Helicobacter pylori lipopolysaccharide-mediated gastric and extragastric pathology.

Lipopolysaccharides (LPS) are a family of toxic phosphorylated glycolipids in the outer membrane of Gram-negative bacteria, including Helicobacter pylori, and are composed of a lipid moiety (termed lipid A), a core oligosaccharide, and a polymeric O-specific polysaccharide chain. Compared with LPS of other bacteria, H. pylori LPS and lipid A induce low immunological activities in a range of test systems. Nevertheless, these reduced levels of LPS-induced cytokines and toxic oxygen radicals can contribute, with those induced by bacterial proteins, to the H. pylori-associated inflammatory response. Whether the ability of H. pylori LPS to induce low production of both procoagulant activity and plasminogen activator inhibitor type 2 by human mononuclear cells contributes to localized inflammatory responses alone and, in addition, play a role in extragastric pathology remains an open question. The core oligosaccharide of H. pylori LPS, in part with a 25 kDa protein adhesin, mediates the binding of the bacterium to the host glycoprotein laminin, and hence interferes with gastric cell receptor-laminin interaction in the basement membrane. Also affecting mucosal integrity, the core sugars of certain H. pylori strains, particularly those associated with gastric ulceration, have been implicated in pepsinogen induction, but this is a strain-dependent phenomenon. Of particular interest, the O-chains of a large proportion of H. pylori strains mimic Lewis (Le) antigens. Although investigations have focussed on the role of these antigens in H. pylori-associated autoimmunity, which remains to be unequivocally established, other pathogenic consequences of Lewis mimicry are becoming apparent. Expression of Lewis antigens may be crucial for H. pylori colonization and adherence and, by aiding bacterial interaction with the gastric mucosa, thereby aid delivery of secreted products, and hence influence the inflammatory response.     

Review: Helicobacter pylori and extragastric diseases

In the last year, many studies have demonstrated a potential role of Helicobacter pylori in the pathogenic mechanisms of different extragastric diseases. While the role of H pylori in idiopathic thrombocytopenic purpura, idiopathic iron deficiency anemia, and vitamin B12 deficiency has already been demonstrated, there is growing evidence of other related conditions, especially cardiovascular, metabolic, and neurologic disorders, including neurodegenerative diseases. A summary of the results of the most relevant studies published over the last year on this attractive topic is presented in this review.     

Helicobacter pylori and extragastric diseases -- other helicobacters

The role of Helicobacter pylori infection is explored in more and more extragastric diseases without definite proof in most of the studies, except possibly some hematologic diseases. In cardiovascular diseases, including stroke, the presence of CagA positive strains may be involved. The possible role of helicobacters in hepatobiliary diseases goes beyond that of H. pylori to involve enterohepatic helicobacters. New Helicobacter species are regularly described and molecular methods are developed to improve their detection. Helicobacter felis remains the major species to be used in animal models of Helicobacter infection.     

Helicobacter pylori CagA transfection of gastric epithelial cells induces interleukin-8

To determine the effect of Helicobacter pylori CagA expression on interleukin-8 (IL-8) induction in AGS cells, cagA and five of its fragments from strains 147A and 147C that vary in the 3' repeat region were cloned into the eukaryotic expression plasmid pSP65SRalpha. IL-8, but not RANTES or IL-Ibeta, levels were increased in AGS cells transfected with 147A-cagA and to a greater extent with 147C-cagA, compared with negative controls. The 5' b fragment from the two strains had similar effects, but the 3' d and e fragments from 147C CagA had greater effects than those from 147A-CagA. When the Western CagA-specific sequence (WSS) of 147C-cagA was replaced with East Asian CagA-specific sequence (ESS) and cloned into pSP65SRalpha as an East/West chimera, there was no significant effect on IL-8 production. Use of specific inhibitors indicates that Src kinase activation, and the mitogen-activated protein (MAP) kinase and NF-kappaB pathways are the major intermediates for CagA effects on IL-8 induction, but the p38 MAP kinase pathway has little effect. These results indicate a direct CagA effect on IL-8 induction by gastric epithelial cells, and indicate signal pathway loci that can be targeted for amelioration.     

SagA of CagA in Helicobacter pylori pathogenesis

Much attention has recently been given to the role of the Helicobacter pylori CagA protein, the only as yet identified H. pylori protein that is delivered into the host gastric epithelial cells by a type IV secretion system, in the development of H. pylori-associated diseases, including gastric carcinoma. This review summarizes the latest advances in our understanding of pathogenic actions of H. pylori CagA, particularly focusing on the molecular mechanisms underlying CagA entry into the host cells as well as CagA-mediated perturbation of host cell signaling involved in proliferation, motility, differentiation, and polarity, which contributes malignant transformation of mammalian cells.     

Epidemiology of Helicobacter pylori: transmission, translocation and extragastric reservoirs.

Although H. pylori infection is endemic and despite more than 10 years of research, the mode and route of transmission remain elusive. This may, in part, be due to the inherent problems of detecting H. pylori noninvasively. The prevalence of infection varies between countries and is closely related to Growth Domestic Product. An age-cohort effect and data from longitudinal studies suggest that the incidence of infection is much higher in children than adults. In developing countries the prevalence of infection is often more than 80% in young adults, in contrast to less than 10% for similar age groups in developed countries. The observations of mosaicism (in the VacA gene) and a panmycytic population structure imply exchange of genetic material either in or outside of the host, which is supported by the increasing recognition of polyclonal infection and suggests that secondary infection occurs after primary acquisition. In addition, in children persistent primary infection may sometimes occur only after previous (repeated) exposure and/or transient colonisation of the gastric mucosa. H. pylori and other gastric Helicobacter spp are always noninvasive, but other human nongastric Helicobacter spp have sometimes been isolated from the systemic circulation in immunocompromised patients. For nonhuman hosts, intestinal Helicobacter spp are thought to translocate more frequently from the colon to the liver. Within the human host, the oral cavity is the principal extragastric reservoir, although case reports suggest that H. pylori may sometimes be found beyond the 2nd part of the duodenum. The hypothesis that H. pylori is a zoonosis or transmitted as coccoid forms by a vector (pets, houseflies) is not supported by recent research showing that H. pylori is entirely unable to support an aerobic or anaerobic metabolism and that coccoid forms are non-viable. H. pylori is primarily acquired in infancy, most probably via the oroorogastric route, from other family members or close contacts encountered after weaning or socialisation. Further studies to support or refute this hypothesis are required.     

Combined serum IgG response to Helicobacter pylori VacA and CagA predicts gastric cancer

Helicobacter pylori is a major factor for the development of gastric cancer. The aim of this study was to define serum antibody patterns associated with H. pylori infection in patients with gastric cancer using a Western blot technique. Serum samples collected from 115 patients with gastric cancer and 110 age- and gender-matched patients without gastrointestinal diseases were tested for IgG antibodies to H. pylori antigens (outer membrane proteins and whole cell preparations). No significant differences were found between patients with and without gastric cancer using outer membrane proteins (82% and 73%, P>0.05) or whole cell antigens (84% and 76%, P>0.05), respectively. The significant differences between patients with and without gastric cancer were associated with bands of 94 kDa (54% and 20%, P<0.001) and 30 kDa (65% and 44%, P<0.01). A combination of antibodies to 85 kDa (VacA) and 120 kDa (CagA) was significantly (P<0.01) more frequent in gastric cancer patients than in patients without gastric cancer. The detection of antibodies to 94- and 30-kDa bands, in association with the determination of serum antibodies to CagA+/VacA+, may have a prospective value in assessment of the risk of developing of gastric cancer.     

CagA tyrosine phosphorylation in gastric epithelial cells caused by Helicobacter pylori in patients with gastric adenocarcinoma

Background. Tyrosine phosphorylation of Helicobacter pylori cytotoxin‐associated protein of in gastric epithelial cells is reported. The goals of this study are first to examine the occurrence of CagA tyrosine phosphorylation in H. pylori strains isolated from patients with gastric adenocarcinoma and gastritis, and second to clarify the relationship between the diversity of tyrosine phosphorylation motifs and the presence of CagA tyrosine phosphorylation. Methods. Fifty‐eight clinical isolates of H. pylori from patients with gastric adenocarcinoma (29 cases) and gastritis (29 cases) were studied for CagA tyrosine phosphorylation by Western blotting. Sequence diversity of tyrosine phosphorylation motifs was analysed among positive‐ or negative‐CagA tyrosine phosphorylation isolates. Results. Positive CagA tyrosine phosphorylation was found in 93.1% (27 of 29) of strains from gastric adenocarcinoma patients and 51.7% (15 of 29) of strains from gastritis patients (p < 0.001). Intact motifs were found in H. pylori isolates with CagA tyrosine phosphorylation. Of the 16 negative CagA tyrosine phosphorylation isolates, intact tyrosine phosphorylation motifs were found in 15 isolates. Conclusions. CagA tyrosine phosphorylation, which is significantly greater in strains from gastric adenocarcinoma patients, may play a role in gastric carcinogenesis, and could be a better marker of more virulent strains than the cag pathogenicity island in Asia, where the cag pathogenicity island is present in nearly all H. pylori strains. Sequence diversity of tyrosine phosphorylation motifs on CagA was not related to the presence of tyrosine phosphorylation. The absence of tyrosine phosphorylation motif might result in negative tyrosine phosphorylation phenotypes, but such motifs are not the sole factors associated with CagA tyrosine phosphorylation.     

Helicobacter pylori infection and CagA protein translocation in human primary gastric epithelial cell culture

BACKGROUND: Increasing evidence has shown that Helicobacter pylori CagA protein translocation into gastric epithelial cells plays an important role in the development of gastric inflammation and malignancy. Translocated CagA undergoes tyrosine phosphorylation in gastric adenocarcinoma cell line cells, and CagA involves disruption of cellular apical-junction complex in Madin-Darby canine kidney cells. METHODS: To elucidate whether these events take place in normal human gastric epithelium, we infected human primary gastric epithelial cells with H. pylori. RESULTS: Our results demonstrate that CagA protein was translocated into primary gastric epithelial cells and tyrosine phosphorylated. The translocated CagA induces cytoskeletal rearrangement and the disruption of tight junctions in primary gastric epithelial cells. CONCLUSIONS: This study provides direct evidence of the modulation of gastric epithelial cells by CagA protein translocation, and advances our understanding of the pathogenesis of H. pylori infection.     

The Helicobacter pylori CagA protein disrupts matrix adhesion of gastric epithelial cells by dephosphorylation of vinculin

Helicobacter pylori colonizes the human stomach, contributing to or causing several diseases. Translocation of the CagA bacterial protein into gastric epithelial cells has been linked to an increased risk of peptic ulcer disease and gastric carcinoma. Upon translocation, CagA is tyrosine phosphorylated by Src family kinases (SFKs), which themselves become inactivated via a negative feedback loop. Here, we show that tyrosine-phosphorylated CagA disrupts adhesion of AGS cells to the extracellular matrix. Owing to the inactivation of c-Src via CagA interaction, vinculin is dephosphorylated at tyrosine residues, 100 and 1065, by corresponding phosphatases. Vinculin dephosphorylation disturbs the interaction and recruitment of the actin-related protein 2/3 (Arp2/3) complex by p34Arc, resulting in a reduction of focal adhesion complexes. These defects can be mimicked by downregulating vinculin using RNA interference in non-infected cells. Tyrosine dephosphorylation of vinculin results in severe cellular deficiencies in cell-matrix adhesion, cell spreading and wound repair. We hypothesize that CagA-mediated inactivation of vinculin is a key step in the mechanism by which H. pylori induces damage to the gastric epithelium and represents an important step in disease development.     

The Helicobacter pylori CagA protein induces tyrosine dephosphorylation of ezrin

Helicobacter pylori is one of the most wide-spread bacterial pathogens and infects the human stomach to cause diseases, such as gastritis, gastric ulceration, and gastric cancer. A major virulence determinant is the H. pylori CagA protein (encoded by the cytotoxin-associated gene A) which is translocated from the bacteria into the cytoplasm of host cells by a type IV secretion system. In the host cell, CagA is phosphorylated on tyrosine residues and induces rearrangements of the actin cytoskeleton. We have previously shown that tyrosine-phosphorylated CagA inhibits the catalytic activity of Src family kinases and induces tyrosine dephosphorylation of several host cell proteins. Here, we identified one of these proteins as ezrin by a combination of preparative gel electrophoresis, two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Specific pharmacological inhibition of Src family kinases also induces ezrin dephosphorylation. Therefore, ezrin dephosphorylation appears to be induced by CagA-mediated Src inactivation. Ezrin is the founding member of the ezrin-radixin-moesin (ERM) family of proteins which are signalling integrators at the cell cortex. Since ezrin is a component of microvilli and a linker protein between actin filaments and membrane proteins, this observation has important implications for H. pylori pathogenesis and might also help to explain the development of gastric cancer.     

MicroRNAs up-regulated by CagA of Helicobacter pylori induce intestinal metaplasia of gastric epithelial cells

CagA of Helicobacter pylori is a bacterium-derived oncogenic protein closely associated with the development of gastric cancers. MicroRNAs (miRNAs) are a class of widespread non-coding RNAs, many of which are involved in cell growth, cell differentiation and tumorigenesis. The relationship between CagA protein and miRNAs is unclear. Using mammalian miRNA profile microarrays, we found that miRNA-584 and miRNA-1290 expression was up-regulated in CagA-transformed cells, miRNA-1290 was up-regulated in an Erk1/2-dependent manner, and miRNA-584 was activated by NF-κB. miRNA-584 sustained Erk1/2 activities through inhibition of PPP2a activities, and miRNA-1290 activated NF-κB by knockdown of NKRF. Foxa1 was revealed to be an important target of miRNA-584 and miRNA-1290. Knockdown of Foxa1 promoted the epithelial-mesenchymal transition significantly. Overexpression of miRNA-584 and miRNA-1290 induced intestinal metaplasia of gastric epithelial cells in knock-in mice. These results indicate that miRNA-584 and miRNA-1290 interfere with cell differentiation and remodel the tissues. Thus, the miRNA pathway is a new pathogenic mechanism of CagA.     

Potential role of CagA in the inhibition of T cell reactivity in Helicobacter pylori infections

The pathogenicity of chronic gastroduodenal diseases is very often related to Helicobacter pylori infections. Most H. pylori strains carry the cagA gene encoding an immunodominant 120- to 128-kDa protein which is considered a virulence marker. The majority of CagA-positive H. pylori isolates also produce a 95-kDa protein cytotoxin (VacA) causing vacuolation and degradation of mammalian cells. In our previous study we have shown that live H. pylori bacteria and their sonicates inhibit PHA-driven proliferation of human T lymphocytes. The H. pylori CagA and VacA proteins were suspected of a paralyzing effect of H. pylori on T cell proliferation. In this report, by using isogenic H. pylori mutant strains defective in CagA and VacA proteins, we determined that CagA is responsible for the inhibition of PHA-induced proliferation of T cells.     

Helicobacter pylori CagA tertiary structure reveals functional insights

CagA is a major disease-associated factor injected by the gastric pathogen Helicobacter pylori. In this issue, Hayashi et al. (2012) report the crystallographic structure of the CagA N terminus (residues 24-876) at 3.19 ? resolution. This study revealed three distinct domains, giving novel insights into intramolecular and intermolecular protein and phosphatidylserine interactions.