Helicobacter pylori stimulates gastric epithelial cell MMP-1 secretion via CagA-dependent and -independent ERK activation

Because the mechanisms of Helicobacter pylori-induced gastric injury are incompletely understood, we examined the hypothesis that H. pylori induces matrix metalloproteinase-1 (MMP-1) secretion, with potential to disrupt gastric stroma. We further tested the role of CagA, an H. pylori virulence factor, in MMP-1 secretion. Co-incubation of AGS cells with Tx30a, an H. pylori strain lacking the cagA virulence gene, stimulated MMP-1 secretion, confirming cagA-independent secretion. Co-incubation with strain 147C (cagA(+)) resulted in CagA translocation into AGS cells and increased MMP-1 secretion relative to Tx30a. Transfection of cells with the recombinant 147C cagA gene also induced MMP-1 secretion, indicating that CagA can independently stimulate MMP-1 secretion. Co-incubation with strain 147A, containing a cagA gene that lacks an EPIYA tyrosine phosphorylation motif, as well as transfection with 147A cagA, yielded an MMP-1 secretion intermediate between no treatment and 147C, indicating that CagA tyrosine phosphorylation regulates cellular signaling in this model system. H. pylori induced activation of the MAP kinase ERK, with CagA-independent (early) and dependent (later) components. MEK inhibitors UO126 and PD98059 inhibited both CagA-independent and -dependent MMP-1 secretion, whereas p38 inhibition enhanced MMP-1 secretion and ERK activation, suggesting p38 negative regulation of MMP-1 and ERK. These data indicate H. pylori effects on host epithelial MMP-1 expression via ERK, with p38 playing a potential regulatory role.     

幽门螺杆菌临床分离株CagA的序列比对及其对胃上皮细胞形态与功能的影响

[背景]细胞毒素相关基因A蛋白(Cytotoxin Associated Gene A Protein,CagA)是幽门螺杆菌(Helicobacter pylori)重要的效应蛋白,CagA的多态性与胃癌的发生发展密切相关.[目的]比较幽门螺杆菌临床分离株的CagA结构差异,探讨不同CagA对胃上皮细胞形态及功能的影...     

Helicobacter pylori picB, a homologue of the Bordetella pertussis toxin secretion protein, is required for induction of IL-8 in gastric epithelial cells

Approximately 60% of Helicobacter pylori strains are cagA ⁺ and this genotype is more frequently associated with duodenal ulcer disease. Although most wild-type cagA ⁺ strains are both cytotoxigenic and induce enhanced Interleukin-8 (IL-8) secretion in gastric epithelial cells, isogenic cagA ⁻ mutants retain full activity in these assays; thus, cagA appears to be a marker of enhanced virulence. Delineation of the nucleotide sequence of a 4 kb region upstream of cagA allowed the identification of 966 bp ( picA ) and 2655 bp ( picB ) open reading frames encoding 36 kDa and 101 kDa polypeptides, respectively. picA and picB constitute an operon in opposite orientation to cagA . The deduced picB product showed significant homology (26% identity and 50% similarity) with the Bordetella pertussis toxin secretion protein (PtlC). Of 55 H. pylori clinical isolates, the picA and picB segment was conserved exclusively in cagA ⁺ strains and present in all isolates from patients with duodenal ulceration, versus 59% of isolates from patients with gastritis alone ( P =0.01). Using gene-replacement techniques, we constructed picA and picB mutant H. pylori strains and demonstrated that the picB gene product is involved in the induction of IL-8 expression in gastric epithelial cells. Further, Northern blot hybridization and RT-PCR data showed that picA and picB are co-transcribed and an insertional mutation in picA ablates picB expression. These studies indicate a role of picA and picB in the induction of an inflammatory response in gastric epithelial cells either directly or by enabling secretion of an unidentified product, and suggest a mechanism for the overrepresentation of strains possessing these genes in patients with peptic ulceration.     

Helicobacter pylori induces gastric epithelial cell invasion in a c-Met and type IV secretion system-dependent manner

Helicobacter pylori interacts with gastric epithelial cells, activating signaling pathways important for carcinogenesis. In this study we examined the role of H. pylori on cell invasion and the molecular mechanisms underlying this process. The relevance of H. pylori cag pathogenicity island-encoded type IV secretion system (T4SS), CagA, and VacA for cell invasion was also investigated. We found that H. pylori induces AGS cell invasion in collagen type I and in Matrigel invasion assays. H. pylori-induced cell invasion requires the direct contact between bacteria and cancer cells. H. pylori-mediated cell invasion was dependent on the activation of the c-Met receptor and on increased MMP-2 and MMP-9 activity. The abrogation of the c-Met receptor using the specific NK4 inhibitor or the silencing of c-Met expression with small interference RNA suppressed both cell invasion and MMP activity. Studies with different H. pylori strains revealed that cell invasion, c-Met tyrosine phosphorylation, and increased MMP-2 and MMP-9 activity were all dependent on the presence of a functional bacterial T4SS, but not on VacA cytotoxicity. Our findings demonstrate that H. pylori strains with a functional T4SS stimulate gastric epithelial cell invasion through a c-Met-dependent signaling pathway that comprises an increase in MMP-2 and MMP-9 activity.     

Helicobacter pylori CagA-mediated IL-8 induction in gastric epithelial cells is cholesterol-dependent and requires the C-terminal tyrosine phosphorylation-containing domain

Upon infection of the gastric epithelial cells, the Helicobacter pylori cytotoxin-associated gene A (CagA) virulence protein is injected into the epithelial cells via the type IV secretion system (TFSS), which is dependent on cholesterol. Translocated CagA is targeted by the membrane-recruited c-Src family kinases in which a tyrosine residue in the Glu-Pro-Ile-Tyr-Ala (EPIYA)-repeat region, which can be phosphorylated, induces cellular responses, including interleukin-8 (IL-8) secretion and hummingbird phenotype formation. In this study, we explored the role of EPIYA-containing C-terminal domain (CTD) in CagA tethering to the membrane lipid rafts and in IL-8 activity. We found that disruption of the lipid rafts reduced the level of CagA translocation/phosphorylation as well as CagA-mediated IL-8 secretion. By CagA truncated mutagenesis, we identified that the CTD, rather than the N-terminal domain, was responsible for CagA tethering to the plasma membrane and association with detergent-resistant membranes, leading to CagA-induced IL-8 promoter activity. Our results suggest that CagA CTD-containing EPIYAs directly interact with cholesterol-rich microdomains that induce efficient IL-8 secretion in the epithelial cells.     

cag+ Helicobacter pylori induce transactivation of the epidermal growth factor receptor in AGS gastric epithelial cells

The gastric pathogen Helicobacter pylori is known to activate epithelial cell signaling pathways that regulate numerous inflammatory response genes. The aim of this study was to elucidate the pathway leading to extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in H. pylori-infected AGS gastric epithelial cells. We find that H. pylori, via activation of the epidermal growth factor (EGF) receptor activates the small GTP-binding protein Ras, which in turn, mediates ERK1/2 phosphorylation. cag+ strains of H. pylori are able to induce greater EGF receptor activation than cag- strains, and studies with isogenic mutants indicate that an intact type IV bacterial secretion system is required for this effect. Blockade of EGF receptor activation using tyrphostin AG1478 prevents H. pylori-mediated Ras activation, inhibits ERK1/2 phosphorylation, and substantially decreases interleukin-8 gene expression and protein production. Investigations into the mechanism of EGF receptor activation, using heparin, a metalloproteinase inhibitor and neutralizing antibodies reveal that H. pylori transactivates the EGF receptor via activation of the endogenous ligand heparin-binding EGF-like growth factor. Transactivation of gastric epithelial cell EGF receptors may be instrumental in regulating both proliferative and inflammatory responses induced by cag+ H. pylori infection.     

Helicobacter pylori lipopolysaccharide effect on the synthesis and secretion of gastric sulfomucin.

The effect of H. pylori lipopolysaccharide on the synthesis and secretion of sulfated mucin in gastric mucosa was investigated using mucosal segments incubated in the presence of [3H]proline, [3H]glucosamine and [35S]Na2SO4. The lipopolysaccharide, while showing no discernible effect on the apomucin synthesis was found to inhibit the process of mucin glycosylation and sulfation, which at 100 micrograms/ml lipopolysaccharide reached the optimal inhibition of 65%. The analysis of mucin secretory responses revealed that the lipopolysaccharide by first 15 min caused a 57% stimulation in sulfomucin secretion followed thereafter by inhibition, which reached maximum of 32% by 45 min. The results suggest that colonization of gastric mucosa by H. pylori may be detrimental to the process of gastric sulfomucin synthesis and secretion.     

Helicobacter pylori CagA transfection of gastric epithelial cells induces interleukin-8

To determine the effect of Helicobacter pylori CagA expression on interleukin-8 (IL-8) induction in AGS cells, cagA and five of its fragments from strains 147A and 147C that vary in the 3' repeat region were cloned into the eukaryotic expression plasmid pSP65SRalpha. IL-8, but not RANTES or IL-Ibeta, levels were increased in AGS cells transfected with 147A-cagA and to a greater extent with 147C-cagA, compared with negative controls. The 5' b fragment from the two strains had similar effects, but the 3' d and e fragments from 147C CagA had greater effects than those from 147A-CagA. When the Western CagA-specific sequence (WSS) of 147C-cagA was replaced with East Asian CagA-specific sequence (ESS) and cloned into pSP65SRalpha as an East/West chimera, there was no significant effect on IL-8 production. Use of specific inhibitors indicates that Src kinase activation, and the mitogen-activated protein (MAP) kinase and NF-kappaB pathways are the major intermediates for CagA effects on IL-8 induction, but the p38 MAP kinase pathway has little effect. These results indicate a direct CagA effect on IL-8 induction by gastric epithelial cells, and indicate signal pathway loci that can be targeted for amelioration.     

Putative effect of Helicobacter pylori and gastritis on gastric acid secretion in cat

Helicobacter pylori may increase or inhibit gastric acid. We studied acid variations and plasma gastrin in cats harboring Helicobacter felis, harboring H. pylori, or free of gastric pathogens with reference to thioperamide (H(3) receptor antagonist) and SR-27417A (PAF receptor antagonist). In cats harboring H. felis, gastric mucosa were histologically normal. After H. felis eradication, pentagastrin-stimulated acid secretion was increased (40%) compared with the situation before eradication. Thioperamide abolished this inhibitory effect of H. felis, whereas SR-27417A did not. Basal and meal-stimulated plasma gastrin levels were not affected by eradication therapy. Acid secretion was inhibited (-80%) in week 3, increased from weeks 5 to 9, and remained constant for up to 42 weeks after H. pylori infection. SR-27417A had no effect on acid secretion before week 8 but inhibited it thereafter, and thioperamide increased it (20%) only before week 7 in those cats. Helicobacter inhibits gastric acid via an H(3) receptor pathway. Inflammatory mediators are thus involved in adaptation to the inhibitory effects of H. pylori on acid secretion.     

Histidine-rich protein Hpn from Helicobacter pylori forms amyloid-like fibrils in vitro and inhibits the proliferation of gastric epithelial AGS cells

Helicobacter pylori causes various gastric diseases, such as gastritis, peptic ulcerations, gastric cancer and mucosa-associated lymphoid tissue lymphoma. Hpn is a histidine-rich protein abundant in this bacterium and forms oligomers in physiologically relevant conditions. In this present study, Hpn oligomers were found to develop amyloid-like fibrils as confirmed by negative stain transition electron microscopy, thioflavin T and Congo red binding assays. The amyloid-like fibrils of Hpn inhibit the proliferation of gastric epithelial AGS cells through cell cycle arrest in the G2/M phase, which may be closely related to the disruption of mitochondrial bioenergetics as reflected by the significant depletion of intracellular ATP levels and the mitochondrial membrane potential. The collective data presented here shed some light on the pathologic mechanisms of H. pylori infections.     

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.     

幽门螺杆菌ureB基因转染胃上皮细胞及其对细胞的作用

研究幽门螺杆菌 (Helicobacterpylori,Hp)ureB基因重组子转染胃上皮细胞后对胃上皮细胞的作用。用PCR方法从Hp标准株NCTC116 37中获取ureB全长基因 ,将其开放读码框架定向克隆入真核表达载体pcDNA3 1;获得的重组子转染SGC 790 1细胞 ,筛选耐潮霉素的细胞克隆 ,用RT PCR方法检测细胞内ureB基因在转录水平的表达 ;分别用荧光染色技术、MTT、流式细胞术检测UreB对细胞表型、增殖、凋亡及细胞周期的影响。UreB阳性表达的细胞 (SureB)胞膜出芽、细胞皱缩 ;用MTT法检测细胞增殖 ,结果表明 ,SureB细胞与SpcDNA3 1细胞比较 (pcDNA3 1转染的细胞 ) ,生长增殖无显著性差异 (P >0 0 5 ) ,流式细胞术检测细胞凋亡结果显示 ,SureB的凋亡率显著高于SpcDNA3 1(P值为 0 0 0 7) ;细胞周期分析显示 ,SureB细胞有S期比率增高、G2 M、G0 G1 期比率下降的趋势。ureB在培养细胞内的表达可促进细胞凋亡     

Helicobacter pylori stimulates epithelial cell migration via CagA-mediated perturbation of host cell signaling

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.     

Helicobacter pylori genes involved in avoidance of mutations induced by 8-oxoguanine

Chromosomal rearrangements and base substitutions contribute to the large intraspecies genetic diversity of Helicobacter pylori. Here we explored the base excision repair pathway for the highly mutagenic 8-oxo-7,8-dihydroguanine (8-oxoG), a ubiquitous form of oxidized guanine. In most organisms, 8-oxoG is removed by a specific DNA glycosylase (Fpg in bacteria or OGG1 in eukaryotes). In the case where replication of the lesion yields an A/8-oxoG base pair, a second DNA glycosylase (MutY) can excise the adenine and thus avoid the fixation of the mutation in the next round of replication. In a genetic screen for H. pylori genes complementing the hypermutator phenotype of an Escherichia coli fpg mutY strain, open reading frame HP0142, a putative MutY coding gene, was isolated. Besides its capacity to complement E. coli mutY strains, HP0142 expression resulted in a strong adenine DNA glycosylase activity in E. coli mutY extracts. Consistently, the purified protein also exhibited such an activity. Inactivation of HP0142 in H. pylori resulted in an increase in spontaneous mutation frequencies. An Mg-dependent AP (abasic site) endonuclease activity, potentially allowing the processing of the abasic site resulting from H. pylori MutY activity, was detected in H. pylori cell extracts. Disruption of HP1526, a putative xth homolog, confirmed that this gene is responsible for the AP endonuclease activity. The lack of evidence for an Fpg/OGG1 functional homolog is also discussed.     

双歧杆菌对肠上皮细胞生长和白介素-8分泌功能的影响

目的探讨双歧杆菌对人肠上皮细胞株HT29生长及其IL-8分泌水平的影响。方法HT29细胞在96孔板上生长24h后分为正常细胞对照组、高剂量双歧杆菌共培养组（细菌终浓度为1×10^10CFU／m1）、低剂量双歧杆菌共培养组（细菌终浓度为1×10^6CFU／ml）、轮状病毒感染对照组,分别加入不同剂量双歧杆菌和感染轮状病毒共培养,继续培养24h,光镜下观察细胞生长状态,MTT比色法检测细胞活性情况,ELISA检测细胞培养上清中IL-8表达水平。结果光镜下观察到双歧杆菌与HT29细胞共培养后细胞形态无明显改变,共培养24h后MTT检测双歧杆菌对HT29细胞增殖和调亡无明显影响,但轮状病毒感染对照组细胞病变脱落,活细胞数量明显减少。共培养6h,其余3组细胞培养上清中IL-8分泌较正常细胞对照组增加（P〈0．05）,高剂量双歧杆菌组增加较低剂量双歧杆菌组差异有显著性（P〈0．05）,但两个剂量组均明显低于轮状病毒感染阳性对照组的IL-8分泌增加水平（P〈0．05）;感染后24h,细胞培养上清中IL-8分泌水平高于正常细胞对照组（P〈0．05）,但高、低剂量双歧杆菌组之间差异无显著性（P〉0．05）,两个剂量组IL-8分泌增加水平均明显低于轮状病毒感染阳性对照组（P〈0．01）。结论两歧双歧杆菌共培养不影响HT29细胞的生长,双歧杆菌能够促进HT29细胞分泌细胞因子IL-8,但明显低于致病微生物刺激引起的细胞因子分泌水平改变,这种促进作用无时间-剂量依赖关系,提示双歧杆菌与肠道内致病微生物对肠道免疫功能的影响不同,双歧杆菌促进肠上皮细胞分泌IL-8可能与其参与的肠道黏膜免疫系统发育成熟相关。