scholarly journals Helicobacter pylori ribBA-Mediated Riboflavin Production Is Involved in Iron Acquisition

1998 ◽  
Vol 180 (6) ◽  
pp. 1473-1479 ◽  
Author(s):  
Dennis J. Worst ◽  
Monique M. Gerrits ◽  
Christina M. J. E. Vandenbroucke-Grauls ◽  
Johannes G. Kusters
Keyword(s):  
Helicobacter Pylori ◽  
Iron Reduction ◽  
Ferric Iron ◽  
Iron Acquisition ◽  
Cosmid Library ◽  
Dot Blot ◽  
E Coli ◽  
Reduction Activity ◽  
Dna Fragment ◽  
H Pylori

ABSTRACT In this study, we cloned and sequenced a DNA fragment from an ordered cosmid library of Helicobacter pylori NCTC 11638 which confers to a siderophore synthesis mutant of Escherichia coli (EB53 aroB hemA) the ability to grow on iron-restrictive media and to reduce ferric iron. Sequence analysis of the DNA fragment revealed the presence of an open reading frame with high homology to the ribA gene of Bacillus subtilis. This gene encodes a bifunctional enzyme with the activities of both 3,4-dihydroxy-2-butanone 4-phosphate (DHBP) synthase and GTP cyclohydrolase II, which catalyze two essential steps in riboflavin biosynthesis. Expression of the gene (designatedribBA) resulted in the formation of one translational product, which was able to complement both the ribA and theribB mutation in E. coli. Expression ofribBA was iron regulated, as was suggested by the presence of a putative FUR box in its promotor region and as shown by RNA dot blot analysis. Furthermore, we showed that production of riboflavin inH. pylori cells is iron regulated. E. coliEB53 containing the plasmid with H. pylori ribBAexcreted riboflavin in the culture medium, and this riboflavin excretion also appeared to be iron regulated. We postulate that the iron-regulated production of riboflavin and ferric-iron-reduction activity by E. coli EB53 transformed with the H. pylori ribBA gene is responsible for the survival of EB53 on iron-restrictive medium. Because disruption of ribBA inH. pylori eliminates its ferric-iron-reduction activity, we conclude that ribBA has an important role in ferric-iron reduction and iron acquisition by H. pylori.

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

2007 ◽  
Vol 73 (22) ◽  
pp. 7380-7387 ◽  
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.

scholarly journals Antibacterial Activity of Geraniol in Combination with Standard Antibiotics against Staphylococcus aureus, Escherichia coli and Helicobacter pylori

2018 ◽  
Vol 13 (7) ◽  
pp. 1934578X1801300 ◽  
Author(s):  
Subrat Kumar Bhattamisra ◽  
Chew Hui Kuean ◽  
Lee Boon Chieh ◽  
Vivian Lee Yean Yan ◽  
Chin Koh Lee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Helicobacter Pylori ◽  
Antibacterial Activity ◽  
Synergistic Effect ◽  
Inhibitory Concentration ◽  
Therapeutic Potential ◽  
E Coli ◽  
Checkerboard Assay ◽  
H Pylori

The antibacterial activity of geraniol and its effect in combination with ampicillin, amoxicillin and clarithromycin against Staphylococcus aureus, Escherichia coli and Helicobacter pylori was tested. The minimum inhibitory concentrations (MICs) and combinatory effects of geraniol against the bacteria were assessed by using the modified broth microdilution and checkerboard assay, respectively. The combinatory effect is expressed as fractional inhibitory concentration index (FICI). The MIC of geraniol against S. aureus, E. coli and H. pylori was found to be 11200, 5600, and 7325 μg/mL, respectively. A significant synergistic effect was observed with geraniol and ampicillin against S. aureus with FICI in the range 0.19 to 0.32. Geraniol and ampicillin exhibited a partial synergistic effect against E. coli. A similar effect was observed with geraniol and clarithromycin against S. aureus. A partial synergistic effect was observed with clarithromycin and geraniol against H. pylori with the FICI value in the range 0.86 to 0.89. An additive effect was observed with geraniol and amoxicillin combination against H. pylori. However, the amoxicillin and clarithromycin dose was reduced by thirty-two fold when combined with geraniol against H. pylori. The anti- H. pylori effect of geraniol with clarithromycin and amoxicillin could be of potential interest in the treatment of H. pylori infection and associated ulcers in humans. Further, geraniol, in combination with other antibiotics, has substantial therapeutic potential against S. aureus and E.coli infection.

Protection against Helicobacter pylori infection in mice by intragastric vaccination with H. pylori antigens is achieved using a non-toxic mutant of E. coli heat-labile enterotoxin (LT) as adjuvant

Vaccine ◽  
1998 ◽  
Vol 16 (1) ◽  
pp. 33-37 ◽  
Author(s):  
Marta Marchetti ◽  
Michela Rossi ◽  
Valentina Giannelli ◽  
Marzia M Giuliani ◽  
Mariagrazia Pizza ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Helicobacter Pylori Infection ◽  
E Coli ◽  
Heat Labile ◽  
H Pylori

scholarly journals Bacterial killing in gastric juice – effect of pH and pepsin on Escherichia coli and Helicobacter pylori

2006 ◽  
Vol 55 (9) ◽  
pp. 1265-1270 ◽  
Author(s):  
H. Zhu ◽  
C. A. Hart ◽  
D. Sales ◽  
N. B. Roberts
Keyword(s):  
Escherichia Coli ◽  
Helicobacter Pylori ◽  
Gastric Juice ◽  
Bacterial Killing ◽  
E Coli ◽  
Test Organisms ◽  
H Pylori ◽  
Ph Range ◽  
K 12 ◽  
Rough Mutant

The susceptibility of Escherichia coli and Helicobacter pylori to pH and the effect of pepsin-mediated proteolysis were investigated. This was to establish the relative importance of their bacterial killing properties in gastric juice. Solutions in the pH range 1.5–7.4 with or without pig pepsin A were used, together with seven gastric juice samples obtained from patients undergoing routine gastric collection. Escherichia coli C690 (a capsulate strain), E. coli K-12 (a rough mutant) and Helicobacter pylori E5 were selected as the test organisms. Suspensions of bacteria (1×106 E. coli ml−1 and 1×108 H. pylori ml−1) were pre-incubated with test solutions at 37 °C for up to 2 h, and then cultured to establish the effect on subsequent growth. Survival of bacteria was diminished at pHs of less than 3.5, whereas killing required a pH of less than 2.5. Pre-incubation with pig pepsin at 0.5, 1.0 and 2.0 mg ml−1 at pH 3.5 reduced viable counts by 100 % for E. coli 690 and E. coli K-12 after 100 min incubation. With H. pylori, the viable counts decreased to 50 % of the control after 20 min incubation in 1 mg pepsin ml−1 at pH 2.5, 3.0 and 3.5. The gastric juices showed bactericidal activity at pH 3.5, and the rate of killing was juice dependent, with complete death of E. coli 690 occurring between 5 and 40 min post-incubation. Thus, killing of E. coli and H. pylori occurs optimally at pHs of less than 2.5. At pH 3.5, little effect is observed, whereas addition of pepsin alone or in gastric juice causes a marked increase in bacterial susceptibility, suggesting an important role for proteolysis in the killing of bacteria.

scholarly journals 16S rRNA Mutations That Confer Tetracycline Resistance in Helicobacter pylori Decrease Drug Binding in Escherichia coli Ribosomes

2005 ◽  
Vol 187 (11) ◽  
pp. 3708-3712 ◽  
Author(s):  
Lisa Nonaka ◽  
Sean R. Connell ◽  
Diane E. Taylor
Keyword(s):  
Escherichia Coli ◽  
Helicobacter Pylori ◽  
16S Rrna ◽  
Binding Site ◽  
Tetracycline Resistance ◽  
Drug Binding ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
E Coli ◽  
H Pylori

ABSTRACT Tetracycline resistance in clinical isolates of Helicobacter pylori has been associated with nucleotide substitutions at positions 965 to 967 in the 16S rRNA. We constructed mutants which had different sequences at 965 to 967 in the 16S rRNA gene present on a multicopy plasmid in Escherichia coli strain TA527, in which all seven rrn genes were deleted. The MICs for tetracycline of all mutants having single, double, or triple substitutions at the 965 to 967 region that were previously found in highly resistant H. pylori isolates were higher than that of the mutant exhibiting the wild-type sequence of tetracycline-susceptible H. pylori. The MIC of the mutant with the 965TTC967 triple substitution was 32 times higher than that of the E. coli mutant with the 965AGA967 substitution present in wild-type H. pylori. The ribosomes extracted from the tetracycline-resistant E. coli 965TTC967 variant bound less tetracycline than E. coli with the wild-type H. pylori sequence at this region. The concentration of tetracycline bound to the ribosome was 40% that of the wild type. The results of this study suggest that tetracycline binding to the primary binding site (Tet-1) of the ribosome at positions 965 to 967 is influenced by its sequence patterns, which form the primary binding site for tetracycline.

scholarly journals Implication of the Structure of the Helicobacter pylori cag Pathogenicity Island in Induction of Interleukin-8 Secretion

2001 ◽  
Vol 69 (3) ◽  
pp. 1625-1629 ◽  
Author(s):  
C. Audibert ◽  
C. Burucoa ◽  
B. Janvier ◽  
J. L. Fauchère
Keyword(s):  
Helicobacter Pylori ◽  
Clinical Presentation ◽  
Blot Hybridization ◽  
Pathogenicity Island ◽  
Interleukin 8 ◽  
Middle Region ◽  
Dot Blot ◽  
Long Distance ◽  
H Pylori

ABSTRACT Helicobacter pylori virulence is associated with the presence of the cag pathogenicity island (PAI). Thecag PAI is involved in the ability to induce interleukin-8 (IL-8) secretion by human cells, which is implicated in the inflammatory response of the gastric mucosa to H. pyloriinfection. The aim of this study was to determine whether the genetic structure of the cag PAI is conserved and whether it is linked to IL-8 induction ability. Detection of specific markers (cagA, picB, cag13-cag14, virD4, and IS605) by PCR and dot blot hybridization and long-distance PCR determination of the presence of cagI, cagII, and the middle region of thecag PAI were performed on 153 strains isolated from adults suffering from ulcers (n = 79) or gastritis (n = 74). IL-8 induction ability was evaluated by coculture of the strains with HEp-2 cells. Eighty-three strains (54.3%) had an entire cag PAI, 12 strains (7.8%) had thecag PAI split in two, 49 strains (32%) had nocag PAI, and 9 strains exhibited other structural combinations. The presence of an entire cag PAI was statistically correlated with the presence of IS605(P = 0.006) and the ability to induce IL-8 secretion but not with clinical presentation of the infection. The structure of the cag PAI appears to be rather conserved and is related to the proinflammatory power of a strain. The existence of strains inducing IL-8 secretion regardless of the cag PAI structure suggests that this region is not the only requirement for IL-8 secretion.

scholarly journals Cloning, sequencing, expression, purification and preliminary characterization of a type II dehydroquinase from Helicobacter pylori

1996 ◽  
Vol 319 (2) ◽  
pp. 559-565 ◽  
Author(s):  
Joanna R BOTTOMLEY ◽  
Christopher L. CLAYTON ◽  
Peter A. CHALK ◽  
Colin KLEANTHOUS
Keyword(s):  
Helicobacter Pylori ◽  
Sequence Data ◽  
Shikimate Pathway ◽  
Cosmid Library ◽  
Type Ii ◽  
Gene Encoding ◽  
Heat Stable ◽  
Plasmid Construct ◽  
H Pylori ◽  
Type Ii Dehydroquinase

A heat-stable dehydroquinase was purified to near homogeneity from a plate-grown suspension of the Gram-negative stomach pathogen Helicobacter pylori, and shown from both its subunit and native molecular masses to be a member of the type II family of dehydroquinases. This was confirmed by N-terminal amino acid sequence data. The gene encoding this activity was isolated following initial identification, by random sequencing of the H. pylori genome, of a 96 bp fragment, the translated sequence of which showed strong identity to a C-terminal region of other type II enzymes. Southern blot analysis of a cosmid library identified several potential clones, one of which complemented an Escherichia coliaroD point mutant strain deficient in host dehydroquinase. The gene encoding the H. pylori type II dehydroquinase (designated aroQ) was sequenced. The translated sequence was identical to the N-terminal sequence obtained directly from the purified protein, and showed strong identity to other members of the type II family of dehydroquinases. The enzyme was readily expressed in E. coli from a plasmid construct from which several milligrams of protein could be isolated, and the molecular mass of the protein was confirmed by electrospray MS. The aroQ gene in H. pylori may function in the central biosynthetic shikimate pathway of this bacterium, thus opening the way for the construction of attenuated strains as potential vaccines as well as offering a new target for selective enzyme inhibition.

scholarly journals Crystal Structure of Activated CheY1 from Helicobacter pylori

2010 ◽  
Vol 192 (9) ◽  
pp. 2324-2334 ◽  
Author(s):  
Kwok Ho Lam ◽  
Thomas Kin Wah Ling ◽  
Shannon Wing Ngor Au
Keyword(s):  
Helicobacter Pylori ◽  
Structural Rearrangement ◽  
Active Site Residues ◽  
Response Regulators ◽  
E Coli ◽  
Terminal Loop ◽  
Transduction Mechanisms ◽  
Important Virulence Factor ◽  
H Pylori ◽  
Preferential Recognition

ABSTRACT Chemotaxis is an important virulence factor for Helicobacter pylori colonization and infection. The chemotactic system of H. pylori is marked by the presence of multiple response regulators: CheY1, one CheY-like-containing CheA protein (CheAY2), and three CheV proteins. Recent studies have demonstrated that these molecules play unique roles in the chemotactic signal transduction mechanisms of H. pylori. Here we report the crystal structures of BeF3− -activated CheY1 from H. pylori resolved to 2.4 Å. Structural comparison of CheY1 with active-site residues of BeF3− -bound CheY from Escherichia coli and fluorescence quenching experiments revealed the importance of Thr84 in the phosphotransfer reaction. Complementation assays using various nonchemotactic E. coli mutants and pull-down experiments demonstrated that CheY1 displays differential association with the flagellar motor in E. coli. The structural rearrangement of helix 5 and the C-terminal loop in CheY1 provide a different interaction surface for FliM. On the other hand, interaction of the CheA-P2 domain with CheY1, but not with CheY2/CheV proteins, underlines the preferential recognition of CheY1 by CheA in the phosphotransfer reaction. Our results provide the first structural insight into the features of the H. pylori chemotactic system as a model for Epsilonproteobacteria.

scholarly journals Serum Antibody Responses to Helicobacter pylori and the cagA Marker in Patients with Mucosa-Associated Lymphoid Tissue Lymphoma

1999 ◽  
Vol 6 (4) ◽  
pp. 633-638 ◽  
Author(s):  
Anne Taupin ◽  
Alessandra Occhialini ◽  
Agnès Ruskone-Fourmestraux ◽  
Jean-Charles Delchier ◽  
Jean-Claude Rambaud ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Antibody Response ◽  
Malt Lymphoma ◽  
Lymphoid Tissue ◽  
Serum Antibody ◽  
Dot Blot ◽  
Homologous Strain ◽  
H Pylori ◽  
Different Strains ◽  
Dot Blot Analysis

ABSTRACT The lymphoma of the mucosa-associated lymphoid tissue (MALT) of the stomach has been linked to Helicobacter pylori infection, but the mechanisms involved in B-cell proliferation remain elusive. In a search for putative H. pylori-specific monoclonal immunoglobulin production, an H. pylori strain was isolated from 10 patients with MALT lymphoma and used to detect the specific serum antibody response to the homologous strain by immunoblotting. Moreover, the antigenicity of the different strains was compared by using each of the 10 sera. We found that the different strains induced highly variable patterns of systemic immunoglobulin G antibody response, although several bacterial antigens, such as the 60-kDa urease B, were often recognized by the different sera. ThecagA marker was detected in the strains by PCR with specific primers and by dot blot analysis, and the CagA protein was found in the sera of 4 of the 10 patients by immunoblotting. In conclusion, MALT lymphoma patients, like other patients with H. pylori gastritis, exhibit a polymorphic systemic antibody response, despite an apparently similar antigenic profile. The CagA marker of pathogenicity is not associated with this disease.

scholarly journals Curcumin Oxidation Is Required for Inhibition of Helicobacter pylori Growth, Translocation and Phosphorylation of Cag A

2021 ◽  
Vol 11 ◽  
Author(s):  
Ashwini Kumar Ray ◽  
Paula B. Luis ◽  
Surabhi Kirti Mishra ◽  
Daniel P. Barry ◽  
Mohammad Asim ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Growth Inhibition ◽  
Mrna Expression ◽  
Host Protein ◽  
Dependent Manner ◽  
Gene Encoding ◽  
E Coli ◽  
H Pylori

Curcumin is a potential natural remedy for preventing Helicobacter pylori-associated gastric inflammation and cancer. Here, we analyzed the effect of a phospholipid formulation of curcumin on H. pylori growth, translocation and phosphorylation of the virulence factor CagA and host protein kinase Src in vitro and in an in vivo mouse model of H. pylori infection. Growth of H. pylori was inhibited dose-dependently by curcumin in vitro. H. pylori was unable to metabolically reduce curcumin, whereas two enterobacteria, E. coli and Citrobacter rodentium, which efficiently reduced curcumin to the tetra- and hexahydro metabolites, evaded growth inhibition. Oxidative metabolism of curcumin was required for the growth inhibition of H. pylori and the translocation and phosphorylation of CagA and cSrc, since acetal- and diacetal-curcumin that do not undergo oxidative transformation were ineffective. Curcumin attenuated mRNA expression of the H. pylori virulence genes cagE and cagF in a dose-dependent manner and inhibited translocation and phosphorylation of CagA in gastric epithelial cells. H. pylori strains isolated from dietary curcumin-treated mice showed attenuated ability to induce cSrc phosphorylation and the mRNA expression of the gene encoding for IL-8, suggesting long-lasting effects of curcumin on the virulence of H. pylori. Our work provides mechanistic evidence that encourages testing of curcumin as a dietary approach to inhibit the virulence of CagA.

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