Hummingbird Phenotype Research Articles

Development of gastric cancer associated with Helicobacter pylori infection.

Helicobacter pylori infection is associated with histological gastritis, gastric atrophy, gastric cancer and mucosa-associated lymphoid tissue lymphoma in the stomach. However, gastric cancer only develops in a minority of infected individuals. Such clinical diversity is caused by variations in the interactions between H. pylori pathogenicity, host susceptibility, and environmental factors. Based on evidence from three prospective epidemiological studies, the International Agency for Research on Cancer and the World Health Organization (IARC/WHO) concluded in 1994 that H. pylori has a causal linkage to gastric carcinogenesis and is a definite carcinogen in humans. Two large-scale, prospective, epidemiological studies have recently been reported in Japan and have confirmed that H. pylori infection constitutes a high risk factor for the development of gastric cancer, at least in males. In order to obtain evidence that eradication of H. pylori leads to a reduction in the occurrence of gastric cancer, reversibility of precancerous lesions, gastric atrophy or intestinal metaplasia should be proven after eradication treatment. A biopsy specimen from the lesser curvature of the corpus is the most sensitive for evaluating the regression of gastric atrophy on histology, and the evaluation needs be conducted at least 13 months after treatment. In a Mongolian gerbil model with or without low-dose chemical carcinogens, it has been demonstrated that H. pylori can lead to the development of gastric cancer. Experimental studies have elucidated that virulence factors of H. pylori interact with gastric epithelial cell signaling related to carcinogenesis. The cag pathogenicity island (cagPAI) is a major virulence gene cluster; it encodes the type IV secretion machinery system forming a cylinder-like structure. The CagA protein is translocated into target cells via this secretion system and induces a hummingbird phenotype, a growth factor-like effect. The other gene products are probably translocated into target cells and accelerate cellular proliferation and apoptosis. The molecular mechanism of the interaction between H. pylori and gastric epithelial cells may provide a new strategy for effective prevention of the development of gastric cancer induced by H. pylori infection.

Conditional gene silencing utilizing the lac repressor reveals a role of SHP‐2 in cagA‐positive Helicobacter pylori pathogenicity

RNA interference (RNAi) is a newly described biological phenomenon mediated by small interfering RNA (siRNA) that targets mRNA for degradation by cellular enzymes and has become a powerful method for studying gene functions in mammalian systems. The development of systems for inducing siRNA expression should enable examination of acute loss-of-function phenotypes in a cell of interest without the need to consider lethality or epigenetic adaptation of cells. We describe in this report an inducible siRNA expression system made by combined utilization of the RNA polymerase III-dependent promoter H1 and the bacterial lac repressor. Using this system, we established AGS gastric epithelial cells in which expression of SHP-2, a cellular tyrosine phosphatase known to specifically bind the Helicobacter pylori virulence factor CagA, is conditionally and reversibly silenced by the lactose analog isopropyl-1-thio-beta-D-galactopyranoside (IPTG). Upon expression in AGS cells, CagA provoked a morphological transformation, termed the hummingbird phenotype, which is associated with CagA virulence. This morphogenetic activity of CagA was totally abolished when SHP-2 expression was silenced by inducible siRNA expression in AGS cells. Our results indicate that SHP-2 is a critical downstream effector of H. pylori CagA. The conditional gene silencing system described here should become a powerful tool for investigating the roles of cancer-related genes through a reversed genetic approach.

Helicobacter pylori CagA Induces Ras-independent Morphogenetic Response through SHP-2 Recruitment and Activation

The CagA protein of Helicobacter pylori, which is injected from the bacteria into bacteria-attached gastric epithelial cells, is associated with gastric carcinoma. CagA is tyrosine-phosphorylated by Src family kinases, binds the SH2 domain-containing SHP-2 phosphatase in a tyrosine phosphorylation-dependent manner, and deregulates its enzymatic activity. We established AGS human gastric epithelial cells that inducibly express wild-type or a phosphorylation-resistant CagA, in which tyrosine residues constituting the EPIYA motifs were substituted with alanines. Upon induction, wild-type CagA, but not the mutant CagA, elicited strong elongation of cell shape, termed the "hummingbird" phenotype. Time-lapse video microscopic analysis revealed that the CagA-expressing cells exhibited a marked increase in cell motility with successive rounds of elongation-contraction processes. Inhibition of CagA phosphorylation by an Src kinase inhibitor, PP2, or knockdown of SHP-2 expression by small interference RNA (siRNA) abolished the CagA-mediated hummingbird phenotype. The morphogenetic activity of CagA also required Erk MAPK but was independent of Ras or Grb2. In AGS cells, CagA prolonged duration of Erk activation in response to serum stimulation. Conversely, inhibition of SHP-2 expression by siRNA abolished the sustained Erk activation. Thus, SHP-2 acts as a positive regulator of Erk activity in AGS cells. These results indicate that SHP-2 is involved in the Ras-independent modification of Erk signals that is necessary for the morphogenetic activity of CagA. Our work therefore suggests a key role of SHP-2 in the pathological activity of H. pylori virulence factor CagA.

Cytoskeletal rearrangements in gastric epithelial cells in response to Helicobacter pylori infection.

Helicobacter pylori causes host epithelial cell cytoskeletal rearrangements mediated by the translocation and tyrosine phosphorylation of an outer-membrane protein, CagA, and by the vacuolating cytotoxin, VacA. However, the mechanisms by which H. pylori mediates cytoskeletal rearrangements in infected host cells need to be more clearly defined. The aim of this study was to determine the effects of H. pylori isolates from children on the architecture of host gastric epithelial cells. Gastric epithelial (AGS) cells were infected with type I (cagE(+), cagA(+), VacA(+)) H. pylori, a type II H. pylori strain (cagE(-), cagA(-), VacA(-)) or a cagE isogenic mutant. Double-labelled immune fluorescence was used to detect adherent H. pylori and the distribution of F-actin, alpha-actinin and Arp3. Both type I and type II H. pylori strains induced stress fibres in gastric epithelial cells that were not observed in uninfected cells. Type I H. pylori also induced cell elongation (hummingbird phenotype) after 4 h of infection, whereas the type II H. pylori strain did not. Less elongation occurred when AGS cells were exposed to a cagE isogenic mutant, compared with the parental strain. Confocal microscopy showed Arp3 accumulation in AGS cells infected with wild-type H. pylori, but not in response to infection with the cagE mutant. These findings indicate that type I H. pylori induce a stress fibre-like phenotype in infected gastric epithelia by a mechanism that is different from the induction of host-cell elongation. In addition to CagA and VacA, cagE also impacts on the morphology of infected gastric epithelial cells.

Attenuation of Helicobacter pylori CagA·SHP-2 Signaling by Interaction between CagA and C-terminal Src Kinase

Helicobacter pylori (H. pylori) is a causative agent of gastric diseases ranging from gastritis to cancer. The CagA protein is the product of the cagA gene carried among virulent H. pylori strains and is associated with severe disease outcomes, most notably gastric carcinoma. CagA is injected from the attached H. pylori into gastric epithelial cells and undergoes tyrosine phosphorylation. The phosphorylated CagA binds and activates SHP-2 phosphatase and thereby induces a growth factor-like morphological change termed the "hummingbird phenotype." In this work, we demonstrate that CagA is also capable of interacting with C-terminal Src kinase (Csk). As is the case with SHP-2, Csk selectively binds tyrosine-phosphorylated CagA via its SH2 domain. Upon complex formation, CagA stimulates Csk, which in turn inactivates the Src family of protein-tyrosine kinases. Because Src family kinases are responsible for CagA phosphorylation, an essential prerequisite of CagA.SHP-2 complex formation and subsequent induction of the hummingbird phenotype, our results indicate that CagA-Csk interaction down-regulates CagA.SHP-2 signaling by both competitively inhibiting CagA.SHP-2 complex formation and reducing levels of CagA phosphorylation. We further demonstrate that CagA.SHP-2 signaling eventually induces apoptosis in AGS cells. Our results thus indicate that CagA-Csk interaction prevents excess cell damage caused by deregulated activation of SHP-2. Attenuation of CagA activity by Csk may enable cagA-positive H. pylori to persistently infect the human stomach for decades while avoiding excess CagA toxicity to the host.

Src Is the Kinase of the Helicobacter pylori CagA Protein in Vitro and in Vivo

The gastric pathogen Helicobacter pylori uses a type IV secretion system to inject the bacterial CagA protein into gastric epithelial cells. Within the host cell, CagA becomes phosphorylated on tyrosine residues and initiates cytoskeletal rearrangements. We demonstrate here that Src-like protein-tyrosine kinases mediate CagA phosphorylation in vitro and in vivo. First, the Src-specific tyrosine kinase inhibitor PP2 specifically blocks CagA phosphorylation and cytoskeletal rearrangements thereby inhibiting the CagA-induced hummingbird phenotype of gastric epithelial cells. Second, CagA is in vivo phosphorylated by transiently expressed c-Src. Third, recombinant c-Src and lysates derived from c-Src-expressing fibroblasts but not lysates derived from Src-, Yes-, and Fyn-deficient cells phosphorylated CagA in vitro. Fourth, a transfected CagA-GFP fusion protein is phosphorylated in vivo in Src-positive fibroblasts but not in Src-, Yes-, and Fyn-deficient cells. Because a CagA-GFP fusion protein mutated in an EPIYA motif is not efficiently phosphorylated in any of these fibroblast cells, the CagA EPIYA motif appears to constitute the major c-Src phosphorylation site conserved among CagA-positive Helicobacter strains.

Hijacking of key signaling pathways by H. pylori

The Gram-negative bacterium Helicobacter pylori colonizes the human gastric epithelium and is strongly associated with peptic ulceration, mucosal-associated lymphoid tissue lymphoma and adenocarcinoma of the stomach. Around 50% of the world's population suffer from H. pylori infection. Virulent strains of the H. pylori infect the stomach lining and cause cells to elongate until they resemble hummingbird beaks. This so-called hummingbird phenotype is also a feature of exposure of cells to hepatocyte growth factor. Virulent, but not benign, strains of the microbe inject a protein termed Cag A into the stomach cells. The function of CagA protein is unknown, although it is known to be a substrate for host-cell tyrosine kinases, suggesting that key phosphorylation events involved in regulating signal transduction cascades are targeted by the CagA protein. A recent paper now reveals that the CagA protein perturbs cellular function by dysregulating the tyrosine phosphatase SHP-2.

Deregulation of SHP-2 tyrosine phosphatase by the Helicobacter pylori virulence factor CagA.

Helicobacter pylori (H. pylori) is estimated to infect about half of the world population. It causes gastric diseases ranging from gastritis to cancer and has been classified as a class I carcinogen by WHO. However, little is known about the molecular mechanisms by which H. pylori induces pathogenesis. CagA is the product of the cagA gene carried among virulent H. pylori strains and is associated with severe clinical outcomes, most notably gastric carcinoma. CagA is injected from the attached H. pylori into gastric epithelial cells and undergoes tyrosine phosphorylation. We found that wild-type, but not phosphorylation-resistant CagA, is capable of inducing a growth factor-like morphological change, termed the hummingbird phenotype, in cells. Furthermore, CagA specifically binds the SH2-containing protein tyrosine phosphatase SHP-2 in a tyrosine phosphorylation-dependent manner and stimulates phosphatase activity. Disruption of the CagA-SHP-2 complex abolishes the CagA-dependent morphological change. Conversely, constitutively active SHP-2 is capable of inducing a CagA-like morphological change when it is plasma membrane-targeted. Our results show that CagA perturbs cellular functions by deregulating SHP-2 phosphatase after translocation from H. pylori into gastric epithelial cells. Given the positive regulatory roles of SHP-2 in both cell proliferation and cell movement, the CagA-SHP-2 interaction may play an important role in the oncogenic transformation that is a hallmark of cagA+ H. pylori infection.

Phosphorylation of tyrosine 972 of the Helicobacter pylori CagA protein is essential for induction of a scattering phenotype in gastric epithelial cells.

Helicobacter pylori colonizes the human stomach and is the causative agent of a variety of gastric diseases. After bacterial attachment, the H. pylori CagA protein is translocated into gastric epithelial cells and tyrosine phosphorylated. This process is associated with characteristic cytoskeletal rearrangements, resulting in a scatter factor-like ('hummingbird') phenotype. In this study, using a cagA mutant complemented with wild-type cagA and transiently expressing CagA in AGS cells, we have demonstrated that translocated CagA is necessary for rearrangements of the actin cytoskeleton to occur. Anti-phosphotyrosine immunoblotting studies and treatment of infected cells with phosphotyrosine kinase inhibitors suggested that not only translocation but also phosphorylation of CagA is important in this process. Transient expression of CagA-green fluorescent protein (GFP) fusion proteins and two-dimensional gel electrophoresis of CagA protein species demonstrated tyrosine phosphorylation in the C-terminus. Site-directed mutagenesis of CagA revealed that tyrosine residue 972 is essential for induction of the cellular phenotype. We have also demonstrated that translocation and phosphorylation of CagA is necessary but not sufficient for induction of the hummingbird phenotype in AGS cells, indicating the involvement of as yet unidentified bacterial factor(s).