Helicobacter pylori cag Pathogenicity Island (cagPAI) Involved in Bacterial Internalization and IL-8 Induced Responses via NOD1- and MyD88-Dependent Mechanisms in Human Biliary Epithelial Cells

Helicobacter pylori infection has been proposed to be associated with various diseases of the hepatobiliary tract, including cancer of the bile duct epithelial cells (cholangiocarcinoma, CCA). The ability of H. pylori bacteria to cause pathogenic effects in these cells has, however, yet to be investigated. Given that the cag pathogenicity island (cagPAI) is required for H. pylori pathogenesis in gastric epithelial cells, we investigated wild-type and cag mutant strains for their ability to adhere, be internalized and induce pro-inflammatory responses in two bile duct epithelial cell lines derived from cases of CCA. The findings from these experiments were compared to results obtained with the well-characterized AGS gastric cancer cell line. We showed that the cagPAI encodes factors involved in H. pylori internalization in CCA cells, but not for adhesion to these cells. Consistent with previous studies in hepatocytes, actin polymerization and α5β1 integrin may be involved in H. pylori internalization in CCA cells. As for AGS cells, we observed significantly reduced levels of NF-κB activation and IL-8 production in CCA cells stimulated with either cagA, cagL or cagPAI bacteria, when compared with wild-type bacteria. Importantly, these IL-8 responses could be inhibited via either pre-treatment of cells with antibodies to α5β1 integrins, or via siRNA-mediated knockdown of the innate immune signaling molecules, nucleotide oligomerization domain 1 (NOD1) and myeloid differentiation response gene 88 (MyD88). Taken together, the data demonstrate that the cagPAI is critical for H. pylori pathogenesis in bile duct cells, thus providing a potential causal link for H. pylori in biliary tract disease.     

Phosphorylation of ATM/ATR substrates in eukaryotic cells after infection with <Emphasis Type="Italic">Helicobacter pylori</Emphasis>

Phosphorylation of ATM-kinase substrates in HeLa and AGS cells in response to Helicobacter pylori infection has been characterized. Infection with wild-type (cagPAI-positive) and corresponding isogenic cagPAI negative mutant induced activation of Chk1 and Chk2 kinases. However, only Chk1 was directly activated by ATM-kinase. Using 2D-electrophoresis and mass spectrometry a group of proteins phosphorylated in AGS cells by ATM1/ATR kinases during H. pylori infection has been identified.     

Decreased adherence of cagG-deleted Helicobacter pylori to gastric epithelial cells in Japanese clinical isolates

Background. The cag pathogenicity island (cag PAI) is a major virulence factor. The ability of Helicobacter pylori to adhere to gastric epithelial cells is an important initial step for virulence. The aim of this study was to evaluate the relationship between genetic variations of cag PAI in Japanese clinical isolates and the ability of H. pylori to adhere to gastric epithelial cells. Materials and Methods. The polymerase chain reaction and Southern blot analysis were used to verify the presence or absence of cagA, cagE, cagG, cagI and cagM in the cag PAI in 236 Japanese clinical isolates. The ability of H. pylori to adhere to KATOIII cells was examined by flow cytometry. Results. Seven (3.0%) cag PAI partial‐deleted strains were found in 236 clinical isolates, and these strains showed three patterns in the deleted region within the cag PAI. All of the cagG‐deleted strains showed decreased adherence to KATOIII cells, in comparison with cagG‐positive strains. These strains had abolished IL‐8 induction despite the presence of cagE, which is essential for IL‐8 induction. Conclusions. Our results suggest that cagG or surrounding genes in the cag PAI has a function related to adhesion to epithelial cells.     

Expression of CEACAM1 or CEACAM5 in AZ‐521 cells restores the type IV secretion deficiency for translocation of CagA by Helicobacter pylori

Helicobacter pylori represents an important pathogen involved in diseases ranging from gastritis, peptic ulceration, to gastric malignancies. Prominent virulence factors comprise the vacuolating cytotoxin VacA and the cytotoxin‐associated genes pathogenicity island (cagPAI)‐encoded type IV secretion system (T4SS). The T4SS effector protein CagA can be translocated into AGS and other gastric epithelial cells followed by phosphorylation through c‐Src and c‐Abl tyrosin kinases to hijack signalling networks. The duodenal cell line AZ‐521 has been recently introduced as novel model system to investigate CagA delivery and phosphorylation in a VacA‐dependent fashion. In contrast, we discovered that AZ‐521 cells display a T4SS incompetence phenotype for CagA injection, which represents the first reported gastrointestinal cell line with a remarkable T4SS defect. We proposed that this deficiency may be due to an imbalanced coexpression of T4SS receptor integrin‐β₁ or carcinoembryonic antigen‐related cell adhesion molecules (CEACAMs), which were described recently as novel H. pylori receptors. We demonstrate that AZ‐521 cells readily express integrin‐β₁, but overexpression of integrin‐β₁ constructs did not restore the T4SS defect. We further show that AZ‐521 cells lack the expression of CEACAMs. We demonstrate that genetic introduction of either CEACAM1 or CEACAM5, but not CEACAM6, in AZ‐521 cells is sufficient to permit injection and phosphorylation of CagA by H. pylori to degrees observed in the AGS cell model. Expression of CEACAM1 or CEACAM5 in infected AZ‐521 cells was also accompanied by tyrosine dephosphorylation of the cytoskeletal proteins vinculin and cortactin, a hallmark of H. pylori‐infected AGS cells. Our results suggest the existence of an integrin‐β₁‐ and CEACAM1‐ or CEACAM5‐dependent T4SS delivery pathway for CagA, which is clearly independent of VacA. The presence of two essential host protein receptors during infection with H. pylori represents a unique feature in the bacterial T4SS world. Further detailed investigation of these T4SS functions will help to better understand infection strategies by bacterial pathogens.     

Helicobacter pylori Colonization Ameliorates Glucose Homeostasis in Mice through a PPAR γ-Dependent Mechanism

Background

There is an inverse secular trend between the incidence of obesity and gastric colonization with Helicobacter pylori, a bacterium that can affect the secretion of gastric hormones that relate to energy homeostasis. H. pylori strains that carry the cag pathogenicity island (PAI) interact more intimately with gastric epithelial cells and trigger more extensive host responses than cag⁻ strains. We hypothesized that gastric colonization with H. pylori strains differing in cag PAI status exert distinct effects on metabolic and inflammatory phenotypes.

Methodology/Principal Findings

To test this hypothesis, we examined metabolic and inflammatory markers in db/db mice and mice with diet-induced obesity experimentally infected with isogenic forms of H. pylori strain 26695: the cag PAI wild-type and its cag PAI mutant strain 99–305. H. pylori colonization decreased fasting blood glucose levels, increased levels of leptin, improved glucose tolerance, and suppressed weight gain. A response found in both wild-type and mutant H. pylori strain-infected mice included decreased white adipose tissue macrophages (ATM) and increased adipose tissue regulatory T cells (Treg) cells. Gene expression analyses demonstrated upregulation of gastric PPAR γ-responsive genes (i.e., CD36 and FABP4) in H. pylori-infected mice. The loss of PPAR γ in immune and epithelial cells in mice impaired the ability of H. pylori to favorably modulate glucose homeostasis and ATM infiltration during high fat feeding.

Conclusions/Significance

Gastric infection with some commensal strains of H. pylori ameliorates glucose homeostasis in mice through a PPAR γ-dependent mechanism and modulates macrophage and Treg cell infiltration into the abdominal white adipose tissue.     

A Global Overview of the Genetic and Functional Diversity in the Helicobacter pylori cag Pathogenicity Island

The Helicobacter pylori cag pathogenicity island (cagPAI) encodes a type IV secretion system. Humans infected with cagPAI–carrying H. pylori are at increased risk for sequelae such as gastric cancer. Housekeeping genes in H. pylori show considerable genetic diversity; but the diversity of virulence factors such as the cagPAI, which transports the bacterial oncogene CagA into host cells, has not been systematically investigated. Here we compared the complete cagPAI sequences for 38 representative isolates from all known H. pylori biogeographic populations. Their gene content and gene order were highly conserved. The phylogeny of most cagPAI genes was similar to that of housekeeping genes, indicating that the cagPAI was probably acquired only once by H. pylori, and its genetic diversity reflects the isolation by distance that has shaped this bacterial species since modern humans migrated out of Africa. Most isolates induced IL-8 release in gastric epithelial cells, indicating that the function of the Cag secretion system has been conserved despite some genetic rearrangements. More than one third of cagPAI genes, in particular those encoding cell-surface exposed proteins, showed signatures of diversifying (Darwinian) selection at more than 5% of codons. Several unknown gene products predicted to be under Darwinian selection are also likely to be secreted proteins (e.g. HP0522, HP0535). One of these, HP0535, is predicted to code for either a new secreted candidate effector protein or a protein which interacts with CagA because it contains two genetic lineages, similar to cagA. Our study provides a resource that can guide future research on the biological roles and host interactions of cagPAI proteins, including several whose function is still unknown.     

Analyses of the cag pathogenicity island of Helicobacter pylori

Most strains of Helicobacter pylori from patients with peptic ulcer disease or intestinal-type gastric cancer carry cagA, a gene that encodes an immunodominant protein of unknown function, whereas many of the strains from asymptomatically infected persons lack this gene. Recent studies showed that the cagA gene lies near the right end of a ≈37 kb DNA segment (a pathogenicity island, or PAI) that is unique to cagA⁺ strains and that the cag PAI was split in half by a transposable element insertion in the reference strain NCTC11638. In complementary experiments reported here, we also found the same cag PAI, and sequenced a 39 kb cosmid clone containing the left ‘cagII’ half of this PAI. Encoded in cagII were four proteins each with homology to four components of multiprotein complexes of Bordetella pertussis (‘Ptl’), Agrobacterium tumefaciens (‘Vir’), and conjugative plasmids (‘Tra’) that help deliver pertussis toxin and T (tumour inducing) and plasmid DNA, respectively, to target eukaryotic or prokaryotic cells, and also homologues of eukaryotic proteins that are involved in cytoskeletal structure. To the left of cagII in this cosmid were genes for homologues of HslU (heat-shock protein) and Era (essential GTPase); to the right of cagII were homologues of genes for a type I restriction endonuclease and ion transport functions. Deletion of the cag PAI had no effect on synthesis of the vacuolating cytotoxin, but this deletion and several cag insertion mutations blocked induction of synthesis of proinflammatory cytokine IL-8 in gastric epithelial cells. Comparisons among H. pylori strains indicated that cag PAI gene content and arrangement are rather well conserved. We also identified two genome rearrangements with end-points in the cag PAI. One, in reference strain NCTC11638, involved IS605, a recently described transposable element (as also found by others). Another rearrangement, in 3 of 10 strains tested (including type strain NCTC11637), separated the normally adjacent cagA and picA genes and did not involve IS605. Our results are discussed in terms of how cag-encoded proteins might help trigger the damaging inflammatory responses in the gastric epithelium and possible contributions of DNA rearrangements to genome evolution.     

NOD1 is required for Helicobacter pylori induction of IL‐33 responses in gastric epithelial cells

Helicobacter pylori (H. pylori) causes chronic inflammation which is a key precursor to gastric carcinogenesis. It has been suggested that H. pylori may limit this immunopathology by inducing the production of interleukin 33 (IL‐33) in gastric epithelial cells, thus promoting T helper 2 immune responses. The molecular mechanism underlying IL‐33 production in response to H. pylori infection, however, remains unknown. In this study, we demonstrate that H. pylori activates signalling via the pathogen recognition molecule Nucleotide‐Binding Oligomerisation Domain‐Containing Protein 1 (NOD1) and its adaptor protein receptor‐interacting serine–threonine Kinase 2, to promote production of both full‐length and processed IL‐33 in gastric epithelial cells. Furthermore, IL‐33 responses were dependent on the actions of the H. pylori Type IV secretion system, required for activation of the NOD1 pathway, as well as on the Type IV secretion system effector protein, CagA. Importantly, Nod1^+/+ mice with chronic H. pylori infection exhibited significantly increased gastric IL‐33 and splenic IL‐13 responses, but decreased IFN‐γ responses, when compared with Nod1^?/? animals. Collectively, our data identify NOD1 as an important regulator of mucosal IL‐33 responses in H. pylori infection. We suggest that NOD1 may play a role in protection against excessive inflammation.     

Dynamics of the Cag‐type IV secretion system of Helicobacter pylori as studied by bacterial co‐infections

Many pathogenic Gram‐negative bacteria possess type IV secretion systems (T4SS) to inject effector proteins directly into host cells to modulate cellular processes to their benefit. The human bacterial pathogen Helicobacter pylori, a major aetiological agent in the development of chronic gastritis, duodenal ulcer and gastric carcinoma, harbours the cag‐T4SS to inject the cytotoxin associated Antigen (CagA) into gastric epithelial cells. This results in deregulation of major signalling cascades, actin‐cytoskeletal rearrangements and eventually gastric cancer. We show here that a pre‐infection with live H. pylori has a dose‐dependent negative effect on the CagA translocation efficiency of a later infecting strain. This effect of the ‘first’ strain was independent of any of its T4SS, the vacuolating cytotoxin (VacA) or flagella. Other bacterial pathogens, e.g. pathogenic Escherichia coli, Campylobacter jejuni, Staphylococcus aureus, or commensal bacteria, such as lactobacilli, were unable to interfere with H. pylori's CagA translocation capacity in the same way. This interference was independent of the β1 integrin receptor availability for H. pylori, but certain H. pylori outer membrane proteins, such as HopI, HopQ or AlpAB, were essential for the effect. We suggest that the specific interference mechanism induced by H. pylori represents a cellularresponse to restrict and control CagA translocation into a host cell to control the cellular damage.     

Evaluation of the effect of cagPAI genes of Helicobacter pylori on AGS epithelial cell morphology and IL-8 secretion

Helicobacter pylori cagPAI genes play an important role in pathogenesis, however little is known about their functions in isolates from Turkish patients. We aimed to evaluate the intactness and the effect of the cagPAI genes (cagT, cagM, cagE, cagA) and cagA EPIYA motifs on the AGS morphological changes and IL-8 induction. Of 53 patients 38 were found infected with H. pylori. PCR amplification of the cagPAI genes showed 42.1 % intact, 39.5 % partially deleted and 18.4 % with complete deletions. Isolates from gastritis, duodenal and gastric ulcer patients with intact and partially deleted cagPAI genes induced higher IL-8 secretion than those with complete deletions. Isolates from gastritis patients had higher deletion frequencies of the cagT and cagM genes than the other two genes. Infection of AGS cells with isolates that possess intact cagPAI and EPIYA-ABC resulted in the formation of the hummingbird phenotype. The cagA positive isolates induced higher IL-8 secretion than cagA negative isolates. Isolates from DU patients with more than one EPIYA-C motif induced higher concentrations of IL-8 than those with EPIYA-ABC. In conclusion, the intactness of the cagPAI in our isolates from different patients was not conserved. An intact cagPAI was found to play an important role in the pathogenesis of DU but not GU or gastritis. The cagA gene, but not other cagPAI genes, was associated with the induction of IL-8 and the morphological changes of the AGS cells. An increase in the number of EPIYA-C motifs had noticeable effect on the formation of the hummingbird phenotype.     

Review: Helicobacter: Inflammation,immunology, and vaccines

Chronic inflammation induced by Helicobacter pylori infection is a critical factor in the development of peptic ulcer disease and gastric cancer. Central to this inflammation is the initiation of pro‐inflammatory signaling cascades within epithelial cells, in particular those mediated by two sensors of bacterial cell wall components, nucleotide‐binding oligomerization domain‐containing protein 1 (NOD1) and alpha‐protein kinase 1 (ALPK1). H pylori is, however, also highly adept at mitigating inflammation in the host, thereby restricting tissue damage and favoring bacterial persistence. H pylori modulates host immune responses by altering cytokine signaling in epithelial and myeloid cells, which results in increased proliferation of regulatory T cells and downregulation of effector T‐cell responses. H pylori vacuolating cytotoxin A (VacA) has been shown to play an important role in the dampening of immune responses and induction of immune tolerance capable of protecting against asthma. It is also possible to generate protective immune responses by immunization with various H pylori antigens or their epitopes, in combination with an adjuvant, though this for now has only been shown in mouse models. Novel non‐toxic adjuvants, consisting of modified bacterial enterotoxins or nanoparticles, have recently been developed that may not only enhance vaccine efficacy, but also help translate candidate vaccines to the clinic. This review will summarize the main discoveries in the past year regarding host immune responses to H pylori infection, as well as the design of new vaccine approaches against this infection.     

EPs® 7630, an extract from Pelargonium sidoides roots inhibits adherence of Helicobacter pylori to gastric epithelial cells

Helicobacter pylori specifically adheres to gastric host cells, mainly based on carbohydrate-mediated cell–cell interaction. The extract of Pelargonium sidoides roots (EPs^® 7630), a South African herbal remedy, is currently used to treat acute bronchitis. EPs^® 7630 prevents bacteria from attaching to cell membranes. Therefore, the ability of EPs^® 7630 to interfere with H. pylori growth and adhesion to gastric epithelial cells (AGS cells) was tested in vitro. EPs^® 7630 inhibited H. pylori growth and with higher potency adhesion to gastric AGS cells. EPs^® 7630 (50 and 100 μg/ml) reduced bacterial count attached to AGS cells by 77% and 91%, respectively. The results suggest that the mode of action of EPs^® 7630 is mainly related to its anti-adhesive activity.     

Dual roles of CagA protein in Helicobacterpylori-induced chronic gastritis in mice

Cytotoxin-associated gene A (CagA) acts directly on gastric epithelial cells. However, the roles of CagA in host adaptive immunity against Helicobacter pylori (H. pylori) infection are not fully understood. In this study, to investigate the roles of CagA in the development of H. pylori-induced chronic gastritis, we used an adoptive-transfer model in which spleen cells from C57BL/6 mice with or without H. pylori infection were transferred into RAG2^−/− mice, with gastric colonization of either CagA⁺H. pylori or CagA⁻H. pylori. Colonization of CagA⁺H. pylori but not CagA⁻H. pylori in the host gastric mucosa induced severe chronic gastritis in RAG2^−/− mice transferred with spleen cells from H. pylori-uninfected mice. In addition, when CagA⁺H. pylori-primed spleen cells were transferred into RAG2^−/− mice, CD4⁺ T cell infiltration in the host gastric mucosa were observed only in RAG2^−/− mice infected with CagA⁺H. pylori but not CagA⁻H. pylori, suggesting that colonization of CagA⁺H. pylori in the host gastric mucosa is essential for the migration of H. pylori-primed CD4⁺ T cells. On the other hand, transfer of CagA⁻H. pylori-primed spleen cells into CagA⁺H. pylori-infected RAG2^−/− mice induced more severe chronic gastritis with less Foxp3⁺ regulatory T-cell infiltration as compared to transfer of CagA⁺H. pylori-primed spleen cells. In conclusion, CagA in the stomach plays an important role in the migration of H. pylori-primed CD4⁺ T cells in the gastric mucosa, whereas CagA-dependent T-cell priming induces regulatory T-cell differentiation, suggesting dual roles for CagA in the pathophysiology of H. pylori-induced chronic gastritis.     

The Helicobacter pylori adhesin protein HopQ exploits the dimer interface of human CEACAMs to facilitate translocation of the oncoprotein CagA

Helicobacter pylori infects half of the world's population, and strains that encode the cag type IV secretion system for injection of the oncoprotein CagA into host gastric epithelial cells are associated with elevated levels of cancer. CagA translocation into host cells is dependent on interactions between the H. pylori adhesin protein HopQ and human CEACAMs. Here, we present high‐resolution structures of several HopQ‐CEACAM complexes and CEACAMs in their monomeric and dimeric forms establishing that HopQ uses a coupled folding and binding mechanism to engage the canonical CEACAM dimerization interface for CEACAM recognition. By combining mutagenesis with biophysical and functional analyses, we show that the modes of CEACAM recognition by HopQ and CEACAMs themselves are starkly different. Our data describe precise molecular mechanisms by which microbes exploit host CEACAMs for infection and enable future development of novel oncoprotein translocation inhibitors and H. pylori‐specific antimicrobial agents.