Characteristics of Helicobacter pylori strains isolated from Mauritanian patients.

BACKGROUND: Helicobacter pylori (H pylori) is responsible for various diseases including cancer It co-evolved with humans, and human migrations shaped the expansion and the diversity of strains around the world. The risk of developing a disease depends on virulence factors, mainly the cytotoxin-associated gene A protein (CagA). The aim of this study was to determine the cagA status in H pylori strains from Mauritanian patients and to search for a relationship with endoscopic and histologic findings. MATERIAL AND METHODS: H pylori was searched in gastric biopsies taken during endoscopy in patients with gastro-duodenal symptoms. RT-PCR was used for the diagnosis and resistance to clarithromycin. The cagA status was determined with PCR and the EPIYA-cagA polymorphism with sequencing. RESULTS: At all, 76/78 (97.4%) biopsies were positive. The rate of clarithromycin resistance was 4/76 (5.26%) due to the A2143G mutation, with a mixed population in 2 cases. The cagA gene was present in 23/76 (30.26%) biopsies, and the EPIYA motif was ABC in 21 (91.3%). High bacterial load and inflammation were significantly associated with cagA-positive status (P < .01). Phylogenetic analysis of the glmM and hspA genes highlighted a mixture of African and European genes in strains of H pylori isolated from patients of Moor origin. CONCLUSION: We report a high prevalence of H pylori infection in Mauritanian patients, a low rate of clarithromycin resistance (5.26%) and high bacterial load and inflammation associated with cagA-positive status. The phylogenetic analysis highlights the mix of different populations leading to the Moor ethnicity.

Malignant Helicobacter pylori-Associated Diseases: Gastric Cancer and MALT Lymphoma.

Helicobacter pylori is the first bacterium formally recognized to play a causative role in human malignancies, gastric cancer and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Evidence accumulates that H. pylori cagA-positive strains play a crucial role in the neoplastic transformation of mammalian cells. The cagA-encoded CagA protein is delivered into the host cells via bacterial type IV secretion, where it interacts with and thereby aberrantly activates pro-oncogenic phosphatase SHP2. The CagA-SHP2 interaction requires tyrosine phosphorylation of CagA at the Glu-Pro-Ile-Tyr-Ala (EPIYA) motif. The incidences of gastric cancer in East Asian countries such as Japan, China, and Korea are among the highest worldwide. A vast majority of H. pylori circulating in East Asia produce a CagA variant termed East Asian CagA, which possesses the SHP2-binding EPIYA motif (EPIYA-D) that is substantially diverged in sequence from the SHP2-binding EPIYA motif (EPIYA-C) of CagA isolated in the rest of the world (Western CagA). Tyrosine-phosphorylated EPIYA-D interacts with SHP2 approximately two orders of magnitude stronger than tyrosine-phosphorylated EPIYA-C does. The strong SHP2 binding of East Asian CagA is achieved by a cryptic interaction between the phenylalanine residue located at the +5 position from the phospho-tyrosine in EPIYA-D and a small hollow on the N-SH2 phosphopeptide-binding floor, the latter of which cannot be created by the corresponding aspartic acid in EPIYA-C. Thus, a variation in a single amino-acid residue determines the magnitude for the pathogenic/oncogenic action of CagA, which may influence the worldwide landscape in the incidence of H. pylori-associated malignancies, especially gastric cancer.

Helicobacter pylori CagA EPIYA-C motifs and gastric diseases in Moroccan patients.

BACKGROUND: The pathogenicity of cagA-positive H. pylori strains is associated with the number and type of repeated sequences named EPIYA located in the C-terminal region of the CagA protein. The aim of this study is to determine the polymorphism of the H. pylori cagA 3' region circulating in Morocco and its association with different gastric pathologies. METHODS: A total of 1353 consenting patients, were recruited in this study. The gastric biopsies performed during endoscopy were used for histological examination and for molecular characterization of H. pylori. The study of the type and number of "EPIYA" motif was identified by PCR directly on H. pylori positive biopsies. RESULTS: Of all the biopsies, the infection rate was 61.1%. The cagA gene was amplified in 68.9% of the cases and the analysis of the 3' region of cagA showed the exclusive presence of the "Western CagA" type with a predominance of the EPIYA-ABC motif (71.4%). The number of EPIYA-C motif varies from 0 to 2. The multinomial analysis shows that the infection with strains of H. pylori having two EPIYA-C motifs is a factor that increases the risk of developing gastric cancer compared to gastritis cases with strains lacking this motif (OR = 11.64; CI: 3.34-45.15), whereas this risk is 6 fold higher in comparison with duodenal ulcer cases (OR = 6, CI: 1.29-27.76). CONCLUSIONS: The results of this study suggest that the number of EPIYA-C motifs might be useful as a predictive marker of the infection evolution and will help in the identification of patients at high risk of developing gastric cancer.

CagA and vacA allelic combination of Helicobacter pylori in gastroduodenal disorders.

BACKGROUND: Allelic variation of the virulence genes, vacA and cagA, as the most important virulence associated genes play an important role in the pathogenesis of severe gastrointestinal disease. OBJECTIVE: The aim of the present study was to identify the diversity of the virulence genes in patients with Gastric Cancer (GC), who were referred to the gastro-endoscopy unit of Imam Khomeini Hospital, Ahvaz Jundishapur University of medical science, Ahvaz, Iran. METHODS: Gastric biopsy specimens from 301 patients suspected to gastrointestinal disorders, were analyzed for H. pylori using molecular and phenotypical methods (culture, and biochemical test (catalase, oxidase and urease tests)). RESULTS: Among 201 PCR positive for H. pylori, using histopathological methods, 22 (10.9%) patients had GC. Presence of vacA gene in our H. pylori strains was 100% (201/201), while the most virulent vacA s1 allele was detected in 82.6% isolates, and the mid region vacA m1 was found in 39.8% isolates. The vacA s1/m1 genotype was the most virulent allelic combination in GC and Peptic Ulcer Disease (PUD) in 68.2% and 50%, respectively. The cagA gene was detected in 66.7% isolates. Among the cagA positive isolates, EPIYA-ABC motif was the most common motif in the GC (66.7%), PUD (55.6%) and Erosive Gastroduodenitis (EG) samples (55.2%), while EPIYA-ABCC was the most common motif (58.7%) in the Non-Ulcer Dyspepsia (NUD) samples. The vacA s1m1/cagA+ combination was detected in GC (73.3%) and PUD (51.9%), while vacA s1m2/cagA+ presented in the NUD and EG samples in 77.8% and 62.1%, respectively. CONCLUSION: In this work, Western type (EPIYA-ABC and ABCC motifs) cagA, vacA s1m1 combinations have been demonstrated as the dominant genotype in the tested Ahvazian H. Pylori strains. Also the participation of cagA gene and vacA s1m1 genotype in development and severity of gastric disorder was well evident. Therefore, infection with H. pylori strain containing the cagA gene or the vacA s1m1 genotypes could be associated with increased risk of GC. This is the first study in our area that reports the high incidence and diversity of allelic combination of cagA and vacA genes in gastroduodenitis patients.

Genetic diversity and functional analysis of oipA gene in association with other virulence factors among Helicobacter pylori isolates from Iranian patients with different gastric diseases.

Helicobacter pylori (H. pylori) is one of the most genetically diverse bacterial pathogens that persistently colonizes the human gastric epithelium. This remarkable genomic plasticity may act as a driving force for successful adaptation and persistence of the bacteria in the harsh gastric environment. Outer inflammatory protein A (OipA) encoded by oipA gene (HP0638/hopH) is a member of the outer membrane proteins (OMPs) of H. pylori involved in induction of IL-8 secretion and is associated with development of peptic ulcer and gastric cancer. Expression of OipA is regulated by phase variation within a CT dinucleotide repeat motif of the oipA gene. In this study we carried out direct DNA sequence analysis of 53 amplified fragments to investigate the oipA "On/Off" status among Iranian H. pylori isolates from patients with various gastric diseases. The prevalence of cagL, cagA, EPIYA motifs, vacA alleles, babA2 and sabA genotypes as well as cagPAI integrity of the isolates were determined by PCR. Our results demonstrated a high prevalence of strains with functional oipA status (79%) and significant associations were found between functional oipA and cagA (P = 0.027) and vacA s1m1 (P = 0.022) genotypes. The vacA s1m2 genotype was also found to be statistically associated with PUD (P = 0.0001). Interestingly, we showed that H. pylori strains with intact cagPAI co-expressed oipA gene in a significant synergistic relationship (P < 0.01). However, no significant association was observed between the functional oipA status and clinical outcomes (P > 0.05). In conclusion, our findings denotes great diversity in the number and pattern of CT dinucleotide repeats of oipA among Iranian H. pylori strains. The synergistic link between functional oipA and other important virulence factors is proposed to be critical in the pathogenesis of H. pylori, which needs further studies with a larger number of samples.

Differential Mechanisms for SHP2 Binding and Activation Are Exploited by Geographically Distinct Helicobacter pylori CagA Oncoproteins.

Helicobacter pylori East Asian CagA is more closely associated with gastric cancer than Western CagA. Here we show that, upon tyrosine phosphorylation, the East Asian CagA-specific EPIYA-D segment binds to the N-SH2 domain of pro-oncogenic SHP2 phosphatase two orders of magnitude greater than Western CagA-specific EPIYA-C. This high-affinity binding is achieved via cryptic interaction between Phe at the +5 position from phosphotyrosine in EPIYA-D and a hollow on the N-SH2 phosphopeptide-binding floor. Also, duplication of EPIYA-C in Western CagA, which increases gastric cancer risk, enables divalent high-affinity binding with SHP2 via N-SH2 and C-SH2. These strong CagA bindings enforce enzymatic activation of SHP2, which endows cells with neoplastic traits. Mechanistically, N-SH2 in SHP2 is in an equilibrium between stimulatory "relaxed" and inhibitory "squeezed" states, which is fixed upon high-affinity CagA binding to the "relaxed" state that stimulates SHP2. Accordingly, East Asian CagA and Western CagA exploit distinct mechanisms for SHP2 deregulation.

Patrón EPIYA en cepas de Helicobacter pylori CagA positivas en pacientes del Hospital Regional de Talca / EPIYA pattern in Helicobacter pylori CagA positive strains in patients of the Regional Hospital of Talca

BACKGROUND: The clinical outcome of Helicobacter pylori (H. pylori) infection has been related to the presence of CagA protein. This protein is highly polymorphic and its oncogenic ability depends on the number and type of tyrosine phosphorylation sites in the EPIYAs repeat sequences (A, B, C and D). AIM: To determine the EPIYA patterns of the CagA gene in H. pylori strains and its relationship with gastrointestinal pathology in infected patients of the Regional Hospital of Talca. MATERIAL AND METHODS: The strains were isolated from gastric biopsies and characterized by bacteriological and molecular methods. Gastrointestinal pathology was characterized by histopathological analysis. For the determination of the presence of the cagA gene and the EPIYAs standards, the conventional PCR technique was used. RESULTS: 138 DNA samples from H. pylori strains were analyzed. 92.0% (127/138) of the isolates carried the cagA gene, of which 66 (52.0%) corresponded to the EPIYA-ABC pattern, 43 (33.8%) to the EPIYA-ABCC pattern and 21 16.5%) to the EPIYA-ABCCC phosphorylation pattern. 50.4% (64/127) of cagA positive strains isolated from dyspeptic patients in the Maule region have more than two C sites of phosphorylation. The number of EPIYAs C motifs was associated with the presence of more severe histopathological damage in the gastric mucosa.

Sequence Polymorphism and Intrinsic Structural Disorder as Related to Pathobiological Performance of the Helicobacter pylori CagA Oncoprotein.

CagA, an oncogenic virulence factor produced by Helicobacter pylori, is causally associated with the development of gastrointestinal diseases such as chronic gastritis, peptic ulcers, and gastric cancer. Upon delivery into gastric epithelial cells via bacterial type IV secretion, CagA interacts with a number of host proteins through the intrinsically disordered C-terminal tail, which contains two repeatable protein-binding motifs, the Glu-Pro-Ile-Tyr-Ala (EPIYA) motif and the CagA multimerization (CM) motif. The EPIYA motif, upon phosphorylation by host kinases, binds and deregulates Src homology 2 domain-containing protein tyrosine phosphatase 2 (SHP2), a bona fide oncoprotein, inducing pro-oncogenic mitogenic signaling and abnormal cell morphology. Through the CM motif, CagA inhibits the kinase activity of polarity regulator partitioning-defective 1b (PAR1b), causing junctional and polarity defects while inducing actin cytoskeletal rearrangements. The magnitude of the pathobiological action of individual CagA has been linked to the tandem repeat polymorphisms of these two binding motifs, yet the molecular mechanisms by which they affect disease outcome remain unclear. Recent studies using quantitative techniques have provided new insights into how the sequence polymorphisms in the structurally disordered C-terminal region determine the degree of pro-oncogenic action of CagA in the gastric epithelium.

C-terminal Src kinase-mediated EPIYA phosphorylation of Pragmin creates a feed-forward C-terminal Src kinase activation loop that promotes cell motility.

Pragmin is one of the few mammalian proteins containing the Glu-Pro-Ile-Tyr-Ala (EPIYA) tyrosine-phosphorylation motif that was originally discovered in the Helicobacter pylori CagA oncoprotein. Following delivery into gastric epithelial cells by type IV secretion and subsequent tyrosine phosphorylation at the EPIYA motifs, CagA serves as an oncogenic scaffold/adaptor that promiscuously interacts with SH2 domain-containing mammalian proteins such as the Src homology 2 (SH2) domain-containing protein tyrosine phosphatase-2 (SHP2) and the C-terminal Src kinase (Csk), a negative regulator of Src family kinases. Like CagA, Pragmin also forms a physical complex with Csk. In the present study, we found that Pragmin directly binds to Csk by the tyrosine-phosphorylated EPIYA motif. The complex formation potentiates kinase activity of Csk, which in turn phosphorylates Pragmin on tyrosine-238 (Y238), Y343, and Y391. As Y391 of Pragmin comprises the EPIYA motif, Pragmin-Csk interaction creates a feed-forward regulatory loop of Csk activation. Together with the finding that Pragmin and Csk are colocalized to focal adhesions, these observations indicate that the Pragmin-Csk interaction, triggered by Pragmin EPIYA phosphorylation, robustly stimulates the kinase activity of Csk at focal adhesions, which direct cell-matrix adhesion that regulates cell morphology and cell motility. As a consequence, expression of Pragmin and/or Csk in epithelial cells induces an elongated cell shape with elevated cell scattering in a manner that is mutually dependent on Pragmin and Csk. Deregulation of the Pragmin-Csk axis may therefore induce aberrant cell migration that contributes to tumor invasion and metastasis.

Multiple repeats of Helicobacter pylori CagA EPIYA-C phosphorylation sites predict risk of gastric ulcer in Iran.

Biological activity of Helicobacter pylori oncoprotein CagA is determined by a diversity in the tyrosine phosphorylation motif sites. In the present study, the diversity and the type of the H. pylori CagA EPIYA motifs and their association with gastric ulcer (GU) and duodenal ulcer (DU) in Iranian dyspeptic patients were assessed. PCR amplification, sequencing, and bioinformatic analysis were performed to determine the pattern of CagA EPIYA motifs. Of 168 H. pylori cagA(+) strains, the frequency of ABC was 93.50%, ABCCC 5.40%, ABC + ABCCC 0.6% and ABCC 0.6%. There was no EPIYA-D segment. The ABCCC pattern of EPIYA motif was more frequent in the H. pylori isolates from GU (8/50, 16%) than in those from chronic gastritis (CG) (0/81, 0%) (P = 0). In contrast, The ABC pattern of EPIYA motif was less frequent in the H. pylori isolates from GU (41/50, 82%) than in those from CG (80/81, 98.80%) (Age-sex-adjusted odds ratio (OR) = 0.020, 95% CI = 0.002-0.259; P = 0.003). The distribution of the ABC motif was almost the same in H. pylori isolates from CG (98.80%) and DU diseases (97.30%). There was no significant association between the number of CagA EPIYA-C segment and DU (P > 0.05). We have proposed that CagA from Iranian H. pylori strains were Western type and all strains had active phosphorylation sites. The three EPIYA-C motifs of CagA were more frequently observed in the H. pylori strains from GU; thus it might be an important biomarker for predicting the GU risk in Iran.

Analysis of the intactness of Helicobacter pylori cag pathogenicity island in Iranian strains by a new PCR-based strategy and its relationship with virulence genotypes and EPIYA motifs.

Variants of the Helicobacter pylori cag pathogenicity island (cagPAI) and certain virulence genotypes have been proposed to be associated with different gastric disorders. In the present study, we designed a new PCR-based strategy to investigate the intactness of cagPAI in Iranian patients using highly specific primer sets spanning the cagPAI region. The possible relationship between the cagPAI status of the strains and clinical outcomes was also determined. We also characterized virulence genotypes (cagL, cagA, vacA, babA2 and sabA) and variants of CagA EPIYA motifs in these strains. H. pylori was detected in 61 out of 126 patients with various gastroduodenal diseases. The cagL, cagA, vacA s1m1, vacA s1m2, vacA s2m2, babA2, and sabA genotypes were detected in 96.7%, 85.2%, 29.5%, 45.9%, 24.6%, 96.7%, and 83.6% of the strains, respectively. Among the 52 cagA-positive strains, EPIYA motifs ABC, ABCC, ABCCC, and mixed types were orderly detected in the 39, 7, 1, and 5 strains. The cagPAI positivity included both intact and partially deleted, with the overall frequencies of 70.5% and 26.2%, respectively. The majority of the strains from patients with PUD (87.5%), gastric erosion (83.3%) and cancer (80%) presented an intact cagPAI, while a lower frequency of cagPAI intactness was detected in gastritis patients (61.1%). However, no significant relationship was found between the possession of intact cagPAI and clinical outcomes. Furthermore, we found that cagA and vacA s1m1 genotypes were significantly correlated with intact cagPAI (P=0.015 and P=0.012). A significant correlation was also found between EPIYA-ABC and intact cagPAI (P=0.010). The proposed PCR-based scheme was found to be useful for determining the intactness of cagPAI. Our findings also indicate that the cagPAI appears to be intact and rather conserved in majority of Iranian strains. Finally, our study proposed that H. pylori strains with partially deleted cagPAI were less likely to cause severe diseases in comparison with those carrying intact cagPAI.

Novel effects of Helicobacter pylori CagA on key genes of gastric cancer signal transduction: a comparative transfection study.

Helicobacter pylori (H. pylori) infection is now recognized as a worldwide problem. Helicobacter pylori CagA is the first bacterial oncoprotein to be identified in relation to human cancer. Helicobacter pylori CagA is noted for structural diversity in its C-terminal region (contains EPIYA motifs), with which CagA interacts with numerous host cell proteins. Deregulation of host signaling by translocated bacterial proteins provides a new aspect of microbial-host cell interaction. The aim of this study is to compare the cellular effects of two different CagA EPIYA motifs on identified signaling pathways involve in gastric carcinogenesis. To investigate the effects of CagA protein carboxyl region variations on the transcription of genes involved in gastric epithelial carcinogenesis pathways, the eukaryotic vector carrying the cagA gene (ABC and ABCCC types) was transfected into gastric cancer cell line. The 42 identified key genes of signal transduction involved in gastric cancer were analyzed at the transcription level by real-time PCR. The results of real-time PCR provide us important clue that the ABCCC oncoprotein variant can change the fate of the cell completely different from ABC type. In fact, these result proposed that the ABCCC type can induce the intestinal metaplasia, IL-8, perturbation of Crk adaptor proteins, anti-apoptotic effect and carcinogenic effect more significantly than ABC type. These data support our hypothesis of a complex interaction of host cell and these two different H. pylori effector variants that determines host cellular fate.