TIFA Signaling in Gastric Epithelial Cells Initiates the cag Type 4 Secretion System-Dependent Innate Immune Response to Helicobacter pylori Infection.
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ABSTRACT: Helicobacter pylori is a bacterial pathogen that colonizes the human stomach, causing inflammation which, in some cases, leads to gastric ulcers and cancer. The clinical outcome of infection depends on a complex interplay of bacterial, host genetic, and environmental factors. Although H. pylori is recognized by both the innate and adaptive immune systems, this rarely results in bacterial clearance. Gastric epithelial cells are the first line of defense against H. pylori and alert the immune system to bacterial presence. Cytosolic delivery of proinflammatory bacterial factors through the cag type 4 secretion system (cag-T4SS) has long been appreciated as the major mechanism by which gastric epithelial cells detect H. pylori Classically attributed to the peptidoglycan sensor NOD1, recent work has highlighted the role of NOD1-independent pathways in detecting H. pylori; however, the bacterial and host factors involved have remained unknown. Here, we show that bacterially derived heptose-1,7-bisphosphate (HBP), a metabolic precursor in lipopolysaccharide (LPS) biosynthesis, is delivered to the host cytosol through the cag-T4SS, where it activates the host tumor necrosis factor receptor-associated factor (TRAF)-interacting protein with forkhead-associated domain (TIFA)-dependent cytosolic surveillance pathway. This response, which is independent of NOD1, drives robust NF-κB-dependent inflammation within hours of infection and precedes NOD1 activation. We also found that the CagA toxin contributes to the NF-κB-driven response subsequent to TIFA and NOD1 activation. Taken together, our results indicate that the sequential activation of TIFA, NOD1, and CagA delivery drives the initial inflammatory response in gastric epithelial cells, orchestrating the subsequent recruitment of immune cells and leading to chronic gastritis.IMPORTANCEH. pylori is a globally prevalent cause of gastric and duodenal ulcers and cancer. H. pylori antibiotic resistance is rapidly increasing, and a vaccine remains elusive. The earliest immune response to H. pylori is initiated by gastric epithelial cells and sets the stage for the subsequent immunopathogenesis. This study revealed that host TIFA and H. pylori-derived HBP are critical effectors of innate immune signaling that account for much of the inflammatory response to H. pylori in gastric epithelial cells. HBP is delivered to the host cell via the cag-T4SS at a time point that precedes activation of the previously described NOD1 and CagA inflammatory pathways. Manipulation of the TIFA-driven immune response in the host and/or targeting of ADP-heptose biosynthesis enzymes in H. pylori may therefore provide novel strategies that may be therapeutically harnessed to achieve bacterial clearance.
Project description:Helicobacter pylori infects over half the world's population and causes a wide range of diseases, including gastritis, peptic ulcer, and two forms of gastric cancer. H. pylori infection elicits a variety of phenotypic responses in cultured gastric epithelial cells, including the expression of proinflammatory genes and changes in the actin cytoskeleton. Both of these responses are mediated by the type IV secretion system (TFSS) encoded by the cag pathogenicity island (cag PAI). We used human cDNA microarrays to examine the temporal transcriptional profiles of gastric AGS cells infected with H. pylori strain G27 and a panel of isogenic mutants to dissect the contributions of various genes in the cag PAI. Infection with G27 induced expression of genes involved in the innate immune response, cell shape regulation, and signal transduction. A mutant lacking the cagA gene, which encodes an effector molecule secreted by the TFSS and required for the host cell cytoskeletal response, induced the expression of fewer cytoskeletal genes. A mutant lacking cagE, which encodes a structural component of the TFSS, failed to up-regulate a superset of host genes, including the cagA-dependent genes, and many of the immune response genes. A mutant lacking the entire cag PAI failed to induce both the cagE-dependent genes and several transiently expressed cagE independent genes. Host cell transcriptional profiling of infection with isogenic strains offered a detailed molecular picture of H. pylori infection and provided insight into potential targets of individual virulence determinants such as tyrosine kinase and Rho GTPase signaling molecules.
Project description:Helicobacter pylori colonizes the stomach in about half of the world's population. H. pylori strains containing the cag pathogenicity island (cag PAI) are associated with a higher risk of gastric adenocarcinoma or peptic ulcer disease than cag PAI-negative strains. The cag PAI encodes a type IV secretion system (T4SS) that mediates delivery of the CagA effector protein as well as nonprotein bacterial constituents into gastric epithelial cells. H. pylori-induced nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation and interleukin-8 (IL-8) secretion are attributed to T4SS-dependent delivery of lipopolysaccharide metabolites and peptidoglycan into host cells, and Toll-like receptor 9 (TLR9) activation is attributed to delivery of bacterial DNA. In this study, we analyzed the bacterial energetic requirements associated with these cellular alterations. Mutant strains lacking Cagα, Cagβ, or CagE (putative ATPases corresponding to VirB11, VirD4, and VirB4 in prototypical T4SSs) were capable of T4SS core complex assembly but defective in CagA translocation into host cells. Thus, the three Cag ATPases are not functionally redundant. Cagα and CagE were required for H. pylori-induced NF-κB activation, IL-8 secretion, and TLR9 activation, but Cagβ was dispensable for these responses. We identified putative ATP-binding motifs (Walker-A and Walker-B) in each of the ATPases and generated mutant strains in which these motifs were altered. Each of the Walker box mutant strains exhibited properties identical to those of the corresponding deletion mutant strains. These data suggest that Cag T4SS-dependent delivery of nonprotein bacterial constituents into host cells occurs through mechanisms different from those used for recruitment and delivery of CagA into host cells.
Project description:TC1: gastric epithelial (AGS) cells infected with wild type H. pylori (G27) and isogenic mutants in cagA and vacA for 0, 0.5, 3, 6, and 12 hours. Total RNA was used to make single stranded Cy5 labelled probe and compared to Cy3 labelled probe from uninfected AGS cells. Hybridizations of G27 (trial 4) and cagA- (trial 3) timecourses were done in parallel. A technical replicate of the G27 time course (trial 5) and hybridization of vacA- (trial 3) time course were done in parallel. The cagA 6 and 12 hour time points were technically replicated (trial 4) (the cagA 6 hour sample of trial 3 was lost). TC2: Biological replication and expansion of TC1, using more isogenic mutants and timepoints. AGS cells were mock infected, infected with G27, and isogenic mutants in cagN, cagA, cagE, and a deletion of the cag PAI for 0. 1, 3, 6, 12, and 24 hours. Probe synthesis and hybridization was done as in TC1. Note: there may have been a sample mix-up with PAI 12, swapping it with G27 or cagN 12. Groups of assays that are related as part of a time series. Keywords: time_series_design
Project description:TC1: gastric epithelial (AGS) cells infected with wild type H. pylori (G27) and isogenic mutants in cagA and vacA for 0, 0.5, 3, 6, and 12 hours. Total RNA was used to make single stranded Cy5 labelled probe and compared to Cy3 labelled probe from uninfected AGS cells. Hybridizations of G27 (trial 4) and cagA- (trial 3) timecourses were done in parallel. A technical replicate of the G27 time course (trial 5) and hybridization of vacA- (trial 3) time course were done in parallel. The cagA 6 and 12 hour time points were technically replicated (trial 4) (the cagA 6 hour sample of trial 3 was lost). TC2: Biological replication and expansion of TC1, using more isogenic mutants and timepoints. AGS cells were mock infected, infected with G27, and isogenic mutants in cagN, cagA, cagE, and a deletion of the cag PAI for 0. 1, 3, 6, 12, and 24 hours. Probe synthesis and hybridization was done as in TC1. Note: there may have been a sample mix-up with PAI 12, swapping it with G27 or cagN 12. Groups of assays that are related as part of a time series. Computed
Project description:Chronic Helicobacter pylori infection provokes an inflammation of the gastric mucosa, at high risk for ulcer and cancer development. The most virulent strains harbor the cag pathogenicity island (cagPAI) encoding a type 4 secretion system, which allows delivery of bacterial effectors into gastric epithelial cells, inducing pro-inflammatory responses and phenotypic alterations reminiscent of an epithelial-to-mesenchymal transition (EMT). This study characterizes EMT features in H. pylori-infected gastric epithelial cells, and investigates their relationship with NF-κB activation. Cultured human gastric epithelial cell lines were challenged with a cagPAI+ H. pylori strain or cag isogenic mutants. Morphological changes, epithelial and mesenchymal gene expression and EMT-related microRNAs were studied. H. pylori up-regulates mesenchymal markers, including ZEB1. This transcription factor is prominently involved in the mesenchymal transition of infected cells and its up-regulation depends on cagPAI and NF-κB activation. ZEB1 expression and NF-κB activation were confirmed by immunohistochemistry in gastric mucosa from cagPAI+ H. pylori-infected patients. Gastric epithelial cell lines express high miR-200 levels, which are linked to ZEB1 in a reciprocal negative feedback loop and maintain their epithelial phenotype in non-infected conditions. However, miR-200b/c were increased upon infection, despite ZEB1 up-regulation and mesenchymal morphology. In the miR-200b-200a-429 cluster promoter, we identified a functional NF-κB binding site, recruiting NF-κB upon infection and trans-activating the microRNA cluster transcription. In conclusion, in gastric epithelial cells, cagPAI+ H. pylori activates NF-κB, which transactivates ZEB1, subsequently promoting mesenchymal transition. The unexpected N-FκB-dependent increase of miR-200 levels likely thwarts the irreversible loss of epithelial identity in that critical situation.
Project description:Colonization of the human stomach with Helicobacter pylori strains containing the cag pathogenicity island is a risk factor for development of gastric cancer. The cag pathogenicity island contains genes encoding a secreted effector protein (CagA) and components of a type IV secretion system (Cag T4SS). The molecular architecture of the H. pylori Cag T4SS is substantially more complex than that of prototype T4SSs in other bacterial species. In this review, we discuss recent discoveries pertaining to the structure and function of the Cag T4SS and its role in gastric cancer pathogenesis.
Project description:Helicobacter pylori infection systematically causes chronic gastric inflammation that can persist asymptomatically or evolve toward more severe gastroduodenal pathologies, such as ulcer, mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer. The cag pathogenicity island (cag PAI) of H. pylori allows translocation of the virulence protein CagA and fragments of peptidoglycan into host cells, thereby inducing production of chemokines, cytokines, and antimicrobial peptides. In order to characterize the inflammatory response to H. pylori, a new experimental protocol for isolating and culturing primary human gastric epithelial cells was established using pieces of stomach from patients who had undergone sleeve gastrectomy. Isolated cells expressed markers indicating that they were mucin-secreting epithelial cells. Challenge of primary epithelial cells with H. pylori B128 underscored early dose-dependent induction of expression of mRNAs of the inflammatory mediators CXCL1 to -3, CXCL5, CXCL8, CCL20, BD2, and tumor necrosis factor alpha (TNF-α). In AGS cells, significant expression of only CXCL5 and CXCL8 was observed following infection, suggesting that these cells were less reactive than primary epithelial cells. Infection of both cellular models with H. pylori B128ΔcagM, a cag PAI mutant, resulted in weak inflammatory-mediator mRNA induction. At 24 h after infection of primary epithelial cells with H. pylori, inflammatory-mediator production was largely due to cag PAI substrate-independent virulence factors. Thus, H. pylori cag PAI substrate appears to be involved in eliciting an epithelial response during the early phases of infection. Afterwards, other virulence factors of the bacterium take over in development of the inflammatory response. Using a relevant cellular model, this study provides new information on the modulation of inflammation during H. pylori infection.
Project description:IFN regulatory factor 8 (IRF8) is expressed in many types of blood cells and plays critical roles in cellular differentiation and function. However, the role of IRF8 in nonhematopoietic systems remains poorly understood. In this study, we provide evidence that IRF8 is a transcriptional modulator of the gastric mucosa necessary for limiting Helicobacter pylori colonization. H. pylori infection significantly upregulated expression of IRF8, which, in turn, promoted IFN-γ expression by gastric epithelial cells. Mice deficient in IRF8 exhibited increased H. pylori colonization and aborted induction of mucosal IFN-γ. Genome-wide analyses of IFN-γ-treated gastric epithelial cells by chromatin immunoprecipitation sequencing and RNA sequencing led to the identification of IRF8 target genes, with many belonging to the IFN-regulated gene family that was observed previously in immune cells. Our results identify the IRF8-IFN-γ circuit as a novel gastric innate immune mechanism in the host defense against infection with H. pylori.
Project description:The iron deficiency anaemia that often accompanies infection with Helicobacter pylori may reflect increased uptake of iron into gastric epithelial cells. Here we show an infection-associated increase in total intracellular iron levels was associated with the redistribution of the transferrin receptor from the cell cytosol to the cell surface, and with increased levels of ferritin, an intracellular iron storage protein that corresponded with a significant increase in lysosomal stores of labile iron. In contrast, the pool of cytosolic labile iron was significantly decreased in infected cells. These changes in intracellular iron distribution were associated with the uptake and trafficking of H. pylori through the cells, and enhanced in strains capable of expressing the cagA virulence gene. We speculate that degradation of lysosomal ferritin may facilitate H. pylori pathogenesis, in addition to contributing to bacterial persistence in the human stomach.
Project description:AimSince, contradictory data have been reported about the effect of diverse variants of H. pylori virulence factors on IL-8 induction, we aimed to analyze the effect of this diversity on levels of IL-8 secretion in AGS cell line.BackgroundHelicobacter pylori colonizes the human stomach and induces the activation of inflammatory cytokines, including interleukin (IL)-8, in the gastric mucosa. This induction promotes neutrophil and monocyte recruitment that causes gastric tissue damage.MethodsTo determine whether different strains of H. pylori and their CagA variants have possible roles on IL-8 induction, polarized AGS cell line was infected with CagA+ H. pylori strains carrying different EPIYA motifs (ABCCC and ABC) and CagA- strain for 24 hours. Difference in stimulation of IL-8 was measured by ELISA.ResultsIL-8 secretion was elevated in the treated cells with CagA encoding strains compared with the negative one. Furthermore, a noticeably increased level of IL-8 induction was measured by the CagA-EPIYA type ABCCC encoding strain in compare to that carried EPIYA type ABC.ConclusionResults of this study provide new evidence about different effects of H. pylori strains and possible roles of their CagA variants on IL-8 induction. It seems that not only carriage of cagA and its expression, but also diversity in EPIYA motif be involved in IL-8 induction in the gastric epithelial cells.