ABSTRACT
The initial step in bacterial infection is adherence of the bacterium to the target cell surface. Helicobacter pylori exploits the interaction of bacterial adhesin protein HopQ with human epithelial CEACAMs (CEACAM1, 5, and 6) to stably adhere to gastric epithelial cells, which is necessary for delivery of the H. pylori CagA oncoprotein into the epithelial cells via a type IV secretion system. In contrast to human CEACAMs, however, HopQ does not interact with Ceacam1 (mouse CEACAM1) in vitro or in CHO cells ectopically expressing Ceacam1. Since the mouse genome lacks Ceacam5 and Ceacam6, no significant HopQ-Ceacam interaction may occur in mouse gastric epithelial cells. Here, we found that the mouse stomach has a much lower expression level of Ceacam1 than the expression level of CEACAM1 in the human stomach. Consistently, mouse gastric epithelial cells resist CagA delivery by cagA-positive H. pylori, and the delivery is restored by ectopic expression of human CEACAM1 or CEACAM5 in mouse gastric epithelial cells. Thus, despite the fact that mice are routinely used for H. pylori infection studies, a low expression level of Ceacam1 in the mouse stomach together with the loss or greatly reduced interaction of HopQ with Ceacams make the mouse an inappropriate model for studying the role of H. pylori-delivered CagA in gastric pathogenesis, including the development of gastric cancer.
Subject(s)
Helicobacter Infections , Helicobacter pylori , Adhesins, Bacterial/genetics , Adhesins, Bacterial/metabolism , Animals , Antigens, Bacterial/genetics , Antigens, Bacterial/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cricetinae , Cricetulus , Epithelial Cells/metabolism , Helicobacter pylori/metabolism , Mice , Protein Transport , Stomach , Type IV Secretion Systems/genetics , Type IV Secretion Systems/metabolismABSTRACT
Infection with cagA-positive Helicobacter pylori strains plays an etiological role in the development of gastric cancer. The CagA protein is injected into gastric epithelial cells through a bacterial Type IV secretion system. Inside the host cells, CagA promiscuously associates with multiple host cell proteins including the prooncogenic phosphatase SHP2 that is required for full activation of the Ras-ERK pathway. CagA-SHP2 interaction aberrantly activates SHP2 and thereby deregulates Ras-ERK signaling. Cancer is regarded as a disease of the genome, indicating that H. pylori-mediated gastric carcinogenesis is also associated with genomic alterations in the host cell. Indeed, accumulating evidence has indicated that H. pylori infection provokes DNA double-stranded breaks (DSBs) by both CagA-dependent and CagA-independent mechanisms. DSBs are repaired by either error-free homologous recombination (HR) or error-prone non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ). Infection with cagA-positive H. pylori inhibits RAD51 expression while dampening cytoplasmic-to-nuclear translocalization of BRCA1, causing replication fork instability and HR defects (known as "BRCAness"), which collectively provoke genomic hypermutation via non-HR-mediated DSB repair. H. pylori also subverts multiple DNA damage responses including DNA repair systems. Infection with H. pylori additionally inhibits the function of the p53 tumor suppressor, thereby dampening DNA damage-induced apoptosis, while promoting proliferation of CagA-delivered cells. Therefore, H. pylori cagA-positive strains promote abnormal expansion of cells with BRCAness, which dramatically increases the chance of generating driver gene mutations in the host cells. Once such driver mutations are acquired, H. pylori CagA is no longer required for subsequent gastric carcinogenesis (Hit-and-Run carcinogenesis).
Subject(s)
Helicobacter Infections , Helicobacter pylori , Stomach Neoplasms , Antigens, Bacterial/genetics , Antigens, Bacterial/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Carcinogenesis/genetics , DNA/metabolism , DNA Breaks, Double-Stranded , Helicobacter Infections/microbiology , Helicobacter pylori/genetics , Humans , Stomach Neoplasms/metabolismABSTRACT
The proteins from the Fanconi Anemia (FA) pathway of DNA repair maintain DNA replication fork integrity by preventing the unscheduled degradation of nascent DNA at regions of stalled replication forks. Here, we ask if the bacterial pathogen H. pylori exploits the fork stabilisation machinery to generate double stand breaks (DSBs) and genomic instability. Specifically, we study if the H. pylori virulence factor CagA generates host genomic DSBs through replication fork destabilisation and collapse. An inducible gastric cancer model was used to examine global CagA-dependent transcriptomic and proteomic alterations, using RNA sequencing and SILAC-based mass spectrometry, respectively. The transcriptional alterations were confirmed in gastric cancer cell lines infected with H. pylori. Functional analysis was performed using chromatin fractionation, pulsed-field gel electrophoresis (PFGE), and single molecule DNA replication/repair fiber assays. We found a core set of 31 DNA repair factors including the FA genes FANCI, FANCD2, BRCA1, and BRCA2 that were downregulated following CagA expression. H. pylori infection of gastric cancer cell lines showed downregulation of the aforementioned FA genes in a CagA-dependent manner. Consistent with FA pathway downregulation, chromatin purification studies revealed impaired levels of Rad51 but higher recruitment of the nuclease MRE11 on the chromatin of CagA-expressing cells, suggesting impaired fork protection. In line with the above data, fibre assays revealed higher fork degradation, lower fork speed, daughter strands gap accumulation, and impaired re-start of replication forks in the presence of CagA, indicating compromised genome stability. By downregulating the expression of key DNA repair genes such as FANCI, FANCD2, BRCA1, and BRCA2, H. pylori CagA compromises host replication fork stability and induces DNA DSBs through fork collapse. These data unveil an intriguing example of a bacterial virulence factor that induces genomic instability by interfering with the host replication fork stabilisation machinery.
Subject(s)
Antigens, Bacterial/metabolism , Bacterial Proteins/metabolism , DNA Breaks, Double-Stranded , DNA Replication , Down-Regulation , Fanconi Anemia Complementation Group Proteins/metabolism , Helicobacter Infections/metabolism , Helicobacter pylori/metabolism , Oncogene Proteins/metabolism , Signal Transduction , Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Cell Line , Fanconi Anemia Complementation Group Proteins/genetics , Helicobacter Infections/genetics , Helicobacter pylori/genetics , Humans , Oncogene Proteins/geneticsABSTRACT
Infection with CagA-producing Helicobacter pylori plays a causative role in the development of gastric cancer. Upon delivery into gastric epithelial cells, CagA deregulates prooncogenic phosphatase SHP2 while inhibiting polarity-regulating kinase PAR1b through complex formation. Here, we show that CagA/PAR1b interaction subverts nuclear translocation of BRCA1 by inhibiting PAR1b-mediated BRCA1 phosphorylation. It hereby induces BRCAness that promotes DNA double-strand breaks (DSBs) while disabling error-free homologous recombination-mediated DNA repair. The CagA/PAR1b interaction also stimulates Hippo signaling that circumvents apoptosis of DNA-damaged cells, giving cells time to repair DSBs through error-prone mechanisms. The DSB-activated p53-p21Cip1 axis inhibits proliferation of CagA-delivered cells, but the inhibition can be overcome by p53 inactivation. Indeed, sequential pulses of CagA in TP53-mutant cells drove somatic mutation with BRCAness-associated genetic signatures. Expansion of CagA-delivered cells with BRCAness-mediated genome instability, from which CagA-independent cancer-predisposing cells arise, provides a plausible "hit-and-run mechanism" of H. pylori CagA for gastric carcinogenesis.
Subject(s)
Antigens, Bacterial/metabolism , BRCA1 Protein/metabolism , Bacterial Proteins/metabolism , Epithelial Cells/metabolism , Genomic Instability , Helicobacter Infections/microbiology , Helicobacter pylori/metabolism , Stomach Neoplasms/microbiology , Adult , Aged , Aged, 80 and over , Animals , Carcinogenesis/metabolism , Cell Line , DNA Breaks, Double-Stranded , Epithelial Cells/microbiology , Female , Gene Expression Regulation, Neoplastic , Helicobacter pylori/pathogenicity , Host-Pathogen Interactions , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Middle Aged , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Serine-Threonine Kinase 3 , Signal Transduction , Stomach/microbiology , Tumor Suppressor Protein p53/metabolismABSTRACT
Chronic infection with Helicobacter pylori cagA-positive strains is causally associated with the development of gastric diseases, most notably gastric cancer. The cagA-encoded CagA protein, which is injected into gastric epithelial cells by bacterial type IV secretion, undergoes tyrosine phosphorylation at the Glu-Pro-Ile-Tyr-Ala (EPIYA) segments (EPIYA-A, EPIYA-B, EPIYA-C, and EPIYA-D), which are present in various numbers and combinations in its C-terminal polymorphic region, thereby enabling CagA to promiscuously interact with SH2 domain-containing host cell proteins, including the prooncogenic SH2 domain-containing protein tyrosine phosphatase 2 (SHP2). Perturbation of host protein functions by aberrant complex formation with CagA has been considered to contribute to the development of gastric cancer. Here we show that SHIP2, an SH2 domain-containing phosphatidylinositol 5'-phosphatase, is a hitherto undiscovered CagA-binding host protein. Similar to SHP2, SHIP2 binds to the Western CagA-specific EPIYA-C segment or East Asian CagA-specific EPIYA-D segment through the SH2 domain in a tyrosine phosphorylation-dependent manner. In contrast to the case of SHP2, however, SHIP2 binds more strongly to EPIYA-C than to EPIYA-D. Interaction with CagA tethers SHIP2 to the plasma membrane, where it mediates production of phosphatidylinositol 3,4-diphosphate [PI(3,4)P2 ]. The CagA-SHIP2 interaction also potentiates the morphogenetic activity of CagA, which is caused by CagA-deregulated SHP2. This study indicates that initially delivered CagA interacts with SHIP2 and thereby strengthens H. pylori-host cell attachment by altering membrane phosphatidylinositol compositions, which potentiates subsequent delivery of CagA that binds to and thereby deregulates the prooncogenic phosphatase SHP2.
Subject(s)
Antigens, Bacterial/metabolism , Bacterial Proteins/metabolism , Epithelial Cells/metabolism , Gastric Mucosa/metabolism , Helicobacter Infections/metabolism , Helicobacter pylori/metabolism , Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases/metabolism , Amino Acid Motifs , Animals , Antigens, Bacterial/chemistry , Antigens, Bacterial/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Cell Line , Cell Membrane/metabolism , Epithelial Cells/microbiology , Epithelial Cells/pathology , Epithelial-Mesenchymal Transition , Gastric Mucosa/microbiology , Gastric Mucosa/pathology , Helicobacter Infections/microbiology , Helicobacter pylori/genetics , Humans , Phosphatidylinositol Phosphates/metabolism , Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases/chemistry , Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases/genetics , Phosphorylation , Protein Binding , Protein Transport , Protein Tyrosine Phosphatase, Non-Receptor Type 11/chemistry , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , src Homology DomainsABSTRACT
High-molecular-weight hyaluronan, a major component of the extracellular matrix, is anti-oncogenic, whereas low-molecular-weight hyaluronan is pro-oncogenic, though the mechanisms underlying the size-dependent opposite bioactivities of hyaluronan remain uncertain. We show here that treatment with high-molecular-weight hyaluronan stimulates tumor-suppressive Hippo signaling in breast epithelial cells. Mechanistically, clustering of the CD44 extracellular domain by high-molecular-weight hyaluronan leads to recruitment of the polarity-regulating kinase PAR1b by the CD44 intracellular domain, which results in disruption of the Hippo signaling-inhibitory PAR1b-MST complex. Once liberated from PAR1b, MST activates Hippo signaling. Conversely, low-molecular-weight hyaluronan, which is produced by hyaluronidase-mediated degradation of high-molecular-weight hyaluronan, inhibits Hippo signaling by competing with high-molecular-weight hyaluronan for CD44 binding. Triple-negative breast cancers with higher hyaluronidase-2 expression show poorer prognosis than those with lower hyaluronidase-2 expression. Consistently, decreased hyaluronidase-2 is associated with reduced tumorigenicity in a tumor xenograft model. Hence, perturbation of high-molecular-weight hyaluronan-mediated Hippo signaling activation contributes to cancer aggressiveness.
Subject(s)
Hyaluronan Receptors/metabolism , Hyaluronic Acid/pharmacology , Protein Serine-Threonine Kinases/metabolism , Triple Negative Breast Neoplasms/metabolism , Animals , Cell Adhesion Molecules/metabolism , Cell Count , Cell Line, Tumor , Epithelial Cells/metabolism , Extracellular Matrix/metabolism , Female , GPI-Linked Proteins/metabolism , HEK293 Cells , Heterografts , Hippo Signaling Pathway , Humans , Hyaluronic Acid/metabolism , Hyaluronoglucosaminidase/metabolism , MAP Kinase Kinase Kinases/metabolism , MCF-7 Cells , Mice, Inbred BALB C , Mice, Nude , Phosphorylation , Signal Transduction/drug effects , Triple Negative Breast Neoplasms/pathologyABSTRACT
Recent studies have indicated that increased expression of the M2 isoform of pyruvate kinase (PKM2) is involved in glycolysis and tumor development. However, little is known about the role of PKM2 in gastric cancer (GC). Therefore, we examined the expression and function of PKM2 in human GC. We evaluated PKM1 and PKM2 expression by quantitative RT-PCR in gastric tissues from 10 patients who underwent gastric endoscopic submucosal dissection, 80 patients who underwent gastrectomy, and seven healthy volunteers, and analyzed the correlation with clinicopathological variables. To assess the function of PKM2, we generated PKM2-knockdown GC cells, and investigated the phenotypic changes. Furthermore, we examined the induction of PKM2 expression by cytotoxin-associated gene A (CagA), a pathogenic factor of Helicobacter pylori, using CagA-inducible GC cells. We found that PKM2 was predominantly expressed not only in GC lesions but also in the normal gastric regions of GC patients and in the gastric mucosa of healthy volunteers. The PKM2 expression was significantly higher in carcinoma compared to non-cancerous tissue and was associated with venous invasion. Knockdown of PKM2 in GC cells caused significant decreases in cellular proliferation, migration, anchorage-independent growth, and sphere formation in vitro, and in tumor growth and liver metastasis in vivo. The serine concentration-dependent cell proliferation was also inhibited by PKM2 silencing. Furthermore, we found that PKM2 expression was upregulated by CagA by way of the Erk pathway. These results suggested that enhanced PKM2 expression plays a pivotal role in the carcinogenesis and development of GC in part by regulating cancer-specific metabolism.
Subject(s)
Carrier Proteins/genetics , Carrier Proteins/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Protein Isoforms/genetics , Stomach Neoplasms/genetics , Thyroid Hormones/genetics , Thyroid Hormones/metabolism , Aged , Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Female , Gene Expression Regulation, Neoplastic/genetics , Glycolysis/genetics , Humans , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , MAP Kinase Signaling System/genetics , Male , Protein Isoforms/metabolism , Stomach Neoplasms/metabolism , Stomach Neoplasms/pathology , Up-Regulation/genetics , Thyroid Hormone-Binding ProteinsABSTRACT
Most if not all gastric cancers are associated with chronic infection of the stomach mucosa with Helicobacter pylori cagA-positive strains(1-4). Approximately 10% of gastric cancers also harbour Epstein-Barr virus (EBV) in the cancer cells(5,6). Following delivery into gastric epithelial cells via type IV secretion(7,8), the cagA-encoded CagA protein undergoes tyrosine phosphorylation on the Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs initially by Src family kinases (SFKs) and then by c-Abl(9,10). Tyrosine-phosphorylated CagA binds to the pro-oncogenic protein tyrosine phosphatase SHP2 and thereby deregulates the phosphatase activity(11,12), which has been considered to play an important role in gastric carcinogenesis(13). Here we show that the SHP2 homologue SHP1 interacts with CagA independently of the EPIYA motif. The interaction potentiates the phosphatase activity of SHP1 that dampens the oncogenic action of CagA by dephosphorylating the CagA EPIYA motifs. In vitro infection of gastric epithelial cells with EBV induces SHP1 promoter hypermethylation, which strengthens phosphorylation-dependent CagA action via epigenetic downregulation of SHP1 expression. Clinical specimens of EBV-positive gastric cancers also exhibit SHP1 hypermethylation with reduced SHP1 expression. The results reveal that SHP1 is the long-sought phosphatase that can antagonize CagA. Augmented H. pylori CagA activity, via SHP1 inhibition, might also contribute to the development of EBV-positive gastric cancer.
Subject(s)
Antigens, Bacterial/metabolism , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/metabolism , Helicobacter pylori/pathogenicity , Herpesvirus 4, Human/growth & development , Host-Pathogen Interactions , Protein Tyrosine Phosphatase, Non-Receptor Type 6/antagonists & inhibitors , Protein Tyrosine Phosphatase, Non-Receptor Type 6/metabolism , Carcinogenesis , Cell Line, Tumor , DNA Methylation , Epithelial Cells/microbiology , Epithelial Cells/virology , Humans , Phosphorylation , Promoter Regions, Genetic , Protein Binding , Protein Interaction Mapping , Protein Processing, Post-TranslationalABSTRACT
Early detection of gastric cancers saves lives, but remains a diagnostic challenge. In this study, we aimed to identify cell-surface biomarkers of early gastric cancer. We hypothesized that a subset of plasma membrane proteins induced by the Helicobacter pylori oncoprotein CagA will be retained in early gastric cancers through non-oncogene addiction. An inducible system for expression of CagA was used to identify differentially upregulated membrane protein transcripts in vitro. The top hits were then analyzed in gene expression datasets comparing transcriptome of gastric cancer with normal tissue, to focus on markers retained in cancer. Among the transcripts enriched upon CagA induction in vitro, a significant elevation of CEACAM6 was noted in gene expression datasets of gastric cancer. We used quantitative digital immunohistochemistry to measure CEACAM6 protein levels in tissue microarrays of gastric cancer. We demonstrate an increase in CEACAM6 in early gastric cancers, when compared to matched normal tissue, with an AUC of 0.83 for diagnostic validity. Finally, we show that a fluorescently conjugated CEACAM6 antibody binds avidly to freshly resected gastric cancer xenograft samples and can be detected by endoscopy in real time. Together, these results suggest that CEACAM6 upregulation is a cell surface response to H. pylori CagA, and is retained in early gastric cancers. They highlight a novel link between CEACAM6 expression and CagA in gastric cancer, and suggest CEACAM6 to be a promising biomarker to aid with the fluorescent endoscopic diagnosis of early neoplastic lesions in the stomach.
Subject(s)
Antigens, Bacterial/physiology , Antigens, CD/analysis , Bacterial Proteins/physiology , Biomarkers, Tumor/analysis , Cell Adhesion Molecules/analysis , Stomach Neoplasms/diagnosis , Animals , Fluorescent Antibody Technique , GPI-Linked Proteins/analysis , Helicobacter Infections/metabolism , Humans , Mice , Up-RegulationABSTRACT
Pragmin is one of the few mammalian proteins containing the Glu-Pro-Ile-Tyr-Ala (EPIYA) tyrosine-phosphorylation motif that was originally discovered in the Helicobacter pylori CagA oncoprotein. Following delivery into gastric epithelial cells by type IV secretion and subsequent tyrosine phosphorylation at the EPIYA motifs, CagA serves as an oncogenic scaffold/adaptor that promiscuously interacts with SH2 domain-containing mammalian proteins such as the Src homology 2 (SH2) domain-containing protein tyrosine phosphatase-2 (SHP2) and the C-terminal Src kinase (Csk), a negative regulator of Src family kinases. Like CagA, Pragmin also forms a physical complex with Csk. In the present study, we found that Pragmin directly binds to Csk by the tyrosine-phosphorylated EPIYA motif. The complex formation potentiates kinase activity of Csk, which in turn phosphorylates Pragmin on tyrosine-238 (Y238), Y343, and Y391. As Y391 of Pragmin comprises the EPIYA motif, Pragmin-Csk interaction creates a feed-forward regulatory loop of Csk activation. Together with the finding that Pragmin and Csk are colocalized to focal adhesions, these observations indicate that the Pragmin-Csk interaction, triggered by Pragmin EPIYA phosphorylation, robustly stimulates the kinase activity of Csk at focal adhesions, which direct cell-matrix adhesion that regulates cell morphology and cell motility. As a consequence, expression of Pragmin and/or Csk in epithelial cells induces an elongated cell shape with elevated cell scattering in a manner that is mutually dependent on Pragmin and Csk. Deregulation of the Pragmin-Csk axis may therefore induce aberrant cell migration that contributes to tumor invasion and metastasis.
Subject(s)
Carrier Proteins/metabolism , Cell Movement , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , src-Family Kinases/metabolism , Amino Acid Motifs , Animals , Biocatalysis , CSK Tyrosine-Protein Kinase , Carrier Proteins/chemistry , Cell Shape , Cells, Cultured , Enzyme Activation , Feedback, Physiological , Focal Adhesions , Humans , Intracellular Signaling Peptides and Proteins , Mice , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Neoplasm Invasiveness , Phosphorylation , Phosphotyrosine/metabolism , Protein Binding , Substrate SpecificityABSTRACT
More than 50% of people in the world are infected with Helicobacter pylori (H. pylori), which induces various gastric diseases. Especially, epidemiological studies have shown that H. pylori infection is a major risk factor for gastric cancer. It has been reported that the levels of interleukin (IL)-1ß are upregulated in gastric tissues of patients with H. pylori infection. In this study, we investigated the induction mechanism of IL-1ß during H. pylori infection. We found that IL-1ßmRNA and protein were induced in phorbol-12-myristate-13-acetate (PMA)-differentiated THP-1 cells after H. pylori infection. This IL-1ß production was inhibited by a caspase-1 inhibitor and a ROS inhibitor. Furthermore, K(+) efflux and Ca(2+) signaling were also involved in this process. These data suggest that NOD-like receptor (NLR) family, pyrin domain containing 3 (NLRP3) and its complex, known as NLRP3 inflammasome, are involved in IL-1ß production during H. pylori infection because it is reported that NLRP3 inflammasome is activated by ROS, K(+) efflux and/or Ca(2+) signaling. These findings may provide therapeutic strategy for the control of gastric cancer in H. pylori-infected patients.
Subject(s)
Helicobacter Infections/metabolism , Helicobacter pylori , Inflammasomes/metabolism , Interleukin-1beta/metabolism , Macrophages/metabolism , Adenosine Triphosphate/metabolism , Calcium Signaling , Caspase 1/metabolism , Cell Line , Extracellular Space/metabolism , Helicobacter Infections/genetics , Helicobacter Infections/immunology , Helicobacter pylori/immunology , Humans , Interleukin-1beta/genetics , Intracellular Space/metabolism , Macrophages/immunology , Potassium/metabolism , Reactive Oxygen Species/metabolismABSTRACT
CagA, encoded by cytotoxin-associated gene A (cagA), is a major virulence factor of Helicobacter pylori, a gastric pathogen involved in the development of upper gastrointestinal diseases. Infection with cagA-positive H. pylori may also be associated with diseases outside the stomach, although the mechanisms through which H. pylori infection promotes extragastric diseases remain unknown. Here, we report that CagA is present in serum-derived extracellular vesicles, known as exosomes, in patients infected with cagA-positive H. pylori (n = 4). We also found that gastric epithelial cells inducibly expressing CagA secrete exosomes containing CagA. Addition of purified CagA-containing exosomes to gastric epithelial cells induced an elongated cell shape, indicating that the exosomes deliver functional CagA into cells. These findings indicated that exosomes secreted from CagA-expressing gastric epithelial cells may enter into circulation, delivering CagA to distant organs and tissues. Thus, CagA-containing exosomes may be involved in the development of extragastric disorders associated with cagA-positive H. pylori infection.
Subject(s)
Antigens, Bacterial/metabolism , Bacterial Proteins/metabolism , Exosomes/metabolism , Helicobacter pylori/physiology , Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Biological Transport , Biomarkers , Cell Line , Chromatography, Liquid , Helicobacter Infections/complications , Helicobacter Infections/microbiology , Humans , Protein Transport , Stomach Neoplasms/etiology , Tandem Mass Spectrometry , Virulence FactorsABSTRACT
Helicobacter pylori cagA-positive strain delivers the CagA oncoprotein into gastric epithelial cells and at the same time elicits stomach inflammation. To experimentally investigate the pathophysiological interplay between CagA and inflammation, transgenic mice systemically expressing the bacterial cagA gene were treated with a colitis inducer, dextran sulfate sodium (DSS). Compared with control mice, DSS-induced colitis was markedly deteriorated in cagA-transgenic mice. In the colonic epithelia of cagA-transgenic mice, there was a substantial decrease in the level of IκB, which binds and sequesters NF-κB in the cytoplasm. This IκB reduction was due to CagA-mediated inhibition of PAR1, which may stimulate IκB degradation by perturbing microtubule stability. Whereas the CagA-mediated IκB reduction did not automatically activate NF-κB, it lowered the threshold of NF-κB activation by inflammogenic insults, thereby contributing to colitis exacerbation in cagA-transgenic mice. CagA also activates inflammasomes independently of NF-κB signaling, which further potentiates inflammation. The incidence of colonic dysplasia was elevated in DSS-treated cagA-transgenic mice due to a robust increase in the number of pre-cancerous flat-type dysplasias. Thus, CagA deteriorated inflammation, whereas inflammation strengthened the oncogenic potential of CagA. This work revealed that H. pylori CagA and inflammation reinforce each other in creating a downward spiral that instigates neoplastic transformation.
Subject(s)
Antigens, Bacterial/immunology , Bacterial Proteins/immunology , Carcinogenesis/immunology , Colitis/immunology , Gastrointestinal Neoplasms/immunology , Inflammasomes/immunology , Animals , Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Carcinogenesis/drug effects , Colitis/chemically induced , Dextran Sulfate , Female , Gastrointestinal Neoplasms/chemically induced , Male , Mice , Mice, TransgenicABSTRACT
Helicobacter pylori strains carrying the cagA gene are associated with severe disease outcomes, most notably gastric cancer. CagA protein is delivered into gastric epithelial cells by a type IV secretion system. The translocated CagA undergoes tyrosine phosphorylation at the C-terminal EPIYA motifs by host cell kinases. Tyrosine-phosphorylated CagA acquires the ability to interact with and activate SHP2, thereby activating mitogenic signaling and inducing cell morphological transformation (hummingbird phenotype). CagA also interacts with PAR1b via the CM sequence, resulting in induction of junctional and polarity defects. Furthermore, CagA-PAR1b interaction stabilizes the CagA-SHP2 complex. Because transgenic mice systemically expressing CagA develop gastrointestinal and hematological malignancies, CagA is recognized as a bacterium-derived oncoprotein. Interestingly, the C-terminal region of CagA displays a large diversity among H. pylori strains, which influences the ability of CagA to bind to SHP2 and PAR1b. In the present study, we investigated the biological activity of v225d CagA, an Amerindian CagA of H. pylori isolated from a Venezuelan Piaroa Amerindian subject, because the variant CagA does not possess a canonical CM sequence. We found that v225d CagA interacts with SHP2 but not PAR1b. Furthermore, SHP2-binding activity of v225d CagA was much lower than that of CagA of H. pylori isolated from Western countries (Western CagA). v225d CagA also displayed a reduced ability to induce the hummingbird phenotype than that of Western CagA. Given that perturbation of PAR1b and SHP2 by CagA underlies the oncogenic potential of CagA, the v225d strain is considered to be less oncogenic than other well-studied cagA-positive H. pylori strains.
Subject(s)
Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Protein Serine-Threonine Kinases/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Antigens, Bacterial/chemistry , Antigens, Bacterial/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Base Sequence , COS Cells , Chlorocebus aethiops , Dogs , Epithelial Cells/microbiology , Gastric Mucosa/microbiology , Gastric Mucosa/pathology , Humans , Madin Darby Canine Kidney Cells , Molecular Sequence Data , Oncogene Proteins/chemistry , Oncogene Proteins/genetics , Oncogene Proteins/metabolism , Protein Binding , Protein Interaction Domains and Motifs , Protein Serine-Threonine Kinases/chemistry , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolismABSTRACT
Escherichia coli Orf135 hydrolyzes oxidatively damaged nucleotides such as 2-hydroxy-dATP, 8-oxo-dGTP and 5-hydroxy-CTP, in addition to 5-methyl-dCTP, dCTP and CTP. Nucleotide pool sanitization by Orf135 is important since nucleotides are continually subjected to potential damage by reactive oxygen species produced during respiration. Orf135 is a member of the Nudix family of proteins which hydrolyze nucleoside diphosphate derivatives. Nudix hydrolases are characterized by the presence of a conserved motif, even though they recognize various substrates and possess a variety of substrate binding pockets. We investigated the tertiary structure of Orf135 and its interaction with a 2-hydroxy-dATP analog using NMR. We report on the solution structure of Orf135, which should contribute towards a structural understanding of Orf135 and its interaction with substrates.
Subject(s)
Adenosine Triphosphate/analogs & derivatives , Escherichia coli/enzymology , Pyrophosphatases/chemistry , Adenosine Triphosphate/chemistry , Amino Acid Motifs , Binding Sites , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Substrate SpecificityABSTRACT
BACKGROUND: Most of what is known about the Helicobacter pylori (H. pylori) cytotoxin, CagA, pertains to a much-vaunted role as a determinant of gastric inflammation and cancer. Little attention has been devoted to potential roles of CagA in the majority of H. pylori infected individuals not showing oncogenic progression, particularly in relation to host tolerance. Regenerating islet-derived (REG)3γ encodes a secreted C-type lectin that exerts direct bactericidal activity against Gram-positive bacteria in the intestine. Here, we extend this paradigm of lectin-mediated innate immunity, showing that REG3γ expression is triggered by CagA in the H. pylori-infected stomach. METHODOLOGY/PRINCIPAL FINDINGS: In human gastric mucosal tissues, REG3γ expression was significantly increased in CagA-positive, compared to CagA-negative H. pylori infected individuals. Using transfected CagA-inducible gastric MKN28 cells, we recapitulated REG3γ induction in vitro, also showing that tyrosine phosphorylated, not unphosphorylated CagA triggers REG3γ transcription. In concert with induced REG3γ, pro-inflammatory signalling downstream of the gp130 cytokine co-receptor via the signal transducer and activator of transcription (STAT)3 and transcription of two cognate ligands, interleukin(IL)-11 and IL-6, were significantly increased. Exogenous IL-11, but not IL-6, directly stimulated STAT3 activation and REG3γ transcription. STAT3 siRNA knockdown or IL-11 receptor blockade respectively abrogated or subdued CagA-dependent REG3γ mRNA induction, thus demonstrating a requirement for uncompromised signalling via the IL-11/STAT3 pathway. Inhibition of the gp130-related SHP2-(Ras)-ERK pathway did not affect CagA-dependent REG3γ induction, but strengthened STAT3 activation as well as augmenting transcription of mucosal innate immune regulators, IL-6, IL-8 and interferon-response factor (IRF)1. CONCLUSIONS/SIGNIFICANCE: Our results support a model of CagA-directed REG3γ expression in gastric epithelial cells via activation of the IL-11/gp130/STAT3 pathway. This response might allow Gram-negative H. pylori to manipulate host immunity to favour its own survival, by reducing the fitness of co-habiting Gram-positive bacteria with which it competes for resources in the gastric mucosal niche.
Subject(s)
Antigens, Bacterial/metabolism , Bacterial Proteins/metabolism , Gastric Mucosa/metabolism , Gastric Mucosa/microbiology , Gene Expression Regulation , Helicobacter pylori/physiology , Proteins/genetics , STAT3 Transcription Factor/metabolism , Antigens, Bacterial/chemistry , Bacterial Proteins/chemistry , Cell Line , Cytokine Receptor gp130/metabolism , Epithelial Cells/cytology , Epithelial Cells/metabolism , Epithelial Cells/microbiology , Gastric Mucosa/cytology , Gastric Mucosa/immunology , Helicobacter pylori/metabolism , Humans , Immunity, Innate , Interleukin-11/metabolism , Janus Kinases/metabolism , MAP Kinase Signaling System , Pancreatitis-Associated Proteins , Phosphorylation , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcription, Genetic , Transcriptional Activation , Tyrosine/metabolismABSTRACT
Escherichia coli Orf135 protein is thought to be an enzyme that efficiently hydrolyzes oxidatively damaged nucleotides such as 2-hydroxy-dATP, 8-hydroxy-dGTP and 5-hydroxy-CTP, in addition to 5-methyl-dCTP, dCTP and CTP, thus preventing mutations in cells caused by unfavorable base pairing. Nucleotide pool sanitization by Orf135 is important since organisms are continually subjected to potential damage by reactive oxygen species produced during respiration. It is known that the frequency of spontaneous and H(2)O(2)-induced mutations is two to threefold higher in the orf135(-) strain compared with the wild-type. Orf135 is a member of the Nudix family of proteins which hydrolyze nucleoside diphosphate derivatives. Nudix hydrolases are characterized by the presence of a conserved motif, although they recognize various substrates and possess a variety of substrate binding pockets. We are interested in delineating the mechanism by which Orf135 recognizes oxidatively damaged nucleotides. To this end, we are investigating the tertiary structure of Orf135 and its interaction with substrate using NMR. Herein, we report on the (1)H, (13)C and (15)N resonance assignments of Orf135, which should contribute towards a structural understanding of Orf135 and its interaction with substrate.
Subject(s)
Escherichia coli/enzymology , Nuclear Magnetic Resonance, Biomolecular , Pyrophosphatases/chemistry , Pyrophosphatases/metabolismABSTRACT
Helicobacter pylori CagA is delivered into gastric epithelial cells, where undergoes tyrosine phosphorylation at the Glu-Pro-Ile-Tyr-Ala (EPIYA) motif to interact with Src homology 2-containing protein tyrosine phosphatase-2 (SHP2) oncoprotein. CagA also binds to partitioning-defective 1 (PAR1) polarity-regulating kinase via the CagA multimerization (CM) sequence. To investigate pathophysiological role of CagA-SHP2 and/or CagA-PAR1 interaction in H. pylori infection, we generated H. pylori isogenic strains producing a phosphorylation-resistant CagA and a CagA without CM sequence. Infection studies revealed that deregulation of epithelial cell motility was more prominent in the wild-type strain than in the mutant strains. Thus, both CagA-SHP2 and CagA-PAR1 interactions are involved in the pathogenicity of cagA-positive H. pylori.
Subject(s)
Antigens, Bacterial/metabolism , Bacterial Proteins/metabolism , Cell Movement , Epithelial Cells/physiology , Helicobacter pylori/pathogenicity , Host-Pathogen Interactions , Signal Transduction , Virulence Factors/metabolism , Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Cell Line , Epithelial Cells/microbiology , Helicobacter pylori/genetics , Humans , Protein Serine-Threonine Kinases/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Virulence Factors/geneticsABSTRACT
Partitioning-defective 1b (PAR1b), also known as microtubule affinity-regulating kinase 2 (MARK2), is a member of evolutionally conserved PAR1/MARK serine/threonine kinase family, which plays a key role in the establishment and maintenance of cell polarity at least partly by phosphorylating microtubule-associated proteins (MAPs) that regulate microtubule stability. PAR1b has also been reported to influence actin cytoskeletal organization, raising the possibility that PAR1b functionally interacts with the Rho family of small GTPases, central regulators of the actin cytoskeletal system. Consistent with this notion, PAR1 was recently found to be physically associated with a RhoA-specific guanine nucleotide exchange factor H1 (GEF-H1). This observation suggests a functional link between PAR1b and GEF-H1. Here we show that PAR1b induces phosphorylation of GEF-H1 on serine 885 and serine 959. We also show that PAR1b-induced serine 885/serine 959 phosphorylation inhibits RhoA-specific GEF activity of GEF-H1. As a consequence, GEF-H1 phosphorylated on both of the serine residues loses the ability to stimulate RhoA and thereby fails to induce RhoA-dependent stress fiber formation. These findings indicate that PAR1b not only regulates microtubule stability through phosphorylation of MAPs but also influences actin stress fiber formation by inducing GEF-H1 phosphorylation. The dual function of PAR1b in the microtubule-based cytoskeletal system and the actin-based cytoskeletal system in the coordinated regulation of cell polarity, cell morphology, and cell movement.
Subject(s)
Actins/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Protein Serine-Threonine Kinases/metabolism , Stress Fibers/metabolism , rhoA GTP-Binding Protein/metabolism , Actins/genetics , Animals , COS Cells , Cell Movement/physiology , Cell Polarity/physiology , Chlorocebus aethiops , Guanine Nucleotide Exchange Factors/genetics , Humans , Microtubules/genetics , Microtubules/metabolism , Phosphorylation/physiology , Protein Serine-Threonine Kinases/genetics , Rho Guanine Nucleotide Exchange Factors , Stress Fibers/genetics , rhoA GTP-Binding Protein/geneticsABSTRACT
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.