EPIYA Motifs Research Articles

Helicobacter pylori biofilm: a protective environment for bacterial recombination

The aim of this work was to investigate the interaction between two Helicobacter pylori strains in promoting genetic transfer, when grown in the biofilm mode. Biofilms produced by H. pylori 9/10 (A), H. pylori 15/4 (B) and their mixture (C) were studied for biomass production and cell viability. The genetic heterogeneity of 45 clones, coming from mature biofilm of co-cultured H. pylori strains was studied by both RAPD and cagA (EPIYA motifs)/vacA virulence genes analysis. Helicobacter pylori A, B and C developed a well-structured biofilm without significant differences in viability. No significant differences were recorded between A and B biomass measurement, whereas C biofilm expressed a significant (P < 0.001) higher adhesive capability when compared with A and B biofilms. C-clones DNA-fingerprintings showed an high genetic heterogeneity (mean similarity value = 0.528). The 60% of C-clones displayed vacA allelic combination s1i1m1m2 associated with cagA EPIYA motif pattern P1P2P3P3P3. Biofilms developed by multiple H. pylori strains are more complex than those associated with single strains. Such condition might promote the genetic exchange favouring the generation of more virulent strains. The 'biofilm niche' represents a successful strategy and a suitable environment for promoting bacterial population persistence by recombination events.

Distinct repeat motifs at the C-terminal region of CagA of Helicobacter pylori strains isolated from diseased patients and asymptomatic individuals in West Bengal, India

BackgroundInfection with Helicobacter pylori strains that express CagA is associated with gastritis, peptic ulcer disease, and gastric adenocarcinoma. The biological function of CagA depends on tyrosine phosphorylation by a cellular kinase. The phosphate acceptor tyrosine moiety is present within the EPIYA motif at the C-terminal region of the protein. This region is highly polymorphic due to variations in the number of EPIYA motifs and the polymorphism found in spacer regions among EPIYA motifs. The aim of this study was to analyze the polymorphism at the C-terminal end of CagA and to evaluate its association with the clinical status of the host in West Bengal, India.ResultsSeventy-seven H. pylori strains isolated from patients with various clinical statuses were used to characterize the C-ternimal polymorphic region of CagA. Our analysis showed that there is no correlation between the previously described CagA types and various disease outcomes in Indian context. Further analyses of different CagA structures revealed that the repeat units in the spacer sequences within the EPIYA motifs are actually more discrete than the previously proposed models of CagA variants.ConclusionOur analyses suggest that EPIYA motifs as well as the spacer sequence units are present as distinct insertions and deletions, which possibly have arisen from extensive recombination events. Moreover, we have identified several new CagA types, which could not be typed by the existing systems and therefore, we have proposed a new typing system. We hypothesize that a cagA gene encoding higher number EPIYA motifs may perhaps have arisen from cagA genes that encode lesser EPIYA motifs by acquisition of DNA segments through recombination events.

A Potential Association between Helicobacter pylori CagA EPIYA and Multimerization Motifs with Cytokeratin 18 Cleavage Rate during Early Apoptosis

Helicobacter pylori is a highly diverse pathogen, which encounters epithelial cells as the initial defense barrier during its lifelong infection. The structure of epithelial cells can be disrupted through cleavage of microfilaments. Cytokeratin 18 (CK18) is an intermediate filament, the cleavage of which is considered an early event during apoptosis following activation of effector caspases. Helicobacter pylori strains were isolated from 76 dyspeptic patients. cagA 3' variable region and CagA protein status were analyzed by PCR and western blotting, respectively. Eight hours post-co-culture of AGS cells with different H. pylori strains, flow cytometric analysis was performed using M30 monoclonal antibody specific to CK18 cleavage-induced neo-epitope. Higher rates of CK18 cleavage were detected during co-culture of AGS cells with H. pylori strains bearing greater numbers of cagA EPIYA-C and multimerization (CM) motifs. On the other hand, H. pylori strains with greater numbers of EPIYA-B relative to EPIYA-C demonstrated a decrease in CK18 cleavage rate. Thus, H. pylori-mediated cleavage of CK18 appeared proportional to the number of CagA EPIYA-C and CM motifs, which seemed to be downplayed in the presence of EPIYA-B motifs. Our observation associating the heterogeneity of cagA variants with the potential of H. pylori strains in the induction of CK18 cleavage as an early indication of apoptosis in gastric epithelial cells supports the fact that apoptosis may be a type-specific trait. However, additional cagA-targeted experiments are required to clearly identify the role of EPIYA and CM motifs in apoptosis and/or the responsible effector molecules.

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

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. 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). Characterization of the number of H. pylori EPIYA C motifs is important in better defining gastric carcinoma risk.

CagA structural types of Helicobacter pylori strains isolated from patients with gastric carcinoma and chronic gastritis only

Gastric carcinoma (GC) is an important cause of death and is associated with infection by Helicobacter pylori (H. pylori) strains expressing CagA, an oncoprotein encoded by the cagA gene (for a comprehensive discussion of H. pylori infection, see Ref. [1]). Recent studies have shown that CagA can be injected into the colonized mucocytes through a conjugative apparatus encoded by cag genes upstream cagA. Once inside the cell, CagA undergoes phosphorylation by host cell kinases. Phosphorylated CagA is capable of altering the cell morphology and disturbing host-signaling pathways, which ultimately may lead to the development of GC. The fragment of the cagA gene encoding the carboxylterminal end of CagA is polymorphic, as it presents numerous genomic repetitions that contain the tyrosine EPIYA motif, a 5-amino acid sequence (Glu–Pro–Ile–Tyr– Ala), which is the site where CagA undergoes phosphorylation. In 1998, Yamaoka et al. [2] observed that strains with a high number of genomic repetitions in the cagA variable region were isolated more frequently from patients with GC. They classified the cagA structural types in A, B, C and D types, on the basis of the amplicon sizes obtained with primers spanning the entire cagA variable region. Types B and D have similar sizes and can be differentiated by sequencing the amplicons. Later on, the dissection of the cagA 30 repeat region of numerous H. pylori strains indicated that the occurrence of gastric mucosa atrophy, a lesion considered a pre-cancerous condition, was more likely when patients were infected by strains possessing a high number of repeats, i.e. strains with type C cagA gene [3]. In the scientific literature, there are only few reports about the possible association between clinical features and the CagA structural variability. In the present study, we investigated on the possible relationship existing between the polymorphism of the cagA 30 variable region and the occurrence of GC. We also determined the CagA mass expressed by different strains to ascertain whether the CagA size corresponded to the length of the amplicons. Forty H. pylori strains were isolated from resected gastric mucosa of patients with GC and 46 strains from gastric biopsies of patients with chronic gastritis only (CGO). Strains were identified by the Gram stain and oxidase, catalase and urease tests and stored at -80 C until the characterisation of the cagA polymorphism was performed. The cagA structural types were determined by polymerase chain reaction (PCR) using the following primers spanning the cagA variable region [2]; the different sizes of the amplicons obtained range from 642 to 834 bp:

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.

J-Western Forms of Helicobacter pylori cagA Constitute a Distinct Phylogenetic Group with a Widespread Geographic Distribution

Chronic infection with Helicobacter pylori strains expressing the bacterial oncoprotein CagA confers an increased risk of gastric cancer. While much is known about the ancestry and molecular evolution of Western, East Asian, and Amerindian cagA sequences, relatively little is understood about a fourth group, known as "J-Western," which has been detected mainly in strains from Okinawa, Japan. We show here that J-Western cagA sequences have a more widespread global distribution than previously recognized, occur in strains with multiple different ancestral origins (based on multilocus sequence typing [MLST] analysis), and did not arise recently. As shown by comparisons of Western and J-Western forms of CagA, there are 45 fixed or nearly fixed amino acid differences, and J-Western forms contain a unique 4-amino-acid insertion. The mean nucleotide diversity of synonymous sites (π(s)) is slightly lower in the J-Western group than in the Western and East Asian groups (0.066, 0.086, and 0.083, respectively), which suggests that the three groups have comparable, but not equivalent, effective population sizes. The reduced π(s) of the J-Western group is attributable to ancestral recombination events within the 5' region of cagA. Population genetic analyses suggest that within the cagA region encoding EPIYA motifs, the East Asian group underwent a marked reduction in effective population size compared to the Western and J-Western groups, in association with positive selection. Finally, we show that J-Western cagA sequences are found mainly in strains producing m2 forms of the secreted VacA toxin and propose that these functionally interacting proteins coevolved to optimize the gastric colonization capacity of H. pylori.

Relación entre la integridad del CagPAI y los polimorfismos en el gen CagA con la severidad de la gastritis en pacientes infectados con H. pylori CagA positivo

ObjectivesTo evaluate gastric histopathology in Colombian patients infected with CagA-positive Helicobacter pylori and its association with the integrity of Cag (CagPAI) pathogenicity island and the number of EPIYA-C motifs present in the CagA protein. MethodsThirty-one (31) individuals diagnosed with gastritis were included in the study. Using gastric biopsies, CagA-positive H.pylori was isolated and EPIYA motif makeup was characterized by sequencing. Histopathology was evaluated with updated Sydney system. The CagA, CagT, CagE and Cag10 genes were genotyped using PCR and agarose electrophoresis. ResultsA total of 24 isolates (77% of cases) carried a complete CagPAI. Among the negative isolates for one or more genes with CagPAI, 7 (22%) were considered to be carriers of a defective CagPAI. No significant differences were observed in H. pylori density averages, degree of chronic inflammation, presence of glandular atrophy or intestinal metaplasia between isolates with complete CagPAI and isolates with defective CagPAI. No significant differences were observed in the histopathological parameters between isolates with one EPIYAC motif and those with more than one EPIYA-C motif, neither in antrum nor in body, except for infiltration by neutrophils which was significantly greater in bodies with isolates with more than one EPIYA-C motif (p=0.018). ConclusionsNo association was found between the diversity of virulence CagPAI and CagA factors in Colombian isolates and the histopathological findings in gastritis, or in other host or environmental factors that could affect the histopathological characteristics of gastritis.

Variation in number of cagA EPIYA-C phosphorylation motifs between cultured Helicobacter pylori and biopsy strain DNA

The Helicobacter pylori cagA gene encodes a cytotoxin which is activated by phosphorylation after entering the host epithelial cell. Phosphorylation occurs on specific tyrosine residues within EPIYA motifs in the variable 3′-region. Four different cagA EPIYA motifs have been defined according to the surrounding amino acid sequence; EPIYA-A, -B, -C and -D. Commonly, EPIYA-A and -B are followed by one or more EPIYA-C or -D motif. Due to observed discrepancies in cagA genotypes in cultured H. pylori and the corresponding DNA extracts it has been suggested that genotyping assays preferentially should be performed directly on DNA isolated from biopsy specimens. Gastric biopsies randomly selected from a Swedish cohort were homogenised and used for both direct DNA isolation and for H. pylori specific culturing and subsequent DNA isolation. In 123 of 153 biopsy specimens, the cagA EPIYA genotypes were in agreement with the corresponding cultured H. pylori strains. A higher proportion of mixed cagA EPIYA genotypes were found in the remaining 30 biopsy specimens. Cloning and sequencing of selected cagA EPIYA amplicons revealed variations in number of cagA EPIYA-C motifs in the mixed amplicons. The study demonstrates that culturing of H. pylori introduces a bias in the number of EPIYA-C motif. Consistent with other H. pylori virulence genotyping studies, we suggest that cagA EPIYA analysis should be performed using total DNA isolated from biopsy specimens.

EPIYA motif patterns among Cuban Helicobacter pylori CagA positive strains

Introduction. It is known that polymorphisms in C-terminal region of CagA influence gastric disease development on Helicobacter pylori infection. Additionally, the geographic distribution of these polymorphisms has been associated with the appearance of more severe gastroduodenal pathologies.Objective. To determine the CagA phosphorylation motifs pattern (EPIYA pattern) in Cuban H. pylori isolates, and to study its association with patient’s pathologies. Materials and methods. DNAs from 95 H. pylori cagA-positive strains were used to amplify the 3’ variable region of cagA gene by PCR using two different strategies. Additionally, new primers were designed to identify either Western or Eastern CagA EPIYA motif type by PCR. To confirm the PCR results, PCR products from 14 representative isolates were purified and sequencedResults. The distribution of the EPIYA motif found was: 2 AB (2.1 %), 1 AC (1.1 %), 1 BC (1.1 %), 70 ABC (73.6 %), 19 ABCC (20 %), and 2 ABCCC (2.1 %). Sequencing analysis confirmed the PCR classification in the 14 studied strains and showed three strains with unusual nucleotide sequences, not reported before. Distribution of the EPIYA-ABC pattern was equivalent in all pathologies (78.9 % in gastric ulcer, 72.5 % in duodenal ulcer and 72.2 % in non-ulcer dyspepsia).Conclusion. The PCR results using the new primers confirmed that all studied strains carried the Western CagA type. No specific EPIYA motif was associated with peptic ulcer. This is the first report that shows EPIYA motif distribution in H. pylori isolates from the Caribbean region.

EPIYA motif patterns among Cuban Helicobacter pylori CagA positive strains.

It is known that polymorphisms in C-terminal region of CagA influence gastric disease development on Helicobacter pylori infection. Additionally, the geographic distribution of these polymorphisms has been associated with the appearance of more severe gastroduodenal pathologies. Objective. To determine the CagA phosphorylation motifs pattern (EPIYA pattern) in Cuban H. pylori isolates, and to study its association with patient´s pathologies. DNAs from 95 H. pylori cagA-positive strains were used to amplify the 3´ variable region of cagA gene by PCR using two different strategies. Additionally, new primers were designed to identify either Western or Eastern CagAEPIYA motiftype by PCR. To confirm the PCR results, PCR products from 14 representative isolates were purified and sequenced. The distribution of the EPIYA motif found was: 2 AB (2.1 %), 1 AC (1.1 %), 1 BC (1.1 %), 70 ABC (73.6 %), 19 ABCC (20 %), and 2 ABCCC (2.1 %). Sequencing analysis confirmed the PCR classification in the 14 studied strains and showed three strains with unusual nucleotide sequences, not reported before. Distribution of the EPIYA-ABC pattern was equivalent in all pathologies (78.9 % in gastric ulcer, 72.5 % in duodenal ulcer and 72.2 % in non-ulcer dyspepsia). The PCR results using the new primers confirmed that all studied strains carried the Western CagA type. No specific EPIYA motif was associated with peptic ulcer. This is the first report that shows EPIYA motif distribution in H. pylori isolates from the Caribbean region.

Potentiation of Helicobacter pylori CagA Protein Virulence through Homodimerization

Chronic infection with Helicobacter pylori cagA-positive strains is associated with atrophic gastritis, peptic ulceration, and gastric carcinoma. The cagA gene product, CagA, is delivered into gastric epithelial cells via type IV secretion, where it undergoes tyrosine phosphorylation at the EPIYA motifs. Tyrosine-phosphorylated CagA binds and aberrantly activates the oncogenic tyrosine phosphatase SHP2, which mediates induction of elongated cell morphology (hummingbird phenotype) that reflects CagA virulence. CagA also binds and inhibits the polarity-regulating kinase partitioning-defective 1 (PAR1)/microtubule affinity-regulating kinase (MARK) via the CagA multimerization (CM) sequence independently of tyrosine phosphorylation. Because PAR1 exists as a homodimer, two CagA proteins appear to be passively dimerized through complex formation with a PAR1 dimer in cells. Interestingly, a CagA mutant that lacks the CM sequence displays a reduced SHP2 binding activity and exhibits an attenuated ability to induce the hummingbird phenotype, indicating that the CagA-PAR1 interaction also influences the morphological transformation. Here we investigated the role of CagA dimerization in induction of the hummingbird phenotype with the use of a chemical dimerizer, coumermycin. We found that CagA dimerization markedly stabilizes the CagA-SHP2 complex and thereby potentiates SHP2 deregulation, causing an increase in the number of hummingbird cells. Protrusions of hummingbird cells induced by chemical dimerization of CagA are further elongated by simultaneous inhibition of PAR1. This study revealed a role of the CM sequence in amplifying the magnitude of SHP2 deregulation by CagA, which, in conjunction with the CM sequence-mediated inhibition of PAR1, evokes morphological transformation that reflects in vivo CagA virulence.

Mammalian Pragmin regulates Src family kinases via the Glu-Pro-Ile-Tyr-Ala (EPIYA) motif that is exploited by bacterial effectors

Several pathogenic bacteria have adopted effector proteins that, upon delivery into mammalian cells, undergo tyrosine phosphorylation at the Glu-Pro-Ile-Tyr-Ala (EPIYA) or EPIYA-like sequence motif by host kinases such as Src family kinases (SFKs). This EPIYA phosphorylation triggers complex formation of bacterial effectors with SH2 domain-containing proteins that results in perturbation of host cell signaling and subsequent pathogenesis. Although the presence of such an anomalous protein interaction suggests the existence of a mammalian EPIYA-containing protein whose function is mimicked or subverted by bacterial EPIYA effectors, no molecule that uses the EPIYA motif for biological function has so far been reported in mammals. Here we show that mammalian Pragmin/SgK223 undergoes tyrosine phosphorylation at the EPIYA motif by SFKs and thereby acquires the ability to interact with the SH2 domain of the C-terminal Src kinase (Csk), a negative regulator of SFKs. The Pragmin-Csk interaction prevents translocalization of Csk from the cytoplasm to the membrane and subsequent inactivation of membrane-associated SFKs. As a result, SFK activity is sustained in cells where Pragmin is phosphorylated at the EPIYA motif. Because EPIYA phosphorylation of Pragmin is mediated by SFKs, cytoplasmic sequestration of Csk by Pragmin establishes a positive feedback regulation of SFK activation. Remarkably, the Helicobacter pylori EPIYA effector CagA binds to the Csk SH2 domain in place of Pragmin and enforces membrane recruitment of Csk and subsequent inhibition of SFKs. This work identifies Pragmin as a mammalian EPIYA effector and suggests that bacterial EPIYA effectors target Pragmin to subvert SFKs for successful infection.

Evolution of cagA Oncogene of Helicobacter pylori through Recombination

Helicobacter pylori is a gastric pathogen that infects half the human population and causes gastritis, ulcers, and cancer. The cagA gene product is a major virulence factor associated with gastric cancer. It is injected into epithelial cells, undergoes phosphorylation by host cell kinases, and perturbs host signaling pathways. CagA is known for its geographical, structural, and functional diversity in the C-terminal half, where an EPIYA host-interacting motif is repeated. The Western version of CagA carries the EPIYA segment types A, B, and C, while the East Asian CagA carries types A, B, and D and shows higher virulence. Many structural variants such as duplications and deletions are reported. In this study, we gained insight into the relationships of CagA variants through various modes of recombination, by analyzing all known cagA variants at the DNA sequence level with the single nucleotide resolution. Processes that occurred were: (i) homologous recombination between DNA sequences for CagA multimerization (CM) sequence; (ii) recombination between DNA sequences for the EPIYA motif; and (iii) recombination between short similar DNA sequences. The left half of the EPIYA-D segment characteristic of East Asian CagA was derived from Western type EPIYA, with Amerind type EPIYA as the intermediate, through rearrangements of specific sequences within the gene. Adaptive amino acid changes were detected in the variable region as well as in the conserved region at sites to which no specific function has yet been assigned. Each showed a unique evolutionary distribution. These results clarify recombination-mediated routes of cagA evolution and provide a solid basis for a deeper understanding of its function in pathogenesis.

Role of Helicobacter Pylori Virulence Factors for Iron Acquisition from Gastric Epithelial Cells of the Host and Impact on Bacterial Colonization

EVALUATION OF: Tan S, Noto JM, Romero-Gallo J, Peek RM Jr, Amieva MR. Helicobacter pylori perturbs iron trafficking in the epithelium to grow on the cell surface. PLoS Pathog. 7(5), E1002050 (2011). The effects of Helicobacter pylori virulence factors on gastric epithelial cells are topics open to many studies. Major virulence factors, cytotoxin-associated gene A (CagA) and vacuolating cytotoxin (VacA), predict severe infection outcomes in many countries. H. pylori possesses various proteins for iron transport/storage, however, mechanisms of iron acquisition are not fully evaluated. The study by Tan et al. reveals a concurrent CagA/VacA activity for micronutrient acquisition and host tissue colonization. The virulence factors possess new activities, involving VacA-induced apical mislocalization of transferrin receptors to regions of H. pylori attachment and effects of both factors on polarized uptake and recycling of transferrin. The authors used many in vitro methods and an animal model. Iron acquisition by CagA was proven in vitro and in vivo by strain colonization of the gastric mucosa in iron-depleted conditions. CagA EPIYA motifs were associated with increased host internalization of transferrin. Importantly, CagA and VacA were involved in iron acquisition and colonization without severely damaging the host cells, thus favoring the infection chronicity. Further studies should assess molecular mechanisms of H. pylori iron acquisition, comparative activities of contact-dependent/soluble VacA and Eastern/Western CagA on the polarized epithelium and long-term effects of iron deficiency by virulent versus less virulent H. pylori strains. An interesting topic is the association of virulent strains with iron deficiency anemia but also with various H. pylori-induced diseases, in different populations and, possibly, for other bacterial infections. In conclusion, H. pylori iron acquisition is multifaceted. CagA and VacA work concurrently to provide both iron acquisition from interstitial holotransferrin and enhanced bacterial colonization of host cells apically. The new activities of the major virulence factors of adherent H. pylori are important both to research and in a clinical setting.

CagA Phosphorylation-Dependent MMP-9 Expression in Gastric Epithelial Cells

Infection of cagA-positive Helicobacter pylori is associated with increased expression of MMPs in gastric epithelial cells. The role of phosphorylated CagA in the induction of MMP-9, a protease-degrading basement membrane, in gastric epithelial cells has not been clearly defined yet. The aim of this study is to analyze whether the presence of CagA and its phosphorylation status play a role in increased expression of MMP-9 in gastric epithelial cells. Induction of MMP-9 secretion was analyzed in gastric epithelial AGS cells harboring CagA with or without EPIYA motif, which is injected by H. pylori or ectopically expressed. In addition, signaling pathways involved in the CagA-dependent MMP-9 production have been studied. The 147C strain of H. pylori expressing tyrosine-phosphorylated CagA (EPIYA present) induced higher MMP-9 secretion by AGS cells than the 147A strain expressing non-tyrosine-phosphorylated CagA (EPIYA absent). In addition, in bacteria-free CagA-inducible AGS cells, expression of wild-type CagA induced more MMP-9 secretion than phosphorylation-resistant CagA. Inhibition of CagA phosphorylation by the Src family kinase inhibitor PP1 downregulated CagA-mediated MMP-9 secretion. Knockdown of SHP-2 phosphatase dramatically reduced MMP-9 secretion. ERK inhibitors, PD98059 and U0126, and NF-κB pathway inhibitors, sulfasalazine and N-acetyl-l-cysteine, also inhibited MMP-9 expression. These results support a model whereby the EPIYA motif of CagA is phosphorylated by Src family kinases in gastric epithelial cells, which initiates activation of SHP-2. In addition, they suggest that the resultant activation of ERK pathway along with CagA-dependent NF-κB activation is critical for the induction of MMP-9 secretion.

In Vivo Immunohistochemistry With Labeled Bevacizumab Using Confocal Laser Endomicroscopy in Models of Human Colorectal Cancer

Helicobacter pylori CagA is delivered into H. pylori-adherent gastric epithelial cells through a bacterial type IV secretion system. The delivered CagA is tyrosine-phosphorylated at the EPIYA motifs by the host cell kinases, and then binds and aberrantly-activate Src homology 2 domain-containing protein tyrosine phosphatase 2 (SHP-2) and partitioning-defective 1 (PAR1), thereby acting as an epigenetic oncoprotein that promotes gastric cancer development in its early phase. Recently, Ishikawa et al. reported that the biological half-life of CagA is about 200 min in the gastric epithelial cells (FEBS Lett.,583:2414,2009). This report showed that the action of CagA as an epigenetic oncoprotein was not sustained for a prolonged period of time in the normal condition. In fact, the stability of CagA in the gastric epithelial cells is important for its further oncogenesis. The present study was designed to examine the degradation mechanisms of CagA in the gastric epithelial cells. Methods: MKN28 cells were transfected with CagA expression vectors (the pTet-Off and pTRE2hyg-Wild-typecagA-HA) (WT-A10 cells), and then CagA in the WT-A10 cells was induced for 24 h. H. pylori ATCC700392 was infected with AGS cells for 5 h. At 5 h after the infection, the AGS cells were incubated with 400 μg/mL kanamycin and harvested at 4, 12 and 24 h. 50 μM Z-Leu-Leu-Leu-al (MG132) and 20 μM lactacystin were used as the 26S-proteasome inhibitor, 5 mM3-methyladenine and 50 nMwortmannin as the inhibitors of autophagy, and rapamycin as the inducer of autophagy. Intracellular CagA was evaluated by westernblotting and immunocytochemistry. Results: After the induction of CagA in the WT-A10 cells for 24 h, significantly increase by 2-fold of the intracellular level of CagA was observed following treatment with the proteasome inhibitors, but not following that with the inhibitors of autophagy. On the other hand, degradation of CagA in the WT-A10 cells was promoted in a dose-dependent manner by induction of autophagy with rapamycin. In addition, ubiquitinated-CagA was detected in the WT-A10 cells by immunoprecipitation and the TUBE reaction method. Although the levels of intracellular CagA in the AGS cells decreased in a timedependent manner after H. pylori infection, the rate of CagA degradation in the AGS cells was attenuated to 60% under treatment with proteasome inhibitors at 4 h, and further was attenuated to 70% under treatment with the autophagy inhibitors at 24 h. In addition, conversion of LC3-I to LC3-II, which shows autophagy, was detected in the AGS cells at 24 h. Conclusion: Intracellular CagA was initially targeted for proteasomal degradation, and then it was extensively degraded by the induction of autophagy.

Helicobacter pylori CagA Disrupts Epithelial Patterning by Activating Myosin Light Chain

Helicobacter pylori infection is a leading cause of ulcers and gastric cancer. We show that expression of the H. pylori virulence factor CagA in a model Drosophila melanogaster epithelium induces morphological disruptions including ectopic furrowing. We find that CagA alters the distribution and increases the levels of activated myosin regulatory light chain (MLC), a key regulator of epithelial integrity. Reducing MLC activity suppresses CagA-induced disruptions. A CagA mutant lacking EPIYA motifs (CagAEPISA) induces less epithelial disruption and is not targeted to apical foci like wild-type CagA. In a cell culture model in which CagAEPISA and CagA have equivalent subcellular localization, CagAEPISA is equally potent in activating MLC. Therefore, in our transgenic system, CagA is targeted by EPIYA motifs to a specific apical region of the epithelium where it efficiently activates MLC to disrupt epithelial integrity.

Helicobacter pylori strain genotyping based on the amplification and sequencing of cagA gene

Abstract Relevance: H. pylori presence has been described as a high risk factor of suffering gastric cancer, which is the fourth most common cancer in Colombia. Some studies [1, 2]; point out that when a genotypic characterization of different strains of this microorganism is carried out, more than one genotype can be found in the same patient can be found. Purpose: To characterize different H.pylori cagA genotypes that occur in the same patient sample (biopsy) applying molecular and bioinformatic techniques. Methods: cagA positive strains (n=35) from Instituto Nacional de Cancerología (Bogotá-Colombia), bank s samples were included as study material. Tissue DNA was extracted and used to amplify the 3’ region of the cagA gene by PCR [3]. The NTC11637 H. pylori strain was included as a positive control. PCR products were visualized in 2% agarose gels and the amplified fragments were purified using a commercial kit. As a final stage, a precipitation based on ethanol and EDTA was done and reaction products were sequenced. The obtained sequences were edited and submitted to a bioinformatic analysis using AASA, a program designed to characterize the EPIYA phosphorylation motifs localized in the C-terminal of the CagA protein by Rothstain, Yañez and Vivas (Los Andes University Thesis, 2008). Results: The molecular and bioinformatic approach described allowed strain characterization. The applied strategy leads to verify the presence of the A, B and C EPIYA motifs in different combinations. ABC group was the most frequent motif found (48%). These results match those of previous reports in Colombia, nevertheless it is important to remark that amplicons with different molecular sizes can have the same EPIYA motif and that amplicons with similar sizes can have different EPIYA motifs. Conclusions: The current strategy demonstrated to be useful in the identification and posterior characterization of more than one genotype of the different H. pylori strains present in the same biopsy sample. This molecular and bioinformatics approach combined with other techniques seems to be a good alternative to establish if an infection by H. pylori is the result of a coinfection or a microevolution process.

Different cagA and vacA Polymorphisms are Found in the Chinese versus the Malay and Indian Populations: An Analysis of Helicobacter Pylori Virulence Genes in Singapore

Aim: Helicobacter pylori causes peptic ulcer disease and gastric cancer. Western studies suggest that polymorphisms in the virulence factors cagA and vacA may determine the ability of bacteria to cause gastroduodenal diseases. Differences in the cagA EPIYA motifs and polymorphisms of the signal (s), middle (m) and intermediate (i) regions of vacA are thought to be important. The aim of this study was to compare the polymorphisms of cagA and vacA of H. pylori isolated from the Chinese, Malay and Indian populations living in Singapore. Method: A total of 104 H. pylori isolates obtained from patients with dyspeptic symptoms were analysed. Of the 104 patients, 80 were Chinese, 9 Malays and 15 Indians. DNA was extracted from the isolates and the vacA allelic types and cagA EPIYA motifs were determined by polymerase chain reaction (PCR) and sequencing, respectively. Results: Differences in the vacA and cagA polymorphisms were found between the Chinese, Malays and Indians. Significantly more non-Chinese patients carried vacA s1/m1 strains versus Chinese patients ( p &lt; 0.05). All 9 Malay patients, 11/15 (73.3%) Indians and 31/80 (38.8%) Chinese patients carried H. pylori strains with the vacA s1/m1/i1. Significantly more Chinese patients carried isolates with East Asian cagA EPIYA motifs versus non-Chinese patients ( p &lt; 0.05). 79/80 (98.8%) of the Chinese isolates, 2/15 (13%) of Indian isolates, and 5/9(55.6%) of Malay isolates possessed CagA with the East Asian ABD type motif. Conclusion: Results from the current study demonstrated marked differences in the polymorphisms of vacA and CagA EPIYA motifs in strains isolated from Chinese versus non-Chinese patients. Epidemiologically, the Chinese are at the highest risk of developing gastric cancer. Work is ongoing to determine if differences found in the CagA EPIYA motifs of isolates from the Chinese patients can contribute to a subject's risk of developing gastric cancer.