Analysis of the 3′-variable region of the cagA gene from Helicobacter pylori strains infecting patients at New York City hospitals

Helicobacter pylori infects the gastric mucosa in humans and is a causative agent for peptic ulcer disease (PUD) and gastric cancer (GC). CagA is produced by H. pylori and is associated with more severe outcomes. cagA genes vary at the 3′-region with respect to phosphorylation motifs (EPIYA-A, -B, -C, or -D) and CagA multimerization motifs (CM). This variability may be associated with the clinical outcomes. We examined the variable region of cagA genes expressed in H. pylori-infected patients treated at three NYC Hospitals. DNA was isolated from gastric biopsies of patients undergoing upper endoscopy. Most H. pylori-infected patients were Black or Hispanic. The cagA 3′-region of CagA-positive samples was amplified by PCR, purified and sequenced. The patterns of EPIYA and CM motifs were examined and related to clinical outcomes. We obtained 42 CagA sequences from our sample collection. The EPIYA phosphorylation motif pattern was ABC in 81.0% of our samples. Western (W) and Eastern (E) CM motifs have also been defined. CagA proteins lacking an Eastern CM motif and possessing one or two Western CM motifs were observed more frequently in patients with PUD and GC when compared with non-ulcer gastritis (50.0% vs 11.8%, respectively), suggesting that these CM motif patterns are more virulent than those containing at least one Eastern CM motif. We conclude that In H. pylori-infected patients treated at NYC Hospitals, CM motif patterns in the CagA 30-variable region may be more significant than EPIYA motif patterns with respect to clinical outcomes.     

The cag PAI is intact and functional but HP0521 varies significantly in Helicobacter pylori isolates from Malaysia and Singapore

Helicobacter pylori-related disease is at least partially attributable to the genotype of the infecting strain, particularly the presence of specific virulence factors. We investigated the prevalence of a novel combination of H. pylori virulence factors, including the cag pathogenicity island (PAI), and their association with severe disease in isolates from the three major ethnicities in Malaysia and Singapore, and evaluated whether the cag PAI was intact and functional in vitro. Polymerase chain reaction (PCR) was used to detect dupA, cagA, cagE, cagT, cagL and babA, and to type vacA, the EPIYA motifs, HP0521 alleles and oipA ON status in 159 H. pylori clinical isolates. Twenty-two strains were investigated for IL-8 induction and CagA translocation in vitro. The prevalence of cagA, cagE, cagL, cagT, babA, oipA ON and vacA s1 and i1 was >85%, irrespective of the disease state or ethnicity. The prevalence of dupA and the predominant HP0521 allele and EPIYA motif varied significantly with ethnicity (p < 0.05). A high prevalence of an intact cag PAI was found in all ethnic groups; however, no association was observed between any virulence factor and disease state. The novel association between the HP0521 alleles, EPIYA motifs and host ethnicity indicates that further studies to determine the function of this gene are important.     

CagA C-terminal variations in Helicobacter pylori strains from Colombian patients with gastric precancerous lesions

The C-terminus of the Helicobacter pylori CagA protein is polymorphic, bearing different EPIYA sequences (EPIYA-A, B, C or D), and one or more CagA multimerization (CM) motifs. The number of EPIYA-C motifs is associated with precancerous lesions and gastric cancer (GC). The relationship between EPIYA, CM motifs and gastric lesions was examined in H. pylori-infected Colombian patients from areas of high and low risk for GC. Genomic DNA was extracted from H. pylori strains cultured from gastric biopsies from 80 adults with dyspeptic symptoms. Sixty-seven (83.8%) of 80 strains were cagA positive. The 3’ region of cagA was sequenced, and EPIYA and CM motifs were identified. CagA proteins contained one (64.2%), two (34.3%) or three EPIYA-C motifs (1.5%), all with Western type CagA-specific sequences. Strains with one EPIYA-C motif were associated with less severe gastric lesions (non-atrophic and multifocal atrophic gastritis), whereas strains with multiple EPIYA-C motifs were associated with more severe lesions (intestinal metaplasia and dysplasia) (p <0.001). In 54 strains, the CM motifs were identical to those common in Western strains. Thirteen strains from the low-risk area contained two different CM motifs: one of Western type located within the EPIYA-C segment and another following the EPIYA-C segment and resembling the CM motif found in East Asian strains. These strains induced significantly shorter projections in AGS cells and an attenuated reduction in levels of CagA upon immunodepletion of SHP-2 than strains possessing Western/Western motifs. This novel finding may partially explain the difference in GC incidence in these populations.     

The number of Helicobacter pylori CagA EPIYA C tyrosine phosphorylation motifs influences the pattern of gastritis and the development of gastric carcinoma

Ferreira R M, Machado J C, Leite M, Carneiro F & Figueiredo C
(2012) Histopathology  60, 992–998 The number of Helicobacter pylori CagA EPIYA C tyrosine phosphorylation motifs influences the pattern of gastritis and the development of gastric carcinoma Aims: To characterize the variation in virulence of Helicobacter pylori associated with CagA Glu‐Pro‐Ile‐Tyr‐Ala (EPIYA) motifs, and to explore its relationship with the histopathological features of chronic gastritis and with the development of gastric carcinoma. Methods and results: A total of 169 H. pylori‐infected patients with chronic gastritis and gastric carcinoma were studied. The presence of cagA and the number and type of EPIYA motifs were determined by polymerase chain reaction. Infection with strains harbouring two or more CagA EPIYA C motifs was associated with the presence of surface epithelial damage, and with atrophic gastritis and gastric carcinoma. The magnitude of risk for atrophic gastritis and gastric carcinoma increased with increasing number of EPIYA C motifs: strains with one EPIYA C motif conferred a risk (odds ratio [OR]) of 7.3 [95% confidence interval (CI) 2.1–25] for atrophic gastritis, whereas strains with two or more EPIYA C motifs conferred a risk (OR) of 12 (95% CI 2.5–58); strains with one EPIYA C motif conferred a risk (OR) of 17 (95% CI 5.4–55) for gastric carcinoma, whereas strains with two or more EPIYA C motifs conferred a risk (OR) of 51 (95% CI 13–198). Conclusions: Characterization of the number of H. pylori EPIYA C motifs is important in better defining gastric carcinoma risk.     

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.     

Molecular Identification and Detection of Virulent Factors in Helicobacter pylori from Gastric Biopsy Samples of Patients Attended at Assam Medical College and Hospital,Dibrugarh, Assam,India

The present study aimed to detect the major virulence determinants of Helicobacter pylori, the gastric bacteria by polymerase chain reaction from genomic DNA of 314 gastric biopsies from dyspeptic patients in 2015-2016 through upper gastrointestinal endoscopy. In 153 cases out of 314, the high prevalence of oipA gene followed by cagA-vacA s1 m1 combined genotypes was found in mostly gastritis/duodenitis patients followed by the peptic ulcer and normal patients. Therefore, the clinical significance of the virulence markers of H. pylori associated with the severe forms of gastroduodenal diseases is still a matter of controversy since the endoscopically normal patients were found to harbour the virulent genes.     

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.     

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.     

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.     

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

Detection of Helicobacter pylori cagA gene in gastric biopsies, clinical isolates and faeces

Purpose: Helicobacter pylori infection is common in the developing countries. The cagA gene is a marker of pathogenicity island (PAI) in H. pylori. The aim of this study was to determine the prevalence of cagA among dyspeptic patients in Bahrain directly from gastric biopsy and stool specimen. Methods: A total of 100 gastric biopsy samples, 16 clinical isolates and 44 faecal specimens were collected from Bahraini adult dyspeptic patients. cagA gene of H. pylori was assessed using polymerase chain reaction (PCR). Results: The cagA gene was detected in 59 (59%) from biopsy specimens, 10 (62%) clinical isolates and in 10 (22.7%) faecal specimens. The detection of cagA positive H. pylori was significantly higher in patients with duodenal ulcer (80%) compared to those with other endoscopic finding (42%) (P<0.05). Conclusions: Using PCR to detect cagA gene directly from biopsy is a rapid and reliable technique. However, using stool specimen for genotyping in our patients showed reduced sensitivity.     

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.     

Clinical relevance of cagA and vacA gene polymorphisms in Helicobacter pylori isolates from Senegalese patients

The molecular epidemiology of Helicobacter pylori in Africa is poorly documented. From January 2007 to December 2008, we investigated 187 patients with gastric symptoms in one of the main tertiary hospitals in Dakar, Senegal. One hundred and seventeen patients were culture-positive for H. pylori. Polymorphisms in vacA and cagA status were investigated by PCR; the 3′-region of cagA was sequenced, and EPIYA motifs were identified. Bacterial heterogeneity within individuals was extensively assessed by using an approach based on vacA and cagA heterogeneity. Fourteen per cent of H. pylori-positive patients displayed evidence of mixed infection, which may affect disease outcome. Patients with multiple vacA alleles were excluded from subsequent analyses. Among the final study population of 105 patients, 29 had gastritis only, 61 had ulcerated lesions, and 15 had suspicion of neoplasia based on endoscopic findings. All cases of suspected neoplasia were histologically confirmed as gastric cancer (GC). The cagA gene was present in 73.3% of isolates. CagA proteins contained zero (3.7%), one (93.9%) or two (2.4%) EPIYA-C segments, and all were western CagA. Most of the isolates possessed presumed high-vacuolization isotypes (s1i1m1 (57.1%) or s1i1m2 (21.9%)). Despite the small number of cases, GC was associated with cagA (p 0.03), two EPIYA-C segments in the C-terminal region of CagA (p 0.03), and the s1 vacA allele (p 0.002). Multiple EPIYA-C segments were less frequent than reported in other countries, possibly contributing to the low incidence of GC in Senegal.     

Variants of the 3′ Region of the cagA Gene in Helicobacter pylori Isolates from Patients with Different H. pylori-Associated Diseases

The CagA protein of Helicobacter pylori is an immunogenic antigen of variable size and unknown function that has been associated with increased virulence as well as two mutually exclusive diseases, duodenal ulcer and gastric carcinoma. The 3′ region of the cagA gene contains repeated sequences. To determine whether there are structural changes in the 3′ region of cagA that predict outcome of H. pylori infection, we examined 155 cagA gene-positive H. pylori isolates from Japanese patients including 50 patients with simple gastritis, 40 with gastric ulcer, 35 with duodenal ulcer, and 30 with gastric cancer. The 3′ region of the cagA gene was amplified by PCR followed by sequencing. CagA proteins were detected by immunoblotting using a polyclonal antibody against recombinant CagA. One hundred forty-five strains yielded PCR products of 642 to 651 bp; 10 strains had products of 756 to 813 bp. The sequence of the 3′ region of the cagA gene in Japan differs markedly from the primary sequence of cagA genes from Western isolates. Sequence analysis of the PCR products showed four types of primary gene structure (designated types A, B, C, and D) depending on the type and number of repeats. Six of the seven type C strains were found in patients with gastric cancer (P < 0.01 in comparison to noncancer patients). Comparison of type A and type C strains from patients with gastric cancer showed that type C was associated with higher levels of CagA antibody and more severe degrees of atrophy. Differences in cagA genotype may be useful for molecular epidemiology and may provide a marker for differences in virulence among cagA-positive H. pylori strains.     

CagA subtyping in Helicobacter pylori isolates from gastric cancer patients in an ethnic Kashmiri population

The association between gastric cancer and Helicobacter pylori has been well established. Among H. pylori virulence genes the most important determinant is the cytotoxin associated antigen gene (cagA) which is characterized by the presence of repeated EPIYA motifs at the C terminus of the protein. From the alignment and number of these EPIYA motifs, two major types of CagA protein have been identified.AimsThe aim of this study was to classify the CagA into eastern or western type and to determine the number and type of motifs present.MethodsThe CagA subtyping was done by PCR and multiplex PCR for eastern/western classification and determination of EPIYA motifs respectively.ResultsAll the isolates studied were of the western type, with 70% of the isolates having more than one EPIYA-C motifs. No statistically significant association was found between the presence of CagA and more than one EPIYA-C motifs with the clinical outcome (differentiation status of the tumour).     

homB Status of Helicobacter pylori as a Novel Marker To Distinguish Gastric Cancer from Duodenal Ulcer

The hom family of Helicobacter pylori outer-membrane proteins, especially the homB gene, has been suggested as a novel virulence factor; however, the clinical association and function of this gene are still unclear. We evaluated the presence of the homA, homB, and cagA genes in 286 strains isolated from patients in the U.S. and Colombian populations (126 with gastritis, 96 with duodenal ulcer, and 64 with gastric cancer) by PCR. The results were compared with the clinical presentation and gastric injury. The prevalence of the homB gene was significantly higher in strains isolated from gastric-cancer patients (71.9%) than in those from duodenal ulcer patients (52.1%) (P = 0.012). In a multivariate analysis, the presence of the cagA gene significantly increased the risk for developing gastric cancer and duodenal ulcer, with the presence of the homB gene acting as a factor that could distinguish gastric cancer from duodenal ulcer (adjusted odds ratio, 3.033; 95% confidence interval, ∼1.37 to ∼6.73). cagA status was correlated with homB status (r = 0.323; P < 0.01). A histological analysis showed that cagA status was associated with inflammation and atrophy both in the antrum and in the corpus, while homB status was associated with inflammation and atrophy in the corpus. homB gene status might be susceptible to gastric-cancer development such that the homB gene is used as a factor for discriminating the risk of gastric cancer from that of duodenal ulcer.Helicobacter pylori infection is one of the most common infections of mankind and is etiologically associated with gastritis, peptic ulcer disease (PUD), gastric cancer (GC), and gastric mucosa-associated lymphoid tissue lymphoma (19). Most infected people remain asymptomatic. Factors thought to be associated with clinical gastroduodenal diseases include H. pylori virulence, host genetics, and environmental factors, such as diet (11). Putative H. pylori virulence factors associated with an increased risk of a clinical outcome include the cag pathogenicity island, CagA, VacA, BabA, and OipA (5, 9, 22). However, none have been exclusively linked to a specific H. pylori-related disease (e.g., GC).The hom family is a small paralogous family of proteins that contain the C-terminal alternating hydrophobic motif and signal sequences typical of outer-membrane proteins. The homB and homA genes are 90% identical; the differences are confined to the central domain (1). Recent studies suggested that there was a close association between the presence of the homB gene and interleukin-8 secretion from human gastric epithelial cells and that the number of H. pylori isolates binding to gastric cells was related to the number of homB copies present (15). Moreover, the authors proposed that the presence of the homB gene was significantly associated with PUD in Portuguese children and adults less than 40 years of age and that it may be a new H. pylori virulence factor (15, 16). However, there is no study for the association between the homB gene and H. pylori-related diseases in other countries. This study investigated whether there was an association between the homA and homB genes and clinical gastroduodenal diseases and the severity of gastric inflammation in the U.S. and Colombian populations.     

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.