scholarly journals Genomic Methylation: a Tool for Typing Helicobacter pylori Isolates

2007 ◽  
Vol 73 (13) ◽  
pp. 4243-4249 ◽  
Author(s):  
Filipa F. Vale ◽  
Jorge M. B. Vítor
Keyword(s):  
Helicobacter Pylori ◽  
Dna Sequence ◽  
Dna Methyltransferase ◽  
Defense Mechanism ◽  
Methylation Status ◽  
Host Bacterium ◽  
Selfish Genetic Elements ◽  
Genomic Dnas ◽  
H Pylori ◽  
Genomic Methylation

ABSTRACT The genome sequences of three Helicobacter pylori strains revealed an abundant number of putative restriction and modification (R-M) systems within a small genome (1.60 to 1.67 Mb). Each R-M system includes an endonuclease that cleaves a specific DNA sequence and a DNA methyltransferase that methylates either adenosine or cytosine within the same DNA sequence. These are believed to be a defense mechanism, protecting bacteria from foreign DNA. They have been classified as selfish genetic elements; in some instances it has been shown that they are not easily lost from their host cell. Possibly because of this phenomenon, the H. pylori genome is very rich in R-M systems, with considerable variation in potential recognition sequences. For this reason the protective aspect of the methyltransferase gene has been proposed as a tool for typing H. pylori isolates. We studied the expression of H. pylori methyltransferases by digesting the genomic DNAs of 50 strains with 31 restriction endonucleases. We conclude that methyltransferase diversity is sufficiently high to enable the use of the genomic methylation status as a typing tool. The stability of methyltransferase expression was assessed by comparing the methylation status of genomic DNAs from strains that were isolated either from the same patient at different times or from different stomach locations (antrum and corpus). We found a group of five methyltransferases common to all tested strains. These five may be characteristic of the genetic pool analyzed, and their biological role may be important in the host/bacterium interaction.

scholarly journals Functional Analysis of the M.HpyAIV DNA Methyltransferase of Helicobacter pylori

2007 ◽  
Vol 189 (24) ◽  
pp. 8914-8921 ◽  
Author(s):  
Anna Skoglund ◽  
Britta Björkholm ◽  
Christina Nilsson ◽  
Anders F. Andersson ◽  
Cecilia Jernberg ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Restriction Enzyme ◽  
Dna Methyltransferase ◽  
Insertional Mutagenesis ◽  
Phase Variation ◽  
Restriction Enzyme Digestion ◽  
Wild Type ◽  
Gene Mutant ◽  
H Pylori

ABSTRACT A large number of genes encoding restriction-modification (R-M) systems are found in the genome of the human pathogen Helicobacter pylori. R-M genes comprise approximately 10% of the strain-specific genes, but the relevance of having such an abundance of these genes is not clear. The type II methyltransferase (MTase) M.HpyAIV, which recognizes GANTC sites, was present in 60% of the H. pylori strains analyzed, whereof 69% were resistant to restriction enzyme digestion, which indicated the presence of an active MTase. H. pylori strains with an inactive M.HpyAIV phenotype contained deletions in regions of homopolymers within the gene, which resulted in premature translational stops, suggesting that M.HpyAIV may be subjected to phase variation by a slipped-strand mechanism. An M.HpyAIV gene mutant was constructed by insertional mutagenesis, and this mutant showed the same viability and ability to induce interleukin-8 in epithelial cells as the wild type in vitro but had, as expected, lost the ability to protect its self-DNA from digestion by a cognate restriction enzyme. The M.HpyAIV from H. pylori strain 26695 was overexpressed in Escherichia coli, and the protein was purified and was able to bind to DNA and protect GANTC sites from digestion in vitro. A bioinformatic analysis of the number of GANTC sites located in predicted regulatory regions of H. pylori strains 26695 and J99 resulted in a number of candidate genes. katA, a selected candidate gene, was further analyzed by quantitative real-time reverse transcription-PCR and shown to be significantly down-regulated in the M.HpyAIV gene mutant compared to the wild-type strain. This demonstrates the influence of M.HpyAIV methylation in gene expression.

Aberrant CpG Island Hypermethylation in Pediatric Gastric Mucosa in Association With Helicobacter pylori Infection

2011 ◽  
Vol 135 (6) ◽  
pp. 759-765
Author(s):  
So-Hyun Shin ◽  
Seog-Yun Park ◽  
Jae-Sung Ko ◽  
Nayoung Kim ◽  
Gyeong Hoon Kang
Keyword(s):  
Helicobacter Pylori ◽  
Gastric Mucosa ◽  
Epithelial Cells ◽  
Cpg Island ◽  
Methylation Status ◽  
Helicobacter Pylori Infection ◽  
Gastric Epithelial Cells ◽  
Infected Adult ◽  
H Pylori ◽  
Methylight Assay

Abstract Context.—Helicobacter pylori infection is primarily acquired during childhood and persists throughout life in the absence of eradication with antibiotics. Helicobacter pylori infection induces methylation in the promoter CpG island loci in gastric epithelial cells. Thus, aberrant CpG island hypermethylation in gastric epithelial cells likely occurs early in life, although there are no existing data supporting this notion. Objectives.—To identify whether aberrant CpG island hypermethylation occurs in pediatric stomach mucosa in association with H pylori infection and to compare methylation profiles of samples from pediatric and adult stomach tissues. Design.—We analyzed pediatric (n  =  47) and adult (n  =  38) gastric mucosa samples for their methylation status in 12 promoter CpG island loci using the MethyLight assay and compared the number of methylated genes and the methylation levels in individual genes between H pylori–positive and H pylori–negative sample results and between pediatric and adult samples. Results.—The average number of methylated genes was significantly higher in H pylori–infected pediatric samples than in H pylori–negative pediatric samples (3.4 versus 0.3, P < .001) and in H pylori–infected adult samples than in H pylori–negative adult samples (7.6 versus 0.9, P < .001). Seven genes showed significantly higher methylation levels in H pylori–infected pediatric samples than in H pylori–negative pediatric samples (all values were P < .05). Conclusions.—These results indicate that CpG island hypermethylation occurs in pediatric gastric mucosa in association with H pylori infection and that the genes affected by H pylori–associated hypermethylation were similar in pediatric and adult samples.

scholarly journals Differential Helicobacter pylori Plasticity in the Gastric Niche of Subjects at Increased Gastric Cancer Risk

Pathogens ◽  
2019 ◽  
Vol 8 (2) ◽  
pp. 65
Author(s):  
Mariateresa Casarotto ◽  
Chiara Pratesi ◽  
Ettore Bidoli ◽  
Stefania Maiero ◽  
Raffaella Magris ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Helicobacter Pylori ◽  
Precancerous Lesions ◽  
Neutrophil Infiltration ◽  
Host Bacterium ◽  
Gastric Cancer Risk ◽  
Increased Risk ◽  
Cytotoxin Associated Gene A ◽  
Sydney System ◽  
H Pylori

Helicobacter pylori (H. pylori) represents an independent risk factor for Gastric Cancer (GC). First Degree Relatives (FDR) of GC subjects and Autoimmune Gastritis (AG) patients are both at increased risk for GC. H. pylori genetic heterogeneity within the gastric niche of FDR and AG individuals has been little explored. To understand whether they exploit an increased H. pylori stability and virulence, 14 AG, 25 FDR, 39 GC and 13 dyspeptic patients (D) were investigated by a cultural PCR-based approach characterizing single colonies-forming-units. We chose three loci within the Cytotoxin-associated gene-A Pathogenicity Island (CagPAI) (cagA,cagE,virB11), vacA, homA and homB as markers of virulence with reported association to GC. Inflammatory/precancerous lesions were staged according to Sydney System. When compared to D, FDR, similarly to GC patients, were associated to higher atrophy (OR = 6.29; 95% CI:1.23–31.96 in FDR; OR = 7.50; 95% CI:1.67–33.72 in GC) and a lower frequency of mixed infections (OR = 0.16; 95% CI:0.03–0.81 in FDR; OR = 0.10; 95% CI:0.02–0.48 in GC). FDR presented also an increased neutrophil infiltration (OR = 7.19; 95% CI:1.16–44.65). Both FDR and GC carried a higher proportion of CagPAI+vacAs1i1mx+homB+ profiles (OR = 2.71; 95% CI: 1.66–4.41 and OR = 3.43; 95% CI: 2.16–5.44, respectively). Conversely, AG patients presented a lower frequency of subtypes carrying a stable CagPAI and vacAs1i1mx. These results underline different H. pylori plasticity in FDR and AG individuals, and thus, a different host-bacterium interaction capacity that should be considered in the context of eradication therapies.

scholarly journals Helicobacter pylori Urease Suppresses Bactericidal Activity of Peroxynitrite via Carbon Dioxide Production

2000 ◽  
Vol 68 (8) ◽  
pp. 4378-4383 ◽  
Author(s):  
Hideo Kuwahara ◽  
Yoichi Miyamoto ◽  
Takaaki Akaike ◽  
Tatsuo Kubota ◽  
Tomohiro Sawa ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Protective Effect ◽  
High Performance ◽  
Defense Mechanism ◽  
Bacterial Colonization ◽  
Specific Inhibitor ◽  
Continuous Exposure ◽  
Dependent Manner ◽  
Bactericidal Action ◽  
H Pylori

ABSTRACT Helicobacter pylori can produce a persistent infection in the human stomach, where chronic and active inflammation, including the infiltration of phagocytes such as neutrophils and monocytes, is induced. H. pylori may have a defense system against the antimicrobial actions of phagocytes. We studied the defense mechanism of H. pylori against host-derived peroxynitrite (ONOO−), a bactericidal metabolite of nitric oxide, focusing on the role of H. pylori urease, which produces CO2 and NH3 from urea and is known to be an essential factor for colonization. The viability of H. pylori decreased in a time-dependent manner with continuous exposure to 1 μM ONOO−, i.e., 0.2% of the initial bacteria remained after a 5-min treatment without urea. The bactericidal action of ONOO− against H. pyloriwas significantly attenuated by the addition of 10 mM urea, the substrate for urease, whereas ONOO−-induced killing of a urease-deficient mutant of H. pylori or Campylobacter jejuni, another microaerophilic bacterium lacking urease, was not affected by the addition of urea. Such a protective effect of urea was potentiated by supplementation with exogenous urease, and it was almost completely nullified by 10 μM flurofamide, a specific inhibitor of urease. The bactericidal action of ONOO− was also suppressed by the addition of 20 mM NaHCO3 but not by the addition of 20 mM NH3. In addition, the nitration ofl-tyrosine of H. pylori after treatment with ONOO− was significantly reduced by the addition of urea or NaHCO3, as assessed by high-performance liquid chromatography with electrochemical detection. These results suggest that H. pylori-associated urease functions to produce a potent ONOO− scavenger, CO2/HCO3 −, that defends the bacteria from ONOO− cytotoxicity. The protective effect of urease may thus facilitate sustained bacterial colonization in the infected gastric mucosa.

scholarly journals Contraselectable Streptomycin Susceptibility Determinant for Genetic Manipulation and Analysis of Helicobacter pylori

2006 ◽  
Vol 72 (9) ◽  
pp. 5908-5914 ◽  
Author(s):  
Daiva Dailidiene ◽  
Giedrius Dailide ◽  
Dangeruta Kersulyte ◽  
Douglas E. Berg
Keyword(s):  
Helicobacter Pylori ◽  
Dna Sequences ◽  
Genetic Manipulation ◽  
Molecular Genetic ◽  
Gene Cassette ◽  
Erythromycin Resistance ◽  
Conventional Drug ◽  
Genomic Dnas ◽  
H Pylori ◽  
Derivatives Of

ABSTRACT Many Helicobacter pylori genetic studies would benefit from an ability to move DNA sequences easily between strains by transformation and homologous recombination, without needing to leave a conventional drug resistance determinant at the targeted locus. Presented here is a two-gene cassette that can be selected both (i) against, due to a Campylobacter jejuni rpsL gene (dominant streptomycin susceptibility in cells also carrying an rpsL-str r allele), and (ii) for, due to an erm gene (erythromycin resistance). This rpsL,erm cassette's utility was assessed by using it to replace four gene loci (mdaB, frxA, fur, and nikR) in four streptomycin-resistant [Strr] strain backgrounds (derivatives of 26695, SS1, X47, and G27MA). The resultant 16 strains (phenotypically erythromycin resistant [Ermr] and Strs) were each transformed with wild-type genomic DNAs, and Strr derivatives were selected. The desired Erms Strr isolates were obtained at frequencies that ranged from 17 to 96% among Strr transformants, with the Erms yield apparently depending on the strain background and genome location of the targeted locus. The ease of isolating unmarked transformants described here should be valuable for many H. pylori molecular genetic and evolutionary analyses.

scholarly journals Methylation status of CDH1 gene in samples of gastric mucous from brazilian patients with chronic gastritis infected by Helicobacter pylori

2010 ◽  
Vol 47 (1) ◽  
pp. 7-12 ◽  
Author(s):  
Erika Kague ◽  
Cristiane Melissa Thomazini ◽  
Maria Inês de Campo Moura Pardini ◽  
Fabrício de Carvalho ◽  
Celso Vieira Leite ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Tumor Suppressor ◽  
Chronic Gastritis ◽  
Direct Sequencing ◽  
Methylation Status ◽  
Cpg Islands ◽  
Pcr Primers ◽  
Suppressor Gene ◽  
Cdh1 Gene ◽  
H Pylori

CONTEXT: Gastric cancer is one of the top list of cancer types that most leads to death in Brazil and worldwide. Helicobacter pylori(H. pylori) is a class I carcinogen and infect almost 90% of chronic gastritis patients. Some genotypes confer different virulent potential to H. pylori and can increase the risk of gastritis development. Methylation of CpG islands can inactivate tumor suppressor genes and therefore, it can be involved in the tumorigenic process. CDH1 is a tumor suppressor gene that encodes the E-cadherin protein, which is important in maintaining cell-cell contacts. The inactivation of this gene can increase the chance of metastasis. Promoter methylation of CDH1 at early steps of gastric carcinogenesis is not yet completely understood. OBJECTIVE: In this study, we investigated the methylation status of CDH1 in chronic gastritis samples and correlated it with the presence of H. pylori. METHODS: Sixty gastric mucosal biopsies were used in this study. The detection of H. pylori was performed with the PCR primers specific to urease C gene. H. pylori genotyping was performed by PCR to cagA and vacA (s and m region). The methylation status of these gene CDH1 was analyzed using methylation-specific polymerase chain reaction and direct sequencing of the PCR products was performed using primers methylated and unmethylated in both forward and reverse directions. RESULTS: H. pylori was detected in 90% of chronic gastritis samples; among these 33% were cagA positive and 100% vacA s1. The genotype vacA s2/m1 was not detected in any sample analyzed. Methylation of CDH1 was detected in 63.3% of chronic gastritis samples and 95% of them were also H. pylori-positive. CONCLUSION: This work suggests that CDH1 gene methylation and H. pylori infection are frequent events in samples from Brazilian patients with chronic gastritis and reinforces the correlation between H. pylori infection and CDH1 inactivation in early steps of gastric tumorigenesis.

scholarly journals Helicobacter pylori: enemy, commensal or, sometimes, friend?

2015 ◽  
Vol 9 (06) ◽  
pp. 674-678 ◽  
Author(s):  
Michael B Whalen ◽  
Orietta Massidda
Keyword(s):  
Helicobacter Pylori ◽  
Lower Risk ◽  
Wide Distribution ◽  
Host Bacterium ◽  
Peptic Ulcers ◽  
Important Species ◽  
Public Health Officials ◽  
Resistant Strains ◽  
H Pylori ◽  
Mother To Child

Helicobacter pylori is a Gram-negative ε-proteobacterium that colonizes about 50% of humans. Some pertinent characteristics are that it can survive the acid of the stomach, produces urease to neutralize it and is motile due to apical flagella. Not surprisingly given its wide distribution, it has long colonized mankind and its genome encodes many features that allows this. Consequently, it frequently has a persistent lifelong association with humans and, differently from most pathogens that are transmitted horizontally, it is preferentially transmitted vertically, often from mother to child. A variety of genes and polymorphisms, both in H pylori and in humans, mediate the complex host-bacterium relationship, and can also determine if and what pathologies will be triggered by the species. H. pylori is naturally transformable, very recombinogenic and has a high mutation rate. Microbiota studies of the stomach have shown it to be an important species with a potentially regulatory role for the gastric microbial community. Likewise, epidemiological work has suggested that, while it clearly increases the risk of peptic ulcers and gastric cancer in some populations, it is also associated with lower risk of esophageal cancer and several other important pathologies. More recently, antibacterial resistant strains have been isolated, posing a problem for public health officials who called for its eradication. Hence, study of H. pylori and how it interacts with us can help revealing mutualistic or pathogenic interactions and the immune response in the digestive niche.

A nucleotide insertion between two adjacent methyltransferases in Helicobacter pylori results in a bifunctional DNA methyltransferase

2011 ◽  
Vol 433 (3) ◽  
pp. 487-495 ◽  
Author(s):  
Ritesh Kumar ◽  
Desirazu N. Rao
Keyword(s):  
Helicobacter Pylori ◽  
Dna Methyltransferase ◽  
Stop Codon ◽  
Binding Motif ◽  
Modification System ◽  
Restriction Modification System ◽  
Adenine Methylation ◽  
Nucleotide Insertion ◽  
H Pylori ◽  
Catalytic Motif

Helicobacter pylori has a dynamic R-M (restriction–modification) system. It is capable of acquiring new R-M systems from the environment in the form of DNA released from other bacteria or other H. pylori strains. Random mutations in R-M genes can result in non-functional R-M systems or R-M systems with new properties. hpyAVIAM and hpyAVIBM are two solitary DNA MTase (methyltransferase) genes adjacent to each other and lacking a cognate restriction enzyme gene in H. pylori strain 26695. Interestingly, in an Indian strain D27, hpyAVIAM–hpyAVIBM encodes a single bifunctional polypeptide due to insertion of a nucleotide just before the stop codon of hpyAVIBM and, when a similar mutation was made in hpyAVIAM–hpyAVIBM from strain 26695, a functional MTase with an N-terminal C5-cytosine MTase domain and a C-terminal N6-adenine MTase domain was constructed. Mutations in the AdoMet (S-adenosylmethionine)-binding motif or in the catalytic motif of M.HpyAVIA or M.HpyAVIB selectively abrogated the C5-cytosine or N6-adenine methylation activity of M.HpyAVIA–M.HpyAVIB fusion protein. The present study highlights the ability of H. pylori to evolve genes with unique functions and thus generate variability. For organisms such as H. pylori, which have a small genome, these adaptations could be important for their survival in the hostile host environment.

The rod-to-coccoid body transformation in cultures of Helicobacter pylori

1990 ◽  
Vol 48 (3) ◽  
pp. 626-627
Author(s):  
A. R. Crooker ◽  
W. G. Kraft ◽  
T. L. Beard ◽  
M. C. Myers
Keyword(s):  
Helicobacter Pylori ◽  
Growth Cycle ◽  
Negative Bacterium ◽  
Body Formation ◽  
Coccoid Form ◽  
Spiral Form ◽  
Prolonged Culture ◽  
H Pylori ◽  
Upper Gastrointestinal

Helicobacter pylori is a microaerophilic, gram-negative bacterium found in the upper gastrointestinal tract of humans. There is strong evidence that H. pylori is important in the etiology of gastritis; the bacterium may also be a major predisposing cause of peptic ulceration. On the gastric mucosa, the organism exists as a spiral form with one to seven sheathed flagella at one (usually) or both poles. Short spirals were seen in the first successful culture of the organism in 1983. In 1984, Marshall and Warren reported a coccoid form in older cultures. Since that time, other workers have observed rod and coccal forms in vitro; coccoid forms predominate in cultures 3-7 days old. We sought to examine the growth cycle of H. pylori in prolonged culture and the mode of coccoid body formation.

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