Determination of Helicobacter pylori Virulence by Simple Gene Analysis of the cag Pathogenicity Island

Nucleic acid amplification was performed for five loci in the cag pathogenicity island (PAI) of Helicobacter pylori (comprising cagA, the cagA promoter region, cagE, cagT, and the left end of cagII [LEC]), and gastric inflammation in patients was evaluated. Of 204 H. pylori isolates from Japanese patients (53 with peptic ulcer, 55 with gastric cancer, and 96 with chronic gastritis), 197 (96.6%) were positive for all five loci. Two isolates (1%) were negative for all five loci, and five isolates (2.4%) were positive for only cagA and LEC. These latter seven isolates were all from patients with mild chronic gastritis. Neutrophil infiltration in gastric mucosa was significantly milder in patients infected with partially or totally deleted-PAI strains than in those with intact-PAI strains. The cagE gene was a more accurate marker of an intact cag PAI than the cagA gene, and cagE seemed to be more useful in discriminating between H. pylori strains causing different rates of disease progression.     

Study of the oipA genetic diversity and EPIYA motif patterns in cagA-positive Helicobacter pylori strains from Venezuelan patients with chronic gastritis

CagA and OipA are involved, among other virulence factors, in the ability of Helicobacter pylori to colonize the gastric mucosa and to modulate the host environment during the establishment of chronic infection. The number and type of EPIYA phosphorylation motifs and the presence and functional status of oipA have been involved in the induction of cellular transformations playing an important role in the development of H. pylori associated gastric diseases. This work determined the prevalence of the oipA virulence factor and EPIYA motif patterns in cagA-positive H. pylori gastric biopsies from chronic gastritis patients from the Central-Western region of Venezuela. DNA was extracted directly from gastric biopsies collected by upper endoscopy from 113 patients. The EPIYA motif genotyping and oipA gene functional status was determined by PCR and sequencing. Phylogenetic analysis with the 3′ variable region of cagA sequences was performed. Only Western-type EPIYA variants were detected: ABC (68.14%), ABCC (29.20%) and ABCCC (2.66%). High prevalence of strains with the oipA gene (93.8%) and its functional status “ON” (83%) was observed. No significant association between EPIYA motif patterns or oipA functional status with the histological changes in the gastric mucosa was found. Our study demonstrated the absolute predominance of the Western-type cagA gene in a Venezuelan admixed population. This is the first report showing oipA status of H. pylori strains in Venezuela. Further studies with a larger number of samples and including other pathologies are necessary to continue evaluating the role of the H. pylori virulence factors in the prevalence of gastric diseases in our country.     

Comparing genomes of Helicobacter pylori strains from the high-altitude desert of Ladakh, India

The genomic diversity of Helicobacter pylori from the vast Indian subcontinent is largely unknown. We compared the genomes of 10 H. pylori strains from Ladakh, North India. Molecular analysis was carried out to identify rearrangements within and outside the cag pathogenicity island (cag PAI) and DNA sequence divergence in candidate genes. Analyses of virulence genes (such as the cag PAI as a whole, cagA, vacA, iceA, oipA, babB, and the plasticity cluster) revealed that H. pylori strains from Ladakh are genetically distinct and possibly less virulent than the isolates from East Asian countries, such as China and Japan. Phylogenetic analyses based on the cagA-glr motifs, enterobacterial repetitive intergenic consensus patterns, repetitive extragenic palindromic signatures, the glmM gene mutations, and several genomic markers representing fluorescent amplified fragment length polymorphisms revealed that Ladakhi strains share features of the Indo-European, as well as the East Asian, gene pools. However, the contribution of genetic features from the Indo-European gene pool was more prominent.     

Host Cell Responses to Genotypically Similar Helicobacter pylori Isolates from United States and Japan

Associations of Helicobacter pylori genotypes with disease differ between Western countries and Asia. Therefore, we directly compared histopathological and in vitro responses to clinical isolates with similar genotypes. Sixty-three cagA⁺ vacAs1/m1 H. pylori isolates (United States, n = 24; Japan, n = 39) and eight cagA-negative vacAs2/m2 strains were incubated with AGS cells, and supernatants were assayed for interleukin-8 (IL-8) and for DNA fragmentation. CagA tyrosine phosphorylation in AGS cells and the sequence of the putative HP0638 (oipA) signal sequence region were determined for 22 representative strains. HP0638 and/or cag island mutant strains were created and examined in IL-8 and CagA tyrosine phosphorylation assays. Levels of IL-8 induction and DNA fragmentation were similar in the U.S. and Japanese cagA⁺ vacAs1/m1 isolates. All 10 of the isolates with the highest IL-8 induction and 8 of the 10 isolates with the lowest IL-8 induction had an in-frame oipA open reading frame, and all 10 of the isolates with the highest IL-8 induction and 7 of the 10 isolates with the lowest IL-8 induction induced CagA tyrosine phosphorylation in AGS cells. Eight isolates from gastric ulcer patients induced significantly more apoptosis in vitro, and more severe gastritis and atrophy in vivo, than other Japanese isolates. Disruption of HP0638 did not affect IL-8 induction or CagA tyrosine phosphorylation. Thus, H. pylori cagA⁺ vacAs1/m1 isolates from the United States and Japan induce similar IL-8 and apoptosis levels. Inactivation of HP0638 does not alter epithelial responses mediated by the cag island in vitro. Assessment of apoptosis in vitro identified a group of H. pylori isolates that induce more severe gastric inflammation and atrophy.     

Pediatric Helicobacter pylori Isolates Display Distinct Gene Coding Capacities and Virulence Gene Marker Profiles

Helicobacter pylori strains display remarkable genetic diversity, and the presence of strains bearing the toxigenic vacA s1 allele, a complete cag pathogenicity island (PAI), cagA alleles containing multiple EPIYA phosphorylation sites, and expressing the BabA adhesin correlates with development of gastroduodenal disease in adults. To better understand the genetic variability present among pediatric strains and its relationship to disease, we characterized H. pylori strains infecting 47 pediatric North American patients. Prevalence of mixed infection was assessed by random amplified polymorphic DNA analysis of multiple H. pylori clones from each patient. Microarray-based comparative genomic hybridization was used to examine the genomic content of the pediatric strains. The cagA and vacA alleles were further characterized by allele-specific PCR. A range of EPIYA motif configurations were observed for the cagA gene, which was present in strains from 22 patients (47%), but only 19 (41%) patients contained a complete cag PAI. Thirty patients (64%) were infected with a strain having the vacA s1 allele, and 28 patients (60%) had the babA gene. The presence of a functional cag PAI was correlated with ulcer disease (P = 0.0095). In spite of declining rates of H. pylori infection in North America, at least 11% of patients had mixed infection. Pediatric strains differ in their spectrum of strain-variable genes and percentage of absent genes in comparison to adult strains. Most children were infected with H. pylori strains lacking the cag PAI, but the presence of a complete cag PAI, in contrast to other virulence markers, was associated with more severe gastroduodenal disease.It is estimated that >50% of the world''s population is colonized with Helicobacter pylori in the stomach, making it one of the most common bacterial pathogens of humans. H. pylori infection is generally acquired in childhood (24, 33) and can persist for life. Gastritis (inflammation of the gastric mucosa) results in all who are colonized with H. pylori, but some hosts remain asymptomatic, while others develop peptic ulcers, gastric adenocarcinomas, and mucosa-associated lymphoid tissue lymphoma. Gastric cancer is the second leading cause of cancer death worldwide, and 63% of gastric cancer cases in 2002 were attributable to H. pylori infection (38, 49). While severe disease most often presents in adulthood, children display H. pylori-associated gastritis and the incidence of ulcer disease among infected children was 6.8% in a European pediatric population (31). Many studies have examined bacterial, host, and environmental risk factors associated with development of H. pylori-associated diseases in adults, but similar studies in children have been limited.Genetic differences among H. pylori strains contribute to differences in disease outcome among infected individuals in adult populations. The gene encoding VacA, which induces vacuolation of host cells, is present in nearly all H. pylori strains, but a number of allele types have been defined. Strains having the type s1 vacA signal sequence and the m1 vacA middle region allele (vacA s1/m1) are associated with ulcer disease (9). The cag pathogenicity island (PAI) encodes a type IV secretion system (T4SS) (1, 15) that translocates the CagA protein effector, also encoded in the island, into host cells. Presence of the cag PAI is associated with increased inflammation, promoting host cell interleukin-8 (IL-8) production, and cagA-positive strains are associated with peptic ulcers (50) as well as gastric cancer (13). Inside the host cell, CagA protein becomes tyrosine phosphorylated at C-terminal EPIYA (Glu-Pro-Ile-Tyr-Ala) sites by src family kinases, deregulates SHP-2, and induces the hummingbird phenotype (26, 45). Strains having more C-type EPIYA motifs, the major phosphorylation site, induce stronger effects on host cells and are associated with gastric cancer (7, 12, 35). The presence of a functional allele of babA, a gene encoding an adhesin that mediates binding to Lewis B antigens expressed on gastric epithelial cells, is associated with duodenal ulcer and gastric adenocarcinoma (21).While these H. pylori genes and alleles have been associated with disease outcome in adults, studies in children have provided mixed results. A recent study identified two genes (jhp0562, coding for a putative glycosyltransferase, and jhp0870, coding for an outer membrane protein) associated with peptic ulcer disease in children, but not adults, suggesting a different spectrum of genetic risk factors in adults and children (37). Studies using a whole-genome microarray-based approach have been done to investigate the variability in genomic content of H. pylori strains, but these studies have included mostly strains from adult patients (25, 29, 41, 42). Studies of the genetic variability of pediatric H. pylori strains have largely been limited to genes previously associated with virulence in adult populations. To better understand the genetic variability present among pediatric strains, we used whole-genome microarray-based comparative genomic hybridization to examine the genomic content of H. pylori strains isolated from symptomatic North American children and compared the pediatric isolate genetic variability to that observed in adult strains. We then examined the frequency of known virulence genes and virulence alleles among the pediatric H. pylori strains and the associations of strain genotype with the clinical and histological characteristics of the patients.     

Involvement of the <Emphasis Type="Italic">Helicobacter pylori</Emphasis> plasticity region and <Emphasis Type="Italic">cag</Emphasis> pathogenicity island genes in the development of gastroduodenal diseases

Infection by Helicobacter pylori is associated with the development of several gastroduodenal diseases, including gastritis, peptic ulcer disease (gastric ulcers and duodenal ulcers), and gastric adenocarcinoma. Although a number of putative virulence factors have been reported for H. pylori, there are conflicting results regarding their association with specific H. pylori-related diseases. In this work, we investigated the presence of virB11 and cagT, located in the left half of the cag pathogenicity island (cagPAI), and the jhp917–jhp918 sequences, components of the dupA gene located in the plasticity zone of H. pylori, in Brazilian isolates of H. pylori. We also examined the association between these genes and H. pylori-related gastritis, peptic ulcer disease, and gastric and duodenal ulcers in an attempt to identify a gene marker for clinical outcomes related to infection by H. pylori. The cagT gene was associated with peptic ulcer disease and gastric ulcers, whereas the virB11 gene was detected in nearly all of the samples. The dupA gene was not associated with duodenal ulcers or any gastroduodenal disease here analyzed. These results suggest that cagT could be a useful prognostic marker for the development of peptic ulcer disease in the state of São Paulo, Brazil. They also indicate that cagT is associated with greater virulence and peptic ulceration, and that this gene is an essential component of the type IV secretion system of H. pylori.     

Low clarithromycin resistance in virulent Helicobacter pylori from dyspeptic patients at a tertiary care centre in Odisha

PurposeUniversal eradication or use of failing antibiotic can add fuel to the antimicrobial resistance pandemic. Outcome of Helicobacter pylori (HP) infection depends at least partly virulence factors and its eradication as preventive measure against gastric cancer is advocated by some guidelines. There is need to identify candidates at risk for gastric cancer and antimicrobial resistance in HP for rational management. Such candidates could be identified by studying the association of virulence factors with clinical outcome. As this data is lacking from Odisha this study was undertaken.Methods113 consecutive dyspeptic patients who underwent endoscopy at our hospital were recruited to obtain gastric biopsies for culture and antibiotic susceptibility, histological examination, molecular detection of HP, virulence typing (cagA, EPIYA typing, vacA, vacA s1/s2, vacA m1/m2 and babA2) by conventional PCR and identification of clarithromycin resistance by real-time PCR. Cultured isolates were subjected to antibiotic sensitivity using e strips as per EUCAST guidelines.Results93 (82.3%) dyspeptic patients were infected by HP by histology & PCR, while 90 (79.6%) were rapid urea test positive, and HP was cultured from 32 (28.3%) of these patients. Eleven (11.8%) of the 93 samples with HP were resistant to clarithromycin by real-time PCR. Of the 93 patients with HP infection by histopathology and PCR, 62 (66.7%), 87(93.5%) and 43 (46.2%) harboured cagA, vacA and babA2 genes. The western cagA found in 33 (35.5%) samples and vacA s1m1 in 50 (53.8%) samples were the commonest virulence subtypes. No association was found between virulence factors and outcome except vacA s2m2 and vac s1/m1m2, which were significantly associated with peptic ulcers. Phenotypically 11(34.4%), 1(3.1%), 21(65.6%) and 26 (81.2%) isolates were resistant to clarithromycin, amoxicillin, levofloxacin, and metronidazole.ConclusionsThis is the first study that explored the antibiotic resistance of HP, and its virulence factors in dyspeptic patients from this region of India.     

Prevalence of virulence-associated genotypes of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> and correlation with severity of gastric pathology in patients from western Sicily,Italy

In a bacterium like Helicobacter pylori, which is characterized by a recombinant population structure, the associated presence of genes encoding virulence factors might be considered an expression of a selective advantage conferred to strains with certain genotypes and, therefore, a potentially useful tool for predicting the clinical outcome of infections. However, differences in the geographical and ethnic prevalence of the H. pylori virulence-associated genotypes can affect their clinical predictive value and need to be considered in advance. In this study we carried out such an evaluation in a group of patients living in Sicily, the largest and most populous island in the Mediterranean Sea. cagA, vacA, babA2, hopQ, oipA, sabA, and hopZ were the H. pylori virulence-associated genes assayed; their presence, expression status or allelic homologs were detected in H. pylori DNA samples and/or isolated strains, obtained by gastric biopsy from 90 Sicilian patients with chronic gastritis, inactive (n = 37), active (n = 26), or active with peptic ulcer (n = 27). Genotypes cagA ⁺, vacAs1, vacAm1, babA2 ⁺, and hopQ I, I/II were identified in 51.8, 80.4, 35.2, 47.3, and 67.7% of the different samples respectively. Only these genotypes were associated with each other and with the active form of chronic gastritis, irrespective of the presence of a peptic ulcer. In our isolates their prevalence was more similar to values observed in the north of Italy and France than to those observed in Spain or other Mediterranean countries that are closer and climatically more similar to western Sicily.     

<Emphasis Type="Italic">Helicobacter pylori</Emphasis> Virulence Genotypes in Portuguese Children and Adults with Gastroduodenal Pathology

The aim of this study was to evaluate the prevalence of virulence genotypes, namely cagA, vacA and babA2, of Helicobacter pylori strains isolated from Portuguese adults and children presenting gastroduodenal pathology. One hundred thirty-six strains were studied, 82 isolated from adult patients (50 with nonulcerative gastritis and 32 with active peptic ulcer) and 58 isolated from children (54 with nonulcerative gastritis and 4 with duodenal ulcer). Genotyping of cagA, vacA and babA2 was assessed by polymerase chain reaction. Overall, Helicobacter pylori strains carrying more virulent genotypes were much more prevalent in adults than in children, particularly the type I (vacAs1- and cagA-positive) and the triple-positive (vacAs1-, cagA- and babA2-positive) strains (P<0.001). A subpopulation of adults and children with nonulcerative gastritis was also studied, and differences in the prevalence of virulent genotypes were observed, either for individual genotypes (P=0.017 for cagA, P=0.010 for vacAs1) or in combinations, i.e. the type I genotype (P=0.005) and the triple-positive strains (P=0.031). There was no difference between the two populations in the distribution of babA2 and m1/m2 genotypes. Considering the cohort effect in the epidemiology of Helicobacter pylori infection, these results suggest that different strains might circulate during different periods of time, or that, after infection in childhood, individual strains will undergo changes during the course of infection. Electronic Publication     

Virulence factor genotypes of Helicobacter pylori affect cure rates of eradication therapy

The cure rates of Helicobacter pylori infection by using a combination of a proton pump inhibitor (PPI) and antimicrobial agents are mainly influenced by bacterial susceptibility to antimicrobial agents and the magnitude of acid inhibition during the treatment. Currently used empirical triple therapies do not reliably produce a ≥80% cure rate on an intention-to-treat basis. Therefore, tailored regimens based on relevant microbiological findings and pharmacogenomics are recommended for attaining an acceptable ≥95% cure rate. Recently, virulence factors of H. pylori, such as cagA and vacA, are reported to be major factors determining the cure rates. Individuals infected with strains with cagA-negative and vacA s2 genotypes have significantly increased risk of eradication failure of H. pylori infection. These virulence factors enhance gastric mucosal inflammation and are associated with the development of peptic ulcer and gastric cancer. H. pylori virulence factors induce proinflammatory cytokines, such as interleukin (IL)-1, IL-8, and tumor necrosis factor (TNF)-  which influence mucosal inflammation and/or gastric acid secretion. When physicians select an H. pylori eradication regimen with an acceptable cure rate, they might need to consider H. pylori virulence factors, especially cagA and vacA.     

New approaches for genotyping of Helicobacter pylori based on amplification of polymorphisms in intergenic DNA regions and at the insertion site of the cag pathogenicity island

The population of the gastric pathogen Helicobacter pylori shows a high degree of genetic diversity. It is well established that heterogeneity at the isolate level is caused by nucleotide transitions within genes, differences in the gene order, and by genetic instability of single genes as well as of a large virulence-associated genomic DNA region, the cag pathogenicity island (PAI). Analysis of intergenic regions with specific PCR-assays developed in this study, revealed that DNA polymorphisms in the noncoding DNA localized in front of the genes ribA and vacA and at the insertion site of the cag PAI contribute to the genetic diversity of H. pylori and are useful for differentiation of individual isolates. Thirteen individual genotypes were identified by PCR analysis of these polymorphic loci in 487, 241, and 182 clinical H. pylori isolates. Sequence analysis revealed that genetic variability in front of genes ribA and vacA, and in the intergenic region at the PAI insertion site is caused by insertion and deletions of so-far-unknown DNA sequences as well as by parts of the H. pylori IS elements IS605 and IS606, respectively. The new genotypes identified could be used to differentiate antrum and corpus isolates from the same patients. Their combination with vacA allele subtypes and with the cagA status allowed to differentiate 140 isolates in 51 subtypes. In 36 cases the corresponding genotype patterns were isolate specific. In summary, the results confirm that DNA polymorphisms in intergenic regions contribute to the genetic diversity of H. pylori. Although individual H. pylori genotypes were not associated with peptic ulcer disease, the PCR-based approaches for their detection developed here should be of use for further investigation of genetic diversity in H. pylori and for epidemiological purposes. Received: 20 June 2000     

Profile of Helicobacter pylori cagA & vacA genotypes and its association with the spectrum of gastroduodenal disease

PurposeGlobally, H. pylori virulence factors cagA and vacA genotypes and its variation is leading to the austere form of the gastroduodenal disease. Our objectives were to detect H. pylori in dyspeptic patients from biopsy samples with the validation of the various existing diagnostic tools and to screen the cagA, vacA genotypes profile from biopsy specimens and how it impacts in progression of gastroduodenal disease in southern India.Methods374 patients who attended endoscopy unit at Kasturba Hospital, Manipal with their consent obtained their biopsies. H. pylori were detected by HPE, Culture, RUT and PCR and its virulence gene were patterned with PCR.ResultsThe positive rate of H. pylori by HPE, RUT, Culture and PCR were 51.33%, 47.1%, 32.4% and 50.3% respectively and comparison by Bayesian LCMs analysis showed PCR is superior among them. The frequency of H. pylori virulence gene viz cagPAI (cagA) were 80.9%, and vacA alleles-s1m1 (42%), s1m2 (33%) and s2m2 (25%) genotypes by PCR respectively. Four combinations of cagA/vacA genotypes were noted, majority of strains harboured cagA⁺/vacA s1m1 genotypes (42.6%), interestingly this hyper-virulent strain more frequently seen in severe gastroduodenal disease whereas cagPAI negative strains as well as cagA⁻/vacA s2m2 combinations (19.1%) are seen most commonly in functional dyspepsia cases and depicted significant association by Chi-square test.ConclusionsThis study validates and compares the existing diagnostic methods for detecting H. pylori in biopsies. Also, it reveals some pattern of virulence gene combination will play a vital role in disease progression.     

The importance of <Emphasis Type="Italic">Helicobacter pylori tnpA</Emphasis>, <Emphasis Type="Italic">tnpB</Emphasis>, and <Emphasis Type="Italic">cagA</Emphasis> genes in various gastrointestinal diseases

The cag (cytotoxin-associated gene) pathogenicity island (cagPAI) is one of the major virulence determinants of Helicobacter pylori (H. pylori). The purpose of this study was to investigate the association of the three genes (tnpA, tnpB, and cagA) in H. pylori isolated from Azerbaijani patients with the different gastrointestinal disease. A total of 362 gastric biopsies were collected from hospitals of Tabriz University of Medical Sciences, and were cultured on Brucella agar. The tnpA, tnpB, and cagA genes were detected by PCR. Of the total 264 H. pylori isolates, tnpA, tnpB, and cagA genes were detected in 120 (45.5%), 56 (21.2%) and 172 (65.2%), respectively. A significant association between tnpA and tnpB genes and clinical outcomes were found (P < 0.05). The cagA status was not related to clinical outcomes in our subjects. The predominant genotype among cag-PAI is the cagA. The prevalence of tnpA, tnpB, and cagA genes are high in patients with gastric cancer, and a significant association is revealed between tnpA and tnpB with gastric cancer.     

cag Pathogenicity island-dependent upregulation of matrix metalloproteinase-7 in infected patients with Helicobacter pylori

Helicobacter pylori (H. pylori) infection has been involved in the pathogenesis of most important gastroduodenal diseases. Matrix metalloproteinases (MMPs) are a large family of zincendopeptidases which play important roles in degradation of extracellular matrix (ECM) and various inflammatory diseases. Therefore, we examined MMP-7 mRNA levels in the gastric mucosa of patients with H. pylori infection and evaluated the effects of virulence factors, such as vacA (vacuolating cytotoxin A) and cagA (cytotoxin-associated gene), in H. pylori-infected patients upon the MMP-7 mRNA mucosal levels. We also determined the correlation between mucosal MMP-7 mRNA levels and the types of disease. Total RNA was extracted from gastric biopsies of 50 H. pylori-infected patients and 50 uninfected individuals. Mucosal MMP-7 mRNA expression level in H. pylori-infected and non-infected gastric biopsies was determined by real-time polymerase chain reaction (PCR). The presences of cagA and vacA virulence factors was evaluated using PCR. MMP-7 expression was significantly higher in biopsies of patients infected with H .pylori compared to uninfected individuals. In addition, mucosal MMP-7 mRNA expression in H. pylori-infected patients significantly associated with the cagA status and the types of disease. Our results suggest that MMP-7 might be involved in the pathogenesis of H. pylori. Peptic ulcer was associated with cag pathogenicity island-dependent MMP-7 upregulation.     

The association of dupA and Helicobacter pylori-related gastroduodenal diseases

Helicobacter pylori is associated with the development of ulceration and gastric cancer. Recently, a novel virulence factor, duodenal ulcer promoting gene A (dupA), has been identified and found to associate with disease in some populations but not others. We investigated the relationship of dupA genotypes and H. pylori-related clinical outcomes by meta-analysis using previous reports of 2,358 patients from around the world. dupA-positive genotypes was found in 48% and was associated with duodenal ulcer (p?=?0.001, odds ratio [OR]?=?1.4, confidence interval [CI]?=?1.1–1.7). The prevalence of dupA-positivity and its association with disease differed among the various regions around the world. In South America, the highest prevalence was recorded (Colombia and Brazil) and a significant relationship was found between dupA-negative strains and both gastric ulcer (GU) and gastric cancer (GC) (for GU, p?=?0.001, OR?=?0.2, CI?=?0.1–0.4 and for GC, p?=?0.001, OR?=?0.3, CI?=?0.2–0.6). In China, a significant correlation between dupA-positive strains and GU (p?=?0.001, OR?=?5.5, CI?=?2.4–12.4) and GC (p?=?0.009, OR?=?2, Cl?=?1.1–3.1) was found. To conclude, dupA promotes duodenal ulceration in some populations and GU and GC in others. This is typical of other virulence factors, such as cagA. Hence, it was concluded that the H. pylori virulence factor, dupA, is a true virulence factor.     

Helicobacter pylori cagA gene variants in Malaysians of different ethnicity

We have defined DNA repeat variability in the 3′-terminus of the cagA gene of Helicobacter pylori strains from Malaysian patients of different ethnicities. We identified different alleles based on the EPIYA repeats. cagA types A-B-D and A-B-B-D are more similar to the sequence of Japanese strains, whereas cagA types A-B-C, A-B-C-C, A-B and A-C displayed similarity to strain 26695 sequences. A significant association was found between cagA genotypes and patients’ ethnicity, with cagA type A-B-D being predominantly isolated from Chinese patients and cagA type A-B-C from Malays and Indians. Our data further corroborate the possibility that variant biological activity of CagA may affect the host specificity and/or pathogenicity of H. pylori.     

Helicobacter pylori Virulence Factor Genotypes in Children in the United States: Clues about Genotype and Outcome Relationships

The most common Helicobacter pylori genotype among 37 U.S. children was cagA positive, vacA s1m1, and oipA “on” (n = 17, 45.9%), followed by cagA negative, vacA s2m2, and oipA “off” (n = 8, 21.6%), similar to the pattern in adults. cagA positivity was more common in blacks than in whites (i.e., 100% versus 56.5%, P = 0.032).Infection with Helicobacter pylori is etiologically associated with gastritis, peptic ulcer disease, gastric atrophy, and gastric cancer. H. pylori is thought to be typically acquired in childhood, with infection continuing for decades if not lifelong. This long bacterial host association involves countless generations of bacteria which are thought to continually evolve as the intragastric conditions change, such that those best suited for the local conditions outgrow and replace less suited neighbors. There has been considerable interest in the molecular epidemiology of H. pylori''s putative virulence factors, especially CagA, VacA, and OipA (outer inflammatory protein A) (8, 11, 12). However, there are few studies of children and only one previous study investigating the relationship between H. pylori virulence factors and ethnic groups of children in the United States (4-6, 11). This study reports the patterns of H. pylori virulence factor genotypes in children of different ethnic groups in the United States.The biopsy specimens and cultures were obtained as part of a multicenter study from 5 widely dispersed sites in the United States. The study was designed to validate the [¹³C]urea breath test for the diagnosis of H. pylori infection in children aged between 2 years and 17 years and 11 months (2). Symptomatic children scheduled for endoscopy were enrolled. Gastric biopsy specimens were evaluated by histology, rapid urease testing, and culture of H. pylori using established techniques (2). H. pylori culture isolates were evaluated for cagA and vacA genotypes using established PCR assays as previously described (9). The number of EPIYA (Glu-Pro-Ile-Tyr-Ala) repeat motifs in the 3′ region of the cagA gene was evaluated using PCR as previously described (7). OipA is a member of the large outer membrane protein family whose functional status is regulated by slipped-strand mispairing based on the number of CT dinucleotide repeats in the 5′ region of the gene (a switch status of “on” indicates the gene is functional, and a switch status of “off” indicates it is nonfunctional) (10). The 5′ region of the oipA gene was amplified using previously described primers (10), and the PCR fragments were purified and directly sequenced at Macrogen, Ltd., in Seoul, South Korea.Forty-eight of 176 children enrolled were H. pylori infected, based on two positive tests or a positive H. pylori culture. The mean age was 11.5 years (range, 3.2 to 17.9 years). Thirty-seven were H. pylori culture positive. None had atrophic gastritis. Only one patient had a significant endoscopic abnormality, a duodenal ulcer (cagA positive, vacA s1m2, and oipA on). The most common H. pylori genotype was cagA positive, vacA s1m1, and oipA on (n = 17, 45.9%), followed by cagA negative, vacA s2m2, and oipA off (n = 8, 21.6%), cagA positive, vacA s1m2, and oipA on (n = 5), cagA positive, vacA s1m1, and oipA on (n = 3), cagA negative, vacA s2m2, and oipA on (n = 2), cagA negative, vacA s1m1, and oipA on (n = 1), and cagA positive, vacA s1m2, and oipA off (n = 1) (Table ​(Table1).1). Overall, 70% of strains were cagA positive, which is similar to is the proportion found in U.S. adults (6).

TABLE 1.

Relationship between H. pylori genotype and ethnic group

Characterization of the cagA-gene in Helicobacter pylori in Mongolia and detection of two EPIYA-A enriched CagA types

Helicobacter pylori infection is strongly associated with gastritis, gastroduodenal ulcer disease and gastric carcinoma. The virulence of H. pylori strains increases with the presence of the pathogenicity island PAI, which encodes a Type 4 Secretion System and the oncoprotein CagA. Two major CagA types can be distinguished by differences in the repetitive EPIYA region in the C-terminal sequence; the more virulent East Asian CagA type with EPIYA-A, -B, and -D motifs and the Western CagA type with EPIYA-A, -B, and C motifs, the virulence of which is associated with the multitude of EPIYA-C motifs.In this study, the cagA gene was characterized in H. pylori strains isolated from Mongolians suffering from gastritis (80%) or ulcer (20%). The EPIYA region of 53 isolates was determined by PCR-amplification of overlapping cagA regions and subsequent Sanger sequencing. Only one H. pylori isolate carried the East Asian type (ABD) and 52 isolates the Western type of CagA, thereof 30 the EPIYA type ABC, 19 the ABCC type and one each of type ABCCCC, AAABC and AAAAB. An amino acid exchange from EPIYA-B to EPIYT-B was predominantly found in CagA proteins in strains with < 2 EPIYA-C copies (n = 25/32; p = 0.015) including the two EPIYA-A enriched CagA proteins, which have not been described to date. Due to the amino acid triplet preceding the EPIYA motif and strength of predicted phosphorylation, the multiple EPIYA-A motifs A2, A3 and A4 were shown to cluster with EPIYA-B and EPIYT-B with the unique feature of amino acid E in position ? 4 to Y of EPIYA. It has been described that tyrosine-phosphorylated EPIYA-A and -B motifs counteract the EPIYA-C-driven signaling towards host cell transformation and malignancy. Thus, Mongolian H. pylori strains carrying CagA proteins not only with a few EPIYA-C segments but also with multiplied EPIYA-A segments are probably less virulent; a thesis that needs further investigation at the protein level.     

CagA and VacA Polymorphisms Do Not Correlate with Severity of Histopathological Lesions in Helicobacter pylori-Infected Greek Children

The presence of various numbers of EPIYA tyrosine phosphorylation motifs in the CagA protein of Helicobacter pylori has been suggested to contribute to pathogenesis in adults. In this prospective study, we characterized H. pylori isolates from symptomatic children, with reference to the diversity of functional EPIYA motifs in the CagA protein and vacA isotypes, and assessed the potential correlation with the histopathological manifestations of the infection. We analyzed 105 H. pylori isolates from 98 children and determined the diversity of EPIYA motifs in CagA by amplification and sequencing of the 3′ variable region of the cagA gene as well as vacA isotypes for the signal, middle, and intermediate regions. CagA phosphorylation and levels of secreted IL-8 were determined following in vitro infection of AGS gastric epithelial cells. Histopathological evaluation of H. pylori colonization, activity, and severity of the associated gastritis was performed according to the updated Sydney criteria. EPIYA A (GLKN[ST]EPIYAKVNKKK), EPIYA B (Q[V/A]ASPEPIY[A/T]QVAKKVNAKI), and EPIYA C (RS[V/A]SPEPIYATIDDLG) motifs were detected in the ABC (46.6%) and ABCC (17.1%) combinations. No isolates harboring more than two EPIYA C motifs in CagA were found. The presence of isogenic strains with variable numbers of CagA EPIYA C motifs within the same patient was detected in seven cases. Occurrence of increasing numbers of EPIYA C motifs correlated strongly with presence of a high-vacuolation (s1 or s2/i1/m1) phenotype and age. A weak positive correlation was observed between vacuolating vacA genotypes and presence of nodular gastritis. However, CagA- and VacA-dependent pathogenicities were not found to contribute to severity of histopathology manifestations in H. pylori-infected children.Helicobacter pylori infects 50% of the world''s population, and wide differences in prevalence of infection appear to exist between countries with different levels of socioeconomic development. Infection usually occurs in childhood and in the majority of cases remains asymptomatic, although major reasons for endoscopy referral can include recurrent epigastric or abdominal pain, with or without vomiting, neither of which correlates with H. pylori infection (17). Antral nodularity is a well-described endoscopic feature of H. pylori-infected children, and histological observations usually include superficial chronic active gastritis with occasional infiltration of eosinophils; in far fewer cases, they include peptic ulcers; and very rarely, they include gastric atrophy and intestinal metaplasia (13, 27). If the infection is left untreated, it persists through adulthood, and although it can still remain asymptomatic in the vast majority of infected hosts, H. pylori infection is now regarded as the most important etiological risk factor for development of gastric cancer in developed countries (28). H. pylori pathogenesis is manifested through a combined effect of bacterial virulence factors, host genetics, and environmental factors, which orchestrate toward the development of distinct phenotypes in adults, namely, superficial asymptomatic gastritis, duodenal ulcer, and gastric cancer (3). The expression and translocation of cytotoxin-associated gene antigen (CagA), a putative H. pylori virulence factor, inside gastric epithelial cells by cagA-positive H. pylori strains harboring a functional type IV secretion system has been suggested to play an important role in H. pylori pathogenesis (22). Early epidemiological studies of adults associated the presence of the cagA gene with development of peptic ulcer disease (31); gastric cancer (14); and increased inflammation (35), cellular proliferation (36), and intestinal metaplasia (20) of the gastric mucosa. However, in infected children, neither cagA status nor any other putative H. pylori virulence factor has been found to correlate with clinical outcome or severity of histological manifestations. However, recent advances into the fascinating cellular biology of CagA inside the gastric epithelial cell have enhanced its reputation as a potential bacterial oncoprotein (22). Following its translocation inside the gastric epithelial cell via the type IV secretion system (32), CagA has been shown to become at least partly tyrosine phosphorylated (5, 11, 41) by Src family kinases (42, 44) on repeating 5-amino-acid glutamic-proline-isoleucine-tyrosine-alanine (EPIYA) motifs present at the C terminus of the protein. Analysis of EPIYA motifs in CagA has revealed considerable type variation, depending on the peptide sequence surrounding it, namely, EPIYA A (EPIYAKVNKKK), EPIYA B (EPIYAQVAKKV), or EPIYA C (EPIYATIDDLG) in isolates from Western populations or EPIYA D (EPIYATIDFD) in isolates of Asian origin. In addition, considerable variation in number of repeating EPIYA C or D motifs at the carboxyl terminus of the protein (10, 44) has been observed, and biological activity of CagA was suggested to be determined by variation in these motifs (25) in phosphorylation-dependent as well as -independent ways (23). Hence, the number and type of EPIYA phosphorylation motifs may be viewed as putative virulence determinants of CagA activity and therefore become useful clinical markers that may predict the degree of individual H. pylori strain virulence potential. In this context, we proposed a PCR amplification and sequencing-based strategy for accurate characterization of the number and type of EPIYA motifs of CagA in H. pylori clinical isolates (34).A multifactorial role has also been attributed to the secreted VacA virulence factor (16), a protein with multiple cellular activities, as it can disrupt endocytic trafficking of host cells, promote cell death through apoptosis, suppress the local immune system, and possibly potentiate the development of ulcers (6). Although the vacA gene is present in all H. pylori strains, it contains at least three variable parts, the s region, the i region, and the m region, which encode the signal, intermediate, and middle peptides, respectively, which have all been classified as allelic types 1 and 2. The s1-or-s2/i1/m1-or-m2 and s1-or-s2/i1-i2/m1-or-m2 VacA isotypes induce, in general, high and moderate levels of vacuolation, respectively, whereas the s1-or-s2/i2/m1-or-m2 strains induce very little or no vacuolation (39). Consequently, the vacA s/m genotype can also be regarded as a marker of pathogenicity of individual strains (8). Moreover, phylogenetic linkage analysis studies have indicated that there may be a functional basis for the selection of vacA and cagA isotypes (50), although there is substantial distance between vacA loci and cag genes on the bacterial genome. Furthermore, the intermediate region has been associated with development of gastric cancer (39).In the present study, we investigated the potential association of the CagA and VacA virulence factor polymorphisms with clinicopathological manifestations of the disease in symptomatic Greek children. More specifically, H. pylori clinical strains isolated from symptomatic children were characterized with regard to the number and type of repeating EPIYA phosphorylation motifs in CagA protein and the vacA signal, intermediate, and middle region genotypes. Furthermore, these clinical isolates were carefully assessed for their ability to express phosphorylated CagA as well as induce interleukin-8 (IL-8) secretion following infection of gastric epithelial cells. Finally, the potential association of such functional bacterial determinants with H. pylori-associated histopathology in these patients was assessed.