◽

Eradication Therapy ◽

Acid Inhibition ◽

Acid Suppression ◽

Helicobacter Pylori Eradication ◽

Ulcer Disease ◽

Chronic Active Gastritis ◽

H Pylori

Infection of the stomach by the gastric pathogen Helicobacter pylori results in chronic active gastritis and leads to the development of gastric and duodenal ulcer disease and gastric adenocarcinoma. Eradication of H. pylori infection improves or resolves the associated pathology. Current treatments of H. pylori infection rely on acid suppression in combination with at least two antibiotics. The role of acid suppression in eradication therapy has been variously attributed to antibacterial activity of proton pump inhibitors directly or through inhibition of urease activity or increased stability and activity of antibiotics. Here we discuss the effect of acid suppression on enhanced replicative capacity of H. pylori to permit the bactericidal activity of growth-dependent antibiotics. The future of eradication therapy will rely on improvement of acid inhibition along with current antibiotics or the development of novel compounds targeting the organism’s ability to survive in acid.

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