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 Expanding Allelic Diversity of Helicobacter pylori vacA

1998 ◽  
Vol 36 (9) ◽  
pp. 2597-2603 ◽  
Author(s):  
Leen-Jan van Doorn ◽  
Céu Figueiredo ◽  
Ricardo Sanna ◽  
Salvador Pena ◽  
Peter Midolo ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Direct Sequencing ◽  
Allelic Diversity ◽  
Pcr Primers ◽  
Epidemiological Studies ◽  
Sequence Alignments ◽  
Gene Encoding ◽  
The World ◽  
Probe Assay ◽  
H Pylori

The diversity of the gene encoding the vacuolating cytotoxin (vacA) of Helicobacter pylori was analyzed in 98 isolates obtained from different geographic locations. The studies focused on variation in the previously defined s and m regions ofvacA, as determined by PCR and direct sequencing. Phylogenetic analysis revealed the existence of four distinct types of s-region alleles: aside from the previously described s1a, s1b, and s2 allelic types, a novel subtype, designated s1c, was found. Subtype s1c was observed exclusively in isolates from East Asia and appears to be the major s1 allele in that part of the world. Three different allelic forms (m1, m2a, and m2b) were detected in the m region. On the basis of sequence alignments, universal PCR primers that allow effective amplification of the s and m regions from H. pyloriisolates from all over the world were defined. Amplimers were subsequently analyzed by reverse hybridization onto a line probe assay (LiPA) that allows the simultaneous and highly specific hybridization of the different vacA s- and m-region alleles and tests for the presence of the cytotoxin-associated gene (cagA). This PCR-LiPA method permits rapid analysis of the vacA andcagA status of H. pylori strains for clinical and epidemiological studies and will facilitate identification of any further variations.

scholarly journals Characterization of a recombinant type II 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Helicobacter pylori

2005 ◽  
Vol 390 (1) ◽  
pp. 223-230 ◽  
Author(s):  
Celia J. Webby ◽  
Mark L. Patchett ◽  
Emily J. Parker
Keyword(s):  
Helicobacter Pylori ◽  
Sequence Similarity ◽  
Condensation Reaction ◽  
Enzyme Reaction ◽  
Single Type ◽  
Type I ◽  
Type Ii ◽  
Recombinant Type ◽  
H Pylori ◽  
Phosphate Synthase

DAH7P (3-Deoxy-D-arabino-heptulosonate 7-phosphate) synthase catalyses the condensation reaction between phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E4P) as the first committed step in the biosynthesis of aromatic compounds in plants and micro-organisms. Previous work has identified two families of DAH7P synthases based on sequence similarity and molecular mass, with the majority of the mechanistic and structural studies being carried out on the type I paralogues from Escherichia coli. Whereas a number of organisms possess genes encoding both type I and type II DAH7P synthases, the pathogen Helicobacter pylori has only a single, type II, enzyme. Recombinant DAH7P synthase from H. pylori was partially solubilized by co-expression with chaperonins GroEL/GroES in E. coli, and purified to homogeneity. The enzyme reaction follows an ordered sequential mechanism with the following kinetic parameters: Km (PEP), 3 μM; Km (E4P), 6 μM; and kcat, 3.3 s−1. The enzyme reaction involves interaction of the si face of PEP with the re face of E4P. H. pylori DAH7P synthase is not inhibited by phenylalanine, tyrosine, tryptophan or chorismate. EDTA inactivates the enzyme, and activity is restored by a range of bivalent metal ions, including (in order of decreasing effectiveness) Co2+, Mn2+, Ca2+, Mg2+, Cu2+ and Zn2+. Analysis of type II DAH7P synthase sequences reveals several highly conserved motifs, and comparison with the type I enzymes suggests that catalysis by these two enzyme types occurs on a similar active-site scaffold and that the two DAH7P synthase families may indeed be distantly related.

scholarly journals Virulent Strains of Helicobacter pylori Demonstrate Delayed Phagocytosis and Stimulate Homotypic Phagosome Fusion in Macrophages

2000 ◽  
Vol 191 (1) ◽  
pp. 115-128 ◽  
Author(s):  
Lee-Ann H. Allen ◽  
Larry S. Schlesinger ◽  
Byoung Kang
Keyword(s):  
Helicobacter Pylori ◽  
Mononuclear Phagocytes ◽  
Gastric Epithelium ◽  
Type I ◽  
Causative Role ◽  
Type Ii ◽  
Duodenal Ulcers ◽  
H Pylori ◽  
Phagosome Fusion ◽  
Bacterial Protein Synthesis

Helicobacter pylori colonizes the gastric epithelium of ∼50% of the world's population and plays a causative role in the development of gastric and duodenal ulcers. H. pylori is phagocytosed by mononuclear phagocytes, but the internalized bacteria are not killed and the reasons for this host defense defect are unclear. We now show using immunofluorescence and electron microscopy that H. pylori employs an unusual mechanism to avoid phagocytic killing: delayed entry followed by homotypic phagosome fusion. Unopsonized type I H. pylori bound readily to macrophages and were internalized into actin-rich phagosomes after a lag of ∼4 min. Although early (10 min) phagosomes contained single bacilli, H. pylori phagosomes coalesced over the next ∼2 h. The resulting “megasomes” contained multiple viable organisms and were stable for 24 h. Phagosome–phagosome fusion required bacterial protein synthesis and intact host microtubules, and both chloramphenicol and nocodazole increased killing of intracellular H. pylori. Type II strains of H. pylori are less virulent and lack the cag pathogenicity island. In contrast to type I strains, type II H. pylori were rapidly ingested and killed by macrophages and did not stimulate megasome formation. Collectively, our data suggest that megasome formation is an important feature of H. pylori pathogenesis.

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.

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 Construction and Characterization of aHelicobacter pylori clpB Mutant and Role of the Gene in the Stress Response

1998 ◽  
Vol 180 (2) ◽  
pp. 426-429 ◽  
Author(s):  
Elaine Allan ◽  
Peter Mullany ◽  
Soad Tabaqchali
Keyword(s):  
Helicobacter Pylori ◽  
High Temperature ◽  
Stress Response ◽  
Structural Features ◽  
High Temperature Stress ◽  
Gene Encoding ◽  
Increased Sensitivity ◽  
H Pylori

ABSTRACT Antiserum raised against whole Helicobacter pyloricells identified a novel 94-kDa antigen. The nucleotide sequence of the gene encoding the 94-kDa antigen was determined, and analysis of the deduced amino acid sequence revealed structural features typical of the ClpB ATPase family of stress response proteins. An isogenic H. pylori clpB mutant showed increased sensitivity to high-temperature stress, indicating that the clpB gene product functions as a stress response protein in H. pylori.

scholarly journals The fdxA Ferredoxin Gene Can Down-Regulate frxA Nitroreductase Gene Expression and Is Essential in Many Strains of Helicobacter pylori

2003 ◽  
Vol 185 (9) ◽  
pp. 2927-2935 ◽  
Author(s):  
Asish K. Mukhopadhyay ◽  
Jin-Yong Jeong ◽  
Daiva Dailidiene ◽  
Paul S. Hoffman ◽  
Douglas E. Berg
Keyword(s):  
Helicobacter Pylori ◽  
Metabolic Regulation ◽  
Metabolic Networks ◽  
Type I ◽  
Type Ii ◽  
Phenotypic Manifestation ◽  
Great Genetic Diversity ◽  
H Pylori ◽  
High Level ◽  
Homeostatic Mechanisms

ABSTRACT Very few examples of metabolic regulation are known in the gastric pathogen Helicobacter pylori. An unanticipated case was suggested, however, upon finding two types of metronidazole (Mtz)-susceptible strains: type I, in which frxA (which encodes a nitroreductase that contributes to Mtz susceptibility) is quiescent, and type II, in which frxA is well expressed. Here we report that inactivation of the fdxA ferredoxin gene (hp277) in type I strains resulted in high-level frxA expression (in effect, making them type II). However, fdxA null derivatives were obtained from only 6 of 32 type I strains tested that were readily transformed with an frxA::aphA marker. This suggested that fdxA is often essential. This essentiality was overcome in 4 of 20 strains by inactivating frxA, which suggested both that frxA overexpression is potentially deleterious and also that fdxA has additional, often vital roles. With type II strains, in contrast, fdxA null derivatives were obtained in 20 of 23 cases tested. Thus, fdxA is dispensable in most strains that normally exhibit (and tolerate) strong frxA expression. We propose that restraint of frxA expression helps maintain balanced metabolic networks in most type I strains, that other homeostatic mechanisms predominate in type II strains, and that these complex results constitute a phenotypic manifestation of H. pylori's great genetic diversity.

scholarly journals Characterization of the ArsRS Regulon of Helicobacter pylori, Involved in Acid Adaptation

2006 ◽  
Vol 188 (10) ◽  
pp. 3449-3462 ◽  
Author(s):  
Michael Pflock ◽  
Nadja Finsterer ◽  
Biju Joseph ◽  
Hans Mollenkopf ◽  
Thomas F. Meyer ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Response Regulator ◽  
Transcriptional Profiling ◽  
Acid Resistance ◽  
Two Component System ◽  
Gene Encoding ◽  
Acid Adaptation ◽  
Genes Encoding ◽  
H Pylori ◽  
A Site

ABSTRACT The human gastric pathogen Helicobacter pylori is extremely well adapted to the highly acidic conditions encountered in the stomach. The pronounced acid resistance of H. pylori relies mainly on the ammonia-producing enzyme urease; however, urease-independent mechanisms are likely to contribute to acid adaptation. Acid-responsive gene regulation is mediated at least in part by the ArsRS two-component system consisting of the essential OmpR-like response regulator ArsR and the nonessential cognate histidine kinase ArsS, whose autophosphorylation is triggered in response to low pH. In this study, by global transcriptional profiling of an ArsS-deficient H. pylori mutant grown at pH 5.0, we define the ArsR∼P-dependent regulon consisting of 109 genes, including the urease gene cluster, the genes encoding the aliphatic amidases AmiE and AmiF, and the rocF gene encoding arginase. We show that ArsR∼P controls the acid-induced transcription of amiE and amiF by binding to extended regions located upstream of the −10 box of the respective promoters. In contrast, transcription of rocF is repressed by ArsR∼P at neutral, acidic, and mildly alkaline pH via high-affinity binding of the response regulator to a site overlapping the promoter of the rocF gene.

scholarly journals Identification and Analysis of a NewvacA Genotype Variant of Helicobacter pylori in Different Patient Groups in Germany

1998 ◽  
Vol 36 (5) ◽  
pp. 1285-1289 ◽  
Author(s):  
Sonja Strobel ◽  
Stefan Bereswill ◽  
Peter Balig ◽  
Peter Allgaier ◽  
Hans-Günther Sonntag ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Mammalian Cells ◽  
Cell Damage ◽  
Clinical Manifestations ◽  
Peptic Ulceration ◽  
Control Group ◽  
Gene Encoding ◽  
Ulcer Disease ◽  
H Pylori ◽  
Epithelial Lesions

The vacuolating cytotoxin of Helicobacter pylori (VacA) is known to cause cell damage to mammalian cells and is suspected to give rise to gastric epithelial lesions that might lead to peptic ulcer disease. As shown recently, the gene encoding VacA exhibits genetic variation, with three different families of signal sequences (s1a, s1b, and s2) and two families of midregion sequences (m1 and m2). In order to investigate the relationship between the presence of specificvacA genotypes and peptic ulceration, the vacAgenotypes of 158 clinical isolates of H. pylori were determined. The study group consisted of 106 patients with duodenal ulceration; 52 patients with nonulcer dyspepsia (NUD) were used as controls. H. pylori of genotype s1 was isolated from 96% of the patients with ulcerations, whereas genotype s2 was only present in 4%, indicating a strong correlation between thevacA genotype and peptic ulceration (P < 0.001). In contrast, 31% of the patients from the NUD control group were infected with strains of vacA genotype s2. Particular midregion genotypes (m1 and m2) were not associated with clinical manifestations. The midregions from 18% of the isolates could not be classified by the proposed scheme. DNA sequencing revealed high homology between the untypeable midregions and that of genotype m1, with multiple base pair exchanges, some affecting the primer annealing site. Compared to those of m1 and m2 alleles, the divergent midregions from untypeable strains showed clustering, indicating the presence of a further subfamily of sequences in the midregion of vacA in German isolates, for which we propose the term “m1a.” A new specific primer that we designed for typing m1a isolates might be useful in other studies.

scholarly journals Metronidazole Activation Is Mutagenic and Causes DNA Fragmentation in Helicobacter pylori and inEscherichia coli Containing a Cloned H. pylori rdxA+ (Nitroreductase) Gene

2000 ◽  
Vol 182 (18) ◽  
pp. 5091-5096 ◽  
Author(s):  
Gary Sisson ◽  
Jin-Yong Jeong ◽  
Avery Goodwin ◽  
Louis Bryden ◽  
Norma Rossler ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
High Sensitivity ◽  
Wild Type ◽  
Gene Encoding ◽  
E Coli ◽  
Evolution Of Virulence ◽  
Rifampin Resistance ◽  
H Pylori ◽  
Emergence Of Resistance ◽  
Forward Mutation

ABSTRACT Much of the normal high sensitivity of wild-type Helicobacter pylori to metronidazole (Mtz) depends on rdxA(HP0954), a gene encoding a novel nitroreductase that catalyzes the conversion of Mtz from a harmless prodrug to a bactericidal agent. Here we report that levels of Mtz that partially inhibit growth stimulate forward mutation to rifampin resistance inrdxA + (Mtzs) and also inrdxA (Mtzr) H. pylori strains, and that expression of rdxA in Escherichia coliresults in equivalent Mtz-induced mutation. A reversion test using defined lac tester strains of E. coli carryingrdxA + indicated that CG-to-GC transversions and AT-to-GC transitions are induced more frequently than other base substitutions. Alkaline gel electrophoretic tests showed that Mtz concentrations near or higher than the MIC for growth also caused DNA breakage in H. pylori and in E. coli carryingrdxA +, suggesting that this damage may account for most of the bactericidal action of Mtz. Coculture of Mtzs H. pylori with E. coli (highly resistant to Mtz) in the presence of Mtz did not stimulate forward mutation in E. coli, indicating that the mutagenic and bactericidal products of Mtz metabolism do not diffuse significantly to neighboring (bystander) cells. Our results suggest that the widespread use of Mtz against other pathogens in people chronically infected withH. pylori may stimulate mutation and recombination inH. pylori, thereby speeding host-specific adaptation, the evolution of virulence, and the emergence of resistance against Mtz and other clinically useful antimicrobials.

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