Helicobacter pylori sensitizes TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in human gastric epithelial cells through regulation of FLIP

Helicobacter pylori (H. pylori) infection is associated with chronic gastritis, peptic ulcer and gastric cancer. Apoptosis induced by microbial infections is implicated in the pathogenesis of H. pylori infection. Here we show that human gastric epithelial cells sensitized to H. pylori confer susceptibility to TRAIL-mediated apoptosis via modulation of death receptor signaling. Human gastric epithelial cells are intrinsically resistant to TRAIL-mediated apoptosis. The induction of TRAIL sensitivity by H. pylori is dependent on the activation of caspase-8 and its downstream pathway. H. pylori induces caspase-8 activation via enhanced assembly of the TRAIL death-inducing signaling complex (DISC) through downregulation of cellular FLICE-inhibitory protein (FLIP). Overexpression of FLIP abolished the H. pylori-induced TRAIL sensitivity in human gastric epithelial cells. Our study thus demonstrates that H. pylori induces sensitivity to TRAIL apoptosis by regulation of FLIP and assembly of DISC, which initiates caspase activation, resulting in the breakdown of resistance to apoptosis, and provides insight into the pathogenesis of gastric damage in Helicobacter infection. Modulation of host apoptosis signaling by bacterial interaction adds a new dimension to the pathogenesis of Helicobacter.     

Cathepsin X-deficient Gastric Epithelial Cells in Co-culture with Macrophages: CHARACTERIZATION OF CYTOKINE RESPONSE AND MIGRATION CAPABILITY AFTER HELICOBACTER PYLORI INFECTION*

Our previous studies have shown an association between Helicobacter pylori infection, the strong up-regulation of cathepsin X (CTSX, also called cathepsin Z/P), and the development of gastric cancer. In the present study, we analyzed primary and conventional gastric epithelial cell lines to establish an optimal in vitro mouse model system for the examination of H. pylori-induced overexpression of Ctsx in a functional way. Gastric epithelial cells were isolated from stomachs of wild-type C57BL6/N and Ctsx^−/− mice and compared with the gastric cancer cell line CLS103. Indirect co-cultures of epithelial cells and macrophages were infected with H. pylori strain SS1 and analyzed for the expression of cathepsins, cytokines, and adhesion factors. Cellular interactions, migration capability, and adherence of H. pylori were assessed using time-lapse video microscopy and colony-forming assays. Isolated primary cells from wild-type and transgenic mice revealed qualities and expression profiles similar to those of corresponding tissue samples. Adherence of H. pylori was significantly higher in primary compared with commercially cells. Thus, induction of cathepsins, cytokines, and adhesion proteins was detected solely in primary cells and co-cultured macrophages. Microarray and migration experiments indicated that Ctsx is involved in B/T-cell proliferation/migration and adhesion of macrophages. Primary epithelial cells from stomach of Ctsx^−/− mice represent an excellent model of H. pylori gastritis to elaborate the special functions of Ctsx in regulating the immune response to H. pylori.     

Cloning, Expression, Purification and Toxicity Evaluation of Helicobacter pylori Outer Inflammatory Protein A

The Helicobacter pylori outer membrane proteins play an important role in pathogenesis; the outer inflammatory protein A (OipA) is one of these proteins which play the main role in the development of inflammation. In this study, purification of recombinant H. pylori OipA was performed by Ni–NTA affinity chromatography. Gastric carcinoma epithelial cells (AGS cell) were treated by different concentrations of recombinant OipA for various lengths of time and cell viability was evaluated by the viability assay. Statistical analysis showed that OipA had toxic effects on AGS cells in a concentration of 500 ng/ml after 24 and 48 h, and this toxic dose was 256 ng/ml after 72 h. OipA had direct toxic effects on gastric epithelial cells and the toxicity was observed to depend on time and dose of H. pylori exposure. Attachment of H. pylori to gastric epithelial cells is a key part in the pathogenesis and enables H. pylori to damage the epithelial cells with OipA.     

The Host Protein Calprotectin Modulates the Helicobacter pylori cag Type IV Secretion System via Zinc Sequestration

Transition metals are necessary for all forms of life including microorganisms, evidenced by the fact that 30% of all proteins are predicted to interact with a metal cofactor. Through a process termed nutritional immunity, the host actively sequesters essential nutrient metals away from invading pathogenic bacteria. Neutrophils participate in this process by producing several metal chelating proteins, including lactoferrin and calprotectin (CP). As neutrophils are an important component of the inflammatory response directed against the bacterium Helicobacter pylori, a major risk factor for gastric cancer, it was hypothesized that CP plays a role in the host response to H. pylori. Utilizing a murine model of H. pylori infection and gastric epithelial cell co-cultures, the role CP plays in modifying H. pylori -host interactions and the function of the cag Type IV Secretion System (cag T4SS) was investigated. This study indicates elevated gastric levels of CP are associated with the infiltration of neutrophils to the H. pylori-infected tissue. When infected with an H. pylori strain harboring a functional cag T4SS, calprotectin-deficient mice exhibited decreased bacterial burdens and a trend toward increased cag T4SS -dependent inflammation compared to wild-type mice. In vitro data demonstrate that culturing H. pylori with sub-inhibitory doses of CP reduces the activity of the cag T4SS and the biogenesis of cag T4SS-associated pili in a zinc-dependent fashion. Taken together, these data indicate that zinc homeostasis plays a role in regulating the proinflammatory activity of the cag T4SS.     

Compromised autophagy by MIR30B benefits the intracellular survival of Helicobacter pylori

Helicobacter pylori evade immune responses and achieve persistent colonization in the stomach. However, the mechanism by which H. pylori infections persist is not clear. In this study, we showed that MIR30B is upregulated during H. pylori infection of an AGS cell line and human gastric tissues. Upregulation of MIR30B benefited bacterial replication by compromising the process of autophagy during the H. pylori infection. As a potential mechanistic explanation for this observation, we demonstrate that MIR30B directly targets ATG12 and BECN1, which are important proteins involved in autophagy. These results suggest that compromise of autophagy by MIR30B allows intracellular H. pylori to evade autophagic clearance, thereby contributing to the persistence of H. pylori infections.     

Quantitation of Helicobacter pylori ureC gene and its comparison with different diagnostic techniques and gastric histopathology

Numerous diagnostic assays for Helicobacter pylori detection are available. However, these techniques have their own advantages as well as limitations. Here we tried to develop a real-time quantitative (Q) PCR assay to measure ureC copy number to detect H. pylori, based on the fact that there is only one copy of the ureC gene per bacterium. We enrolled 120 adult patients [non-ulcer dyspepsia (NUD) 60, peptic ulcer disease (PUD) 20, gastric cancer (GC) 40] undergoing upper gastrointestinal endoscopies. During each endoscopic examination, antral biopsies from normal region of the antrum were obtained and subjected to the following tests: RUT, culture, histopathology, H. pylori-specific ureC PCR and ureC Q-PCR. Calculation of H. pylori copy number was based on the standard curve generated using 10-fold dilutions of DNA extracted from the H. pylori control strain varying from 10⁵ to 10¹ copies. The prevalence of H. pylori infection in our study population was 54% with no significant difference among disease and control population. The sensitivity of Q-PCR was found to be 100% which was highest among all diagnostic tests. The established Q-PCR is around 10 times more sensitive than the conventional PCR method. The copy number of H. pylori DNA was significantly increased when overall gastritis, H. pylori density, chronic inflammation and intestinal metaplasia were present. In summary, we developed a rapid and sensitive Q-PCR method for detecting H. pylori. This technique offers a significant improvement over other available methods for detecting H. pylori in clinical and research samples.     

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.     

Motility and Chemotaxis Mediate the Preferential Colonization of Gastric Injury Sites by Helicobacter pylori

Helicobacter pylori (H. pylori) is a pathogen contributing to peptic inflammation, ulceration, and cancer. A crucial step in the pathogenic sequence is when the bacterium first interacts with gastric tissue, an event that is poorly understood in vivo. We have shown that the luminal space adjacent to gastric epithelial damage is a microenvironment, and we hypothesized that this microenvironment might enhance H. pylori colonization. Inoculation with 10⁶ H. pylori (wild-type Sydney Strain 1, SS1) significantly delayed healing of acetic-acid induced ulcers at Day 1, 7 and 30 post-inoculation, and wild-type SS1 preferentially colonized the ulcerated area compared to uninjured gastric tissue in the same animal at all time points. Gastric resident Lactobacillus spp. did not preferentially colonize ulcerated tissue. To determine whether bacterial motility and chemotaxis are important to ulcer healing and colonization, we analyzed isogenic H. pylori mutants defective in motility (ΔmotB) or chemotaxis (ΔcheY). ΔmotB (10⁶) failed to colonize ulcerated or healthy stomach tissue. ΔcheY (10⁶) colonized both tissues, but without preferential colonization of ulcerated tissue. However, ΔcheY did modestly delay ulcer healing, suggesting that chemotaxis is not required for this process. We used two-photon microscopy to induce microscopic epithelial lesions in vivo, and evaluated accumulation of fluorescently labeled H. pylori at gastric damage sites in the time frame of minutes instead of days. By 5 min after inducing damage, H. pylori SS1 preferentially accumulated at the site of damage and inhibited gastric epithelial restitution. H. pylori ΔcheY modestly accumulated at the gastric surface and inhibited restitution, but did not preferentially accumulate at the injury site. H. pylori ΔmotB neither accumulated at the surface nor inhibited restitution. We conclude that bacterial chemosensing and motility rapidly promote H. pylori colonization of injury sites, and thereby biases the injured tissue towards sustained gastric damage.     

Crystal structure of HugZ, a novel heme oxygenase from Helicobacter pylori

The crystal structure of a heme oxygenase (HO) HugZ from Helicobacter pylori complexed with heme has been solved and refined at 1.8 Å resolution. HugZ is part of the iron acquisition mechanism of H. pylori, a major pathogen of human gastroenteric diseases. It is required for the adaptive colonization of H. pylori in hosts. Here, we report that HugZ is distinct from all other characterized HOs. It exists as a dimer in solution and in crystals, and the dimer adopts a split-barrel fold that is often found in FMN-binding proteins but has not been observed in hemoproteins. The heme is located at the intermonomer interface and is bound by both monomers. The heme iron is coordinated by the side chain of His²⁴⁵ and an azide molecule when it is present in crystallization conditions. Experiments show that Arg¹⁶⁶, which is involved in azide binding, is essential for HugZ enzymatic activity, whereas His²⁴⁵, surprisingly, is not, implying that HugZ has an enzymatic mechanism distinct from other HOs. The placement of the azide corroborates the observed γ-meso specificity for the heme degradation reaction, in contrast to most known HOs that have α-meso specificity. We demonstrate through sequence and structural comparisons that HugZ belongs to a new heme-binding protein family with a split-barrel fold. Members of this family are widespread in pathogenic bacteria and may play important roles in the iron acquisition of these bacteria.     

Functional characterization of Helicobacter pylori TlyA: Pore-forming hemolytic activity and cytotoxic property of the protein

Helicobacter pylori is a human specific gastric pathogen. H. pylori pathogenesis process involves a number of well-studied virulence factors that include the ‘vacuolating cytotoxin’ and the ‘cytotoxin associated gene A’. Analysis of the H. pylori genome, however, indicates presence of additional virulence factors that are yet to be characterized in molecular detail. For example, H. pylori genome harbors a gene that has potential to encode a protein with sequence similarity to those of the TlyA-like proteins of several pathogenic bacteria. Earlier studies have indicated potential association of this H. pylori tlyA gene in the virulence mechanism of the organism. Despite such notions, however, the TlyA-like protein of H. pylori has not been studied previously in molecular detail. In particular, purified form of H. pylori TlyA has never been studied before toward exploring its functional properties. Here, we report characterization of the H. pylori TlyA protein purified from the recombinant over-expression system in Escherichia coli. Purified form of the recombinant TlyA exhibits prominent hemolytic activity against human erythrocytes, presumably via formation of pores of specific diameter in the cell membrane. Purified TlyA also triggers prominent cytotoxic responses in human gastric adenocarcinoma cells. Altogether, our study establishes H. pylori TlyA as a potential virulence factor of the organism.     

Membrane-associated Activation of Cholesterol ��-Glucosyltransferase, an Enzyme Responsible for Biosynthesis of Cholesteryl-��-d-Glucopyranoside in Helicobacter pylori Critical for Its Survival

Helicobacter pylori (H. pylori) is the causative pathogen underlying gastric diseases such as chronic gastritis and gastric cancer. Previously, the authors revealed that α1,4-linked N-acetylglucosamine-capped O-glycan (αGlcNAc) found in gland mucin suppresses H. pylori growth and motility by inhibiting catalytic activity of cholesterol α-glucosyltransferase (CHLαGcT), the enzyme responsible for biosynthesis of the major cell wall component cholesteryl-α-d-glucopyranoside (CGL). Here, the authors developed a polyclonal antibody specific for CHLαGcT and then undertook quantitative ultrastructural analysis of the enzyme’s localization in H. pylori. They show that 66.3% of CHLαGcT is detected in the cytoplasm beneath the H. pylori inner membrane, whereas 24.7% is present on the inner membrane. In addition, 2.6%, 5.0%, and 1.4% of the protein were detected in the periplasm, on the outer membrane, and outside microbes, respectively. By using an in vitro CHLαGcT assay with fractionated H. pylori proteins, which were used as an enzyme source for CHLαGcT, the authors demonstrated that the membrane fraction formed CGL, whereas other fractions did not. These data combined together indicate that CHLαGcT is originally synthesized in the cytoplasm of H. pylori as an inactive form and then activated when it is associated with the cell membrane. This article contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.     

Structure of the human gastric bacterial community in relation to Helicobacter pylori status

The human stomach is naturally colonized by Helicobacter pylori, which, when present, dominates the gastric bacterial community. In this study, we aimed to characterize the structure of the bacterial community in the stomach of patients of differing H. pylori status. We used a high-density 16S rRNA gene microarray (PhyloChip, Affymetrix, Inc.) to hybridize 16S rRNA gene amplicons from gastric biopsy DNA of 10 rural Amerindian patients from Amazonas, Venezuela, and of two immigrants to the United States (from South Asia and Africa, respectively). H. pylori status was determined by PCR amplification of H. pylori glmM from gastric biopsy samples. Of the 12 patients, 8 (6 of the 10 Amerindians and the 2 non-Amerindians) were H. pylori glmM positive. Regardless of H. pylori status, the PhyloChip detected Helicobacteriaceae DNA in all patients, although with lower relative abundance in patients who were glmM negative. The G2-chip taxonomy analysis of PhyloChip data indicated the presence of 44 bacterial phyla (of which 16 are unclassified by the Taxonomic Outline of the Bacteria and Archaea taxonomy) in a highly uneven community dominated by only four phyla: Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. Positive H. pylori status was associated with increased relative abundance of non-Helicobacter bacteria from the Proteobacteria, Spirochetes and Acidobacteria, and with decreased abundance of Actinobacteria, Bacteroidetes and Firmicutes. The PhyloChip detected richness of low abundance phyla, and showed marked differences in the structure of the gastric bacterial community according to H. pylori status.     

Interaction between gene A-positive Helicobacter pylori and human leukocyte antigen II alleles increase the risk of Graves disease in Chinese Han population: An association study

Objective

The current study explored the correlation of Helicobacter pylori and the polymorphisms of human leukocyte antigen II (HLA-II) alleles with Graves disease (GD).

Methods

A total of 216 patients with GD were recruited. 102 healthy volunteers constituted the control group. Levels of H. pylori immunoglobulin G (IgG) antibodies and H. pylori cytotoxin-associated gene A (CagA) IgG antibodies were detected using enzyme-linked immunosorbent assays. Molecular typing of the HLA-II alleles was conducted using polymerase chain reaction with sequence specific primers.

Results

H. pylori, particularly CagA-positive strains, HLA-DQA1*0201, and HLA-DQA1*0501 were associated with GD (P = 0.015, OR = 1.811; P = 0.000, OR = 3.085; P = 0.000, OR = 0.315; and P = 0.004, OR = 2.844, respectively). Patients with CagA-positive H. pylori and negative HLA-DQA1*0201 or positive HLA-DQA1*0501 were more likely exposed to GD compared with those with only one of these indices.

Conclusion

CagA-positive H. pylori, negative HLA-DQA1*0201, or positive HLA-DQA1*0501 may increase the risk of GD.     

Role of oxygen and the OxyR protein in the response to iron limitation in Rhodobacter sphaeroides

Background

High intracellular levels of unbound iron can contribute to the production of reactive oxygen species (ROS) via the Fenton reaction, while depletion of iron limits the availability of iron-containing proteins, some of which have important functions in defence against oxidative stress. Vice versa increased ROS levels lead to the damage of proteins with iron sulphur centres. Thus, organisms have to coordinate and balance their responses to oxidative stress and iron availability. Our knowledge of the molecular mechanisms underlying the co-regulation of these responses remains limited. To discriminate between a direct cellular response to iron limitation and indirect responses, which are the consequence of increased levels of ROS, we compared the response of the α-proteobacterium Rhodobacter sphaeroides to iron limitation in the presence or absence of oxygen.

Results

One third of all genes with altered expression under iron limitation showed a response that was independent of oxygen availability. The other iron-regulated genes showed different responses in oxic or anoxic conditions and were grouped into six clusters based on the different expression profiles. For two of these clusters, induction in response to iron limitation under oxic conditions was dependent on the OxyR regulatory protein. An OxyR mutant showed increased ROS production and impaired growth under iron limitation.

Conclusion

Some R. sphaeroides genes respond to iron limitation irrespective of oxygen availability. These genes therefore reflect a “core iron response” that is independent of potential ROS production under oxic, iron-limiting conditions. However, the regulation of most of the iron-responsive genes was biased by oxygen availability. Most strikingly, the OxyR-dependent activation of a subset of genes upon iron limitation under oxic conditions, including many genes with a role in iron metabolism, revealed that elevated ROS levels were an important trigger for this response. OxyR thus provides a regulatory link between the responses to oxidative stress and to iron limitation in R. sphaeroides.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-794) contains supplementary material, which is available to authorized users.     

Identification of Chromosomal HP0892-HP0893 Toxin-Antitoxin Proteins in Helicobacter pylori and Structural Elucidation of Their Protein-Protein Interaction

Bacterial chromosomal toxin-antitoxin (TA) systems have been proposed not only to play an important role in the stress response, but also to be associated with antibiotic resistance. Here, we identified the chromosomal HP0892-HP0893 TA proteins in the gastric pathogen, Helicobacter pylori, and structurally characterized their protein-protein interaction. Previously, HP0892 protein was suggested to be a putative TA toxin based on its structural similarity to other RelE family TA toxins. In this study, we demonstrated that HP0892 binds to HP0893 strongly with a stoichiometry of 1:1, and HP0892-HP0893 interaction occurs mainly between the N-terminal secondary structure elements of HP0892 and the C-terminal region of HP0893. HP0892 cleaved mRNA in vitro, preferentially at the 5′ end of A or G, and the RNase activity of HP0892 was inhibited by HP0893. In addition, heterologous expression of HP0892 in Escherichia coli cells led to cell growth arrest, and the cell toxicity of HP0892 was neutralized by co-expression with HP0893. From these results and a structural comparison with other TA toxins, it is concluded that HP0892 is a toxin with intrinsic RNase activity and HP0893 is an antitoxin against HP0892 from a TA system of H. pylori. It has been known that hp0893 gene and another TA antitoxin gene, hp0895, of H. pylori, are both genomic open reading frames that correspond to genes that are potentially expressed in response to interactions with the human gastric mucosa. Therefore, it is highly probable that TA systems of H. pylori are involved in virulence of H. pylori.     

The CagA protein of Helicobacter pylori suppresses the functions of dendritic cell in mice

CagA protein is the most assessed effecter molecule of Helicobacter pylori. In this report, we demonstrate how CagA protein regulates the functions of dendritic cells (DC) against H. pylori infection. In addition, we found that CagA protein was tyrosine-phosphorylated in DC. The responses to cagA-positive H. pylori in DC were reduced in comparison to those induced by cagA-negative H. pylori. CagA-overexpressing DC also exhibited a decline in the responses against LPS stimulation and the differentiation of CD4⁺ T cells toward Th1 type cells compared to wild type DC. In addition, the level of phosphorylated IRF3 decreased in CagA-overexpressing DC stimulated with LPS, indicating that activated SHP-2 suppressed the enzymatic activity of TBK1 and consequently IRF3 phosphorylation. These data suggest that CagA protein negatively regulates the functions of DC via CagA phosphorylation and that cagA-positive H. pylori strains suppress host immune responses resulting in their chronic colonization of the stomach.     

Helicobacter pylori induces direct activation of the lymphotoxin beta receptor and non-canonical nuclear factor-kappa B signaling

The pathogen Helicobacter pylori, which infects half of the world's population, is a major risk factor for the development of gastric diseases including chronic gastritis and gastric cancer. Among H. pylori's virulence factors is the cytotoxin-associated gene pathogenicity island (cagPAI), which encodes for a type IV secretion system (T4SS). The T4SS induces fast canonical nuclear factor-kappa B (NF-κB) signaling, a major factor increasing inflammation, supressing apoptotic cell death and thereby promoting the development of neoplasia. However, H. pylori's capability to mediate fast non-canonical NF-κB signaling is unresolved, despite a contribution of non-canonical NF-κB signaling to gastric cancer has been suggested.We analyzed signaling elements within non-canonical NF-κB in response to H.?pylori in epithelial cell lines by immunoprecipitation, immunoblot, electrophoretic mobility shift assay and RNA interference knockdown. In addition, tissue samples of H. pylori-infected patients were investigated by immunohistochemistry.Here, we provide evidence for a T4SS-dependent direct activation of non-canonical NF-κB signaling. We identified the lymphotoxin beta receptor (LTβR) to elicit the fast release of NF-κB inducing kinase (NIK) from the receptor complex leading to non-canonical NF-κB signaling. Further, NIK expression was increased in human biopsies of H. pylori-associated gastritis. Thus, NIK could represent a novel target to reduce Helicobacter pylori-induced gastric inflammation and pathology.