The effect of the cag pathogenicity island on binding of Helicobacter pylori to gastric epithelial cells and the subsequent induction of apoptosis

BACKGROUND: Helicobacter pylori infection leads to gastritis, peptic ulcer, and gastric cancer, in part due to epithelial damage following bacteria binding to the epithelium. Infection with cag pathogenicity island (PAI) bearing strains of H. pylori is associated with increased gastric inflammation and a higher incidence of gastroduodenal diseases. It is now known that various effector molecules are injected into host epithelial cells via a type IV secretion apparatus, resulting in cytoskeletal changes and chemokine secretion. Whether binding of bacteria and subsequent apoptosis of gastric epithelial cells are altered by cag PAI status was examined in this study. METHODS: AGS, Kato III, and N87 human gastric epithelial cell lines were incubated with cag PAI-positive or cag PAI-negative strains of H. pylori in the presence or absence of clarithromycin. Binding was evaluated by flow cytometry and scanning electron microscopy. Apoptosis was assessed by detection of DNA degradation and ELISA detection of exposed histone residues. RESULTS: cag PAI-negative strains bound to gastric epithelial cells to the same extent as cag PAI-positive strains. Both cag PAI-positive and cag PAI-negative strains induced apoptosis. However, cag PAI-positive strains induced higher levels of DNA degradation. Incubation with clarithromycin inactivated H. pylori but did not affect binding. However, pretreatment with clarithromycin decreased infection-induced apoptosis. CONCLUSIONS: cag PAI status did not affect binding of bacteria to gastric epithelial cells but cag PAI-positive H. pylori induced apoptosis more rapidly than cag PAI-negative mutant strains, suggesting that H. pylori binding and subsequent apoptosis are differentially regulated with regard to bacterial properties.     

Helicobacter pylori cag pathogenicity island-positive strains induce syndecan-4 expression in gastric epithelial cells

Helicobacter pylori is recognized as the main cause of gastritis and is associated with gastric carcinogenesis. Syndecan-4 represents the major source of heparan sulfate (HS) in the gastric cells. HS proteoglycans expressed on the cell surface constitute targets for H. pylori at the early stage of infection. The aim of this study was to determine whether H. pylori induction of syndecan-4 expression is affected by the virulence characteristics of the infecting strain, namely the cytotoxic-associated gene ( cag ) pathogenicity island (PAI). We observed that individuals infected with highly pathogenic H. pylori strains express syndecan-4 in the foveolar epithelium of the gastric mucosa. The association between the cag PAI status of the infecting strain and syndecan-4 expression was further demonstrated by infection of gastric epithelial cell lines with a panel of cag PAI⁺ and cag PAI⁻ H. pylori strains, showing that expression of syndecan-4 was significantly increased in response to infection with the highly pathogenic strains. Moreover, infection of gastric cells with cag A and cag E mutant strains further confirmed that syndecan-4 induction is dependent on an intact cag PAI. The present study shows that highly pathogenic H. pylori strains induce syndecan-4 expression, both in human gastric mucosa and in gastric cell lines, in a cag PAI-dependent manner.     

Cag pathogenicity island-independent up-regulation of matrix metalloproteinases-9 and -2 secretion and expression in mice by Helicobacter pylori infection

Helicobacter pylori cag pathogenicity island (PAI) is a major determinant of gastric injury via induction of several matrix metalloproteinases (MMPs). In the present study, we examined the influence of the cag PAI on gastric infection and MMP-9 production in mice and in cultured cells. A new mouse colonizing Indian H. pylori strain (AM1) that lacks the cag PAI was used to study the cag PAI importance in inflammation. Groups of C57BL/6 mice were inoculated separately with H. pylori strains AM1 and SS1 (cag+), gastric tissues were histologically examined, and bacterial colonization was scored by quantitative culture. Mice infected with either cag+ or cag- H. pylori strains showed gastric inflammation and elevated MMP-3 production. Significant up-regulation of pro-MMP-9 secretion and gene expression in H. pylori infected gastric tissues indicate dispensability of cag PAI for increased pro-MMP-9 secretion and synthesis in mice. In agreement, cell culture studies revealed that both AM1 and SS1 were equipotent in pro-MMP-9 induction in human gastric epithelial cells. Both strains showed moderate increase in MMP-2 activity in vivo and in vitro. In addition, increased secretion of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6 induced pro-MMP-9 secretion and synthesis in AM1 or SS1 strain-infected mice suggesting elicitation of pro-inflammatory cytokines by both cag- and cag+ genotype. Moreover, tissue inhibitors of metalloproteinase-1 expression were decreased with increase in pro-MMP-9 induction. These data show that H. pylori may act through different pathways other than cag PAI-mediated for gastric inflammation and contribute to up-regulation of MMP-9 via pro-inflammatory cytokines.     

Effect of H. pylori on the expression of TRAIL, FasL and their receptor subtypes in human gastric epithelial cells and their role in apoptosis

BACKGROUND AND AIMS: In the human stomach expression of TNF-related apoptosis inducing ligand (TRAIL) and its receptors and the modulatory role of Helicobacter pylori are not well described. Therefore, we investigated the effect of H. pylori on the expression of TRAIL, FasL and their receptors (TRAIL-R1-R4, Fas) in gastric epithelial cells and examined their role in apoptosis. MATERIALS AND METHODS: mRNA and protein expression of TRAIL, FasL and their receptors were analyzed in human gastric epithelial cells using RT-PCR, Western blot, and immunohistochemistry. Gastric epithelial cells were incubated with FasL, TRAIL and/or H. pylori, and effects on expression, cell viability and epithelial apoptosis were monitored. Apoptosis was analyzed by histone ELISA, DAPI staining and immunohistochemistry. RESULTS: TRAIL, FasL and their receptor subtypes were expressed in human gastric mucosa, gastric epithelial cell primary cultures and gastric cancer cells. TRAIL, FasL and H. pylori caused a time- and concentration-dependent induction of DNA fragmentation in gastric cancer cells with synergistic effects. In addition, H. pylori caused a selective up-regulation of TRAIL, TRAIL-R1 and Fas mRNA and protein expression in gastric cancer cells. CONCLUSIONS: Next to FasL and Fas, TRAIL and all of its receptor subtypes are expressed in the human stomach and differentially modulated by H. pylori. TRAIL, FasL and H. pylori show complex interaction mediating apoptosis in human gastric epithelial cells. These findings might be important for the understanding of gastric epithelial cell kinetics in patients with H. pylori infection.     

幽门螺杆菌cag PAI编码的Ⅳ型分泌系统

幽门螺杆菌(Helicobacter pylori,H.pylori)是定植于人胃部特定的病原菌,感染呈全球分布,感染率高达50%以上。现已证实它是轻度胃炎,消化性溃疡及胃癌的主要病因。Ⅰ型H.pylori菌株含有一个约40kb的特殊基因片段,即cag致病岛(cytotoxin associated gene pathogenicity island,cag PAI),该片段只出现于致病相关菌株,基因呈高密度分布并编码一个分泌转运系统称为Ⅳ型分泌系统(type Ⅳ secretion system,TFSS),通过转运相关毒素而参与H.pylori诱导上皮细胞细胞内的酪氨酸磷酸化、细胞骨架重排、基垫结构形成、活化核转录因子NF-κB、诱导促炎细胞因子白细胞介素-8的表达等,故在H.pylori的致病中起着关键作用。近年来,研究者们致力于研究Ⅳ型分泌系统的功能,但是对于这个装置是如何转运蛋白进入宿主细胞的确切机制还是知之甚少,因此,对Ⅳ型分泌系统的研究将有助于进一步明确H.pylori致病机制,并为临床诊断和治疗提供新的靶点。     

Upregulation of Fas ligand expression by human immunodeficiency virus in human macrophages mediates apoptosis of uninfected T lymphocytes.

Apoptosis has been proposed to mediate CD4+ T-cell depletion in human immunodeficiency virus (HIV)-infected individuals. Interaction of Fas ligand (FasL) with Fas (CD95) results in lymphocyte apoptosis, and increased susceptibility to Fas-mediated apoptosis has been demonstrated in lymphocytes from HIV-infected individuals. Cells undergoing apoptosis in lymph nodes from HIV-infected individuals do not harbor virus, and therefore a bystander effect has been postulated to mediate apoptosis of uninfected cells. These data raise the possibility that antigen-presenting cells are a source of FasL and that HIV infection of cells such as macrophages may induce or increase FasL expression. In this report, we demonstrate that HIV infection of monocytic cells not only increases the surface expression of Fas but also results in the de novo expression of FasL. Interference with the FasL-Fas interaction by anti-Fas blocking antibodies abrogates HIV-induced apoptosis of monocytic cells. Human monocyte-derived macrophages from healthy donors contain detectable FasL mRNA, which is further upregulated following HIV infection with monocytotropic strains. HIV-infected human macrophages result in the apoptotic death of Jurkat T cells and peripheral blood T lymphocytes. Interruption of the FasL-Fas interaction abrogates the HIV-infected macrophage-dependent death of T lymphocytes. These results provide evidence that human macrophages can provide a source of FasL, especially following HIV infection, and can thus participate in lymphocyte depletion in HIV-infected individuals.     

Reduced activation of inflammatory responses in host cells by mouse-adapted Helicobacter pylory isolates

Helicobacter pylori strains that harbour the Cag pathogenicity island (Cag PAI) induce interleukin (IL)-8 secretion in gastric epithelial cells, via the activation of NF- kappa B, and are associated with severe inflammation in humans. To investigate the influence of Cag PAI-mediated inflammatory responses on H. pylori adaptation to mice, a selection of H. pylori clinical isolates (n = 12) was cag PAI genotyped and tested in co-culture assays with AGS gastric epithelial cells, and in mouse colonization studies. Six isolates were shown to harbour a complete cag PAI and to induce NF- kappa B activation and IL-8 secretion in AGS cells. Of the eight isolates that spontaneously colonized mice, six had a cag PAI(-) genotype and did not induce pro-inflammatory responses in these cells. Mouse-to-mouse passage of the two cag PAI(+) -colonizing strains yielded host-adapted variants that infected mice with bacterial loads 100-fold higher than those of the respective parental strains (P= 0.001). These mouse-adapted variants were affected in their capacity to induce pro-inflammatory responses in host cells, yet no changes in cag PAI gene content were detected between the strains by DNA microarray analysis. This work provides evidence for in vivo selection of H. pylori bacteria with a reduced capacity to induce inflammatory responses and suggests that such bacteria are better adapted to colonize mice.     

In-Cell Western analysis of Helicobacter pylori-induced phosphorylation of extracellular-signal related kinase via the transactivation of the epidermal growth factor receptor

Helicobacter pylori activates extracellular-signal related (ERK) kinases in gastric epithelial cells, via transactivation of the EGF receptor (EGFR). H. pylori activation of EGFR may be relevant to epithelial hyperproliferation and gastric carcinogenesis. The aim of this study was to develop an 'In-Cell Western' (ICW) assay for quantitative examination of H. pylori-induced epithelial signalling, to enable the role of the EGFR in H. pylori-induced phosphorylation of ERK in epithelial cells to be ascertained. H. pylori strains were co-incubated with A431 and AGS cells. pERK and total ERK were quantified in situ using ICW analysis. H. pylori strains both with, and without a cag PAI, and Helicobacter felis, significantly increased pERK levels in A431 cells. The EGFR inhibitor EKB-569 dose-dependently reduced H. pylori-induced ERK phosphorylation in A431 and AGS cells. A significantly lower reduction was observed with cag+ strains in A431 but not AGS cells. The cag PAI was not necessary for EGFR signal transactivation. These data suggest that H. pylori induces pERK in epithelial cells partly via the EGFR pathway. Additional signalling mechanisms are likely to be involved in H. pylori-induced ERK phosphorylation. ICW analysis is a rapid quantitative method for evaluating the effects of inhibitors on H. pylori-induced cell signalling pathways of relevance to gastric carcinogenesis.     

Induction of human beta-defensin-2 mRNA expression by Helicobacter pylori in human gastric cell line MKN45 cells on cag pathogenicity island.

Helicobacter pylori is an etiological agent of gastritis, peptic ulcer, and gastric cancer. Human beta-defensin-2 (hBD-2) is an antimicrobial peptide which belongs to one of the most important host defense systems against bacterial infection in several epithelial tissues. We studied the effect of H. pylori on the expression of hBD-2 mRNA in MKN45 gastric mucosal cells. H. pylori, but not culture filtrate, increased the hBD-2 mRNA level in MKN45 cells; the inductive effect of H. pylori was not detected with Intestine 407 cells. Among H. pylori strains, strain OHPC0002, which lacks a cag Pathogenicity Island (PAI), did not induce hBD-2 mRNA in MKN45 cells. These results suggested that H. pylori cag PAI is critical for the induction of hBD-2 mRNA in MKN45 cells. Exposure of MKN45 cells to Salmonella typhimurium, S. enteritidis, S. typhi, and S. dublin, but not Escherichia coli ML35, also resulted in induction of hBD-2 mRNA.     

Differential activation of mitogen-activated protein kinases in AGS gastric epithelial cells by cag+ and cag- Helicobacter pylori.

The aim of this study was to determine whether Helicobacter pylori activates mitogen-activated protein (MAP) kinases in gastric epithelial cells. Infection of AGS cells with an H. pylori cag+ strain rapidly (5 min) induced a dose-dependent activation of extracellular signal-regulated kinases (ERK), p38, and c-Jun N-terminal kinase (JNK) MAP kinases, as determined by Western blot analysis and in vitro kinase assay. Compared with cag+ strains, cag- clinical isolates were less potent in inducing MAP kinase, particularly JNK and p38, activation. Isogenic inactivation of the picB region of the cag pathogenicity island resulted in a similar loss of JNK and p38 MAP kinase activation. The specific MAP kinase inhibitors, PD98059 (25 microM; MAP kinase kinase (MEK-1) inhibitor) and SB203580 (10 microM; p38 inhibitor), reduced H. pylori-induced IL-8 production in AGS cells by 78 and 82%, respectively (p < 0.01 for each). Both inhibitors together completely blocked IL-8 production (p < 0.001). However, the MAP kinase inhibitors did not prevent H. pylori-induced IkappaBalpha degradation or NF-kappaB activation. Thus, H. pylori rapidly activates ERK, p38, and JNK MAP kinases in gastric epithelial cells; cag+ isolates are more potent than cag- strains in inducing MAP kinase phosphorylation and gene products of the cag pathogenicity island are required for maximal MAP kinase activation. p38 and MEK-1 activity are required for H. pylori-induced IL-8 production, but do not appear to be essential for H. pylori-induced NF-kappaB activation. Since MAP kinases regulate cell proliferation, differentiation, programmed death, stress, and inflammatory responses, activation of gastric epithelial cell MAP kinases by H. pylori cag+ strains may be instrumental in inducing gastroduodenal inflammation, ulceration, and neoplasia.     

Proteasome inhibitors block Ras/ERK signaling pathway resulting in the downregulation of Fas ligand expression during activation-induced cell death in T cells

Activation-induced cell death (AICD) plays a critical role in the maintenance of homeostasis and peripheral tolerance in the immune system, and is mediated by Fas ligand (FasL) expression and the interaction between Fas and FasL. In the present study, we examined the role of the ubiquitin-proteasome system in AICD using T cell hybridoma N3-6-71 cells. The peptidyl aldehyde proteasome inhibitor carbobenzoxyl-Ile-Glu(O-t-butyl)-Ala-leucinal (PSI) blocked T cell receptor (TCR) stimulation-induced apoptosis in the T cell hybridoma. Fas and FasL gene expression and mouse FasL promoter activity following TCR stimulation were suppressed by PSI pretreatment. Deletion or point mutation of the kappaB site in the FasL promoter region did not suppress inducible FasL promoter activity effectively. PSI blocked extracellular signal-regulated kinase (ERK) activity induced by TCR stimulation, but had no effect on c-jun N-terminal kinase activation. ERK activation was essential for FasL expression and AICD. The initial tyrosine phosphorylation steps following TCR stimulation, i.e., phosphorylation of CD3zeta and Vav, were not altered by PSI. These data suggest that the ubiquitin-proteasome system has some regulatory function at an intermediate step between the initial tyrosine phosphorylation steps and ERK activation in AICD.     

Differential role of tyrosine phosphorylation in the induction of apoptosis in T cell clones via CD95 or the TCR/CD3-complex

Activated T cells undergo apoptosis when the Fas-antigen (APO-1, CD95) is ligated by Fas Ligand (FasL) or agonistic anti-Fas antibodies. Repeated stimulation of T lymphocytes via the TCR/CD3-complex induces activation-induced cell death (AICD) associated with FasL surface expression. FasL binding to Fas molecules triggers the Fas-dependent death signaling cascade. Since it is still controversial whether Fas-induced cell death is associated with tyrosine kinase activity, we investigated the tyrosine kinase activation requirements in anti-Fas antibody-induced cell death and AICD in human T cell clones. We report that cell death triggered by anti-Fas antibody is not accompanied by an increase in tyrosine phosphorylation and cannot be blocked by inhibitors of protein tyrosine kinases (PTK). Under the same conditions, AICD of T cell clones is clearly associated with tyrosine kinase activation. In fact, semiquantitative RT-PCR analysis of FasL mRNA expression triggered in T cell clones via the TCR/CD3-complex revealed that tyrosine phosphorylation is required for functional FasL mRNA and surface expression.     

Significant role for Fas in the pathogenesis of autoimmune diabetes

Programmed cell death represents an important pathogenic mechanism in various autoimmune diseases. Type I diabetes mellitus (IDDM) is a T cell-dependent autoimmune disease resulting in selective destruction of the beta cells of the islets of Langerhans. beta cell apoptosis has been associated with IDDM onset in both animal models and newly diagnosed diabetic patients. Several apoptotic pathways have been implicated in beta cell destruction, including Fas, perforin, and TNF-alpha. Evidence for Fas-mediated lysis of beta cells in the pathogenesis of IDDM in nonobese diabetic (NOD) mice includes: 1) Fas-deficient NOD mice bearing the lpr mutation (NOD-lpr/lpr) fail to develop IDDM; 2) transgenic expression of Fas ligand (FasL) on beta cells in NOD mice may result in accelerated IDDM; and 3) irradiated NOD-lpr/lpr mice are resistant to adoptive transfer of diabetes by cells from NOD mice. However, the interpretation of these results is complicated by the abnormal immune phenotype of NOD-lpr/lpr mice. Here we present novel evidence for the role of Fas/FasL interactions in the progression of NOD diabetes using two newly derived mouse strains. We show that NOD mice heterozygous for the FasL mutation gld, which have reduced functional FasL expression on T cells but no lymphadenopathy, fail to develop IDDM. Further, we show that NOD-lpr/lpr mice bearing the scid mutation (NOD-lpr/lpr-scid/scid), which eliminates the enhanced FasL-mediated lytic activity induced by Fas deficiency, still have delayed onset and reduced incidence of IDDM after adoptive transfer of diabetogenic NOD spleen cells. These results provide evidence that Fas/FasL-mediated programmed cell death plays a significant role in the pathogenesis of autoimmune diabetes.     

Lysophosphatidylcholine-induced apoptosis in H19-7 hippocampal progenitor cells is enhanced by the upregulation of Fas Ligand

Lysophospholipids regulate a wide array of biological processes including apoptosis and neutrophil migration. Fas/Apo-1 and its ligand (FasL) participate in neuronal cell apoptosis causing various neurological diseases. Here, we use hippocampal neuroprogenitor cells to investigate how lysophosphatidylcholine (LPC) induces apoptosis in H19-7 hippocampal progenitor cells via Fas/Fas ligand-mediated apoptotic signaling pathway. Exposed cells with LPC presented on apoptotic morphology, positive TUNEL staining, and DNA fragmentation. We found that the expression of FasL was increased after LPC treatment. Furthermore, LPC-induced H19-7 cell apoptosis was decreased by agonistic anti-FasL antibody. In addition to promotion of caspase cascade activity by LPC, the administration of the caspase inhibitor, DEVD-fmk, prevented H19-7 cell apoptosis. LPC also increased the activation of nuclear factor-κB (NF-κB), which in turn, significantly increased FasL mRNA level. The increase in FasL mRNA level by NF-κB transfection was significantly decreased in the presence of IκB-SR, a super-repressor of IκB. Taken together, these results demonstrate that LPC has the ability to induce apoptosis in H19-7 cells through the upregulation of FasL expression via NF-κB activation.     

Analysis of the type IV secretion system-dependent cell motility of Helicobacter pylori-infected epithelial cells

The pathogenesis of Helicobacter pylori-associated disorders is strongly dependent on a specialized type IV secretion system (T4SS) encoded by the cag pathogenicity island (PAI). Cytotoxin-associated gene A (CagA) is the only known H. pylori protein translocated into the host cell followed by tyrosine phosphorylation through host protein kinases. H. pylori induces cellular processes which are either PAI- or CagA-dependent (e.g., cell motility), PAI-dependent, but CagA-independent (e.g., interleukin-8 release), or PAI- and CagA-independent (e.g., cyclooxygenase-2 release). Here, we investigated H. pylori strains mutated in single PAI genes of the wild type strain Hp26695 and their effects on cell motility. We found 17 gene products out of 27 PAI genes playing a superordinated role and five PAI-encoded proteins exhibiting a clearly critical role in motogenic host cell responses, whereas the remaining five PAI gene products had no significant influence on the motogenic response in reaction to H. pylori infection. This study clearly demonstrated that H. pylori-induced cell motility and invasive growth involve type IV secretion system-dependent signalling as well as translocated and phosphorylated CagA. These findings reveal a deeper insight in to the meaning of the T4SS of H. pylori for host cell motility.