Interaction of CagA with Crk plays an important role in Helicobacter pylori-induced loss of gastric epithelial cell adhesion

CagA protein is a major virulence factor of Helicobacter pylori, which is delivered into gastric epithelial cells and elicits growth factor-like responses. Once within the cells, CagA is tyrosine phosphorylated by Src family kinases and targets host proteins required to induce the cell responses. We show that the phosphorylated CagA binds Crk adaptor proteins (Crk-II, Crk-I, and Crk-L) and that the interaction is important for the CagA-mediated host responses during H. pylori infection. H. pylori-induced scattering of gastric epithelial cells in culture was blocked by overexpression of dominant-negative Crk and by RNA interference-mediated knockdown of endogenous Crk. H. pylori infection of the gastric epithelium induced disruption of E-cadherin/catenin-containing adherens junctions, which was also dependent on CagA/Crk signaling. Furthermore, inhibition of the SoS1/H-Ras/Raf1, C3G/Rap1/B-Raf, or Dock180/Rac1/Wiskott-Aldrich syndrome protein family verprolin homologous protein pathway, all of which are involved downstream of Crk adaptors, greatly diminished the CagA-associated host responses. Thus, CagA targeting of Crk plays a central role in inducing the pleiotropic cell responses to H. pylori infection that cause several gastric diseases, including gastric cancer.     

感染幽门螺杆菌的慢性萎缩性胃炎患者血清hs-CRP水平及其与Cag-A的关系

目的：探讨感染幽门螺杆菌（HP）的慢性萎缩性胃炎（CAG）患者血清超敏C-反应蛋白（hs—CRP）水平及其与HP细胞毒素相关基因CagA的关系。方法：以ELISA法检测CAG组患者血清hsCRP水平和CagA—IgG，并与无临床症状（As）感染者及健康对照者进行比较。结果：CAG组血清hs—CRP平均值（1．30±0．34）mg／L明显高于AS组（0．19±0．03）mg／L和健康对照组（0．11±0．03）mg／L（P均〈0．01），AS组血清hs—CRP平均值显著高于对照组（P〈0．05）；CAG组CagA^＋HP与CagAHP患者之间血清hsCRP水平无明显差异，AS组CagA^＋HP与CagAHP入选者之间血清hsCRP水平亦无明显差异。结论：CAG组与AS组血清hs—CRP水平明显高于对照组。尽管HP感染可导致血清hs～CRP水平升高，但与是否表达CagA无关。     

Impact of CagA status on serum gastrin and pepsinogen I and II concentrations in Japanese children with Helicobacter pylori infection

The study aimed to determine the association between cytotoxin-associated gene product (CagA), serum gastrin and pepsinogen levels in Japanese children infected with Helicobacter pylori. Three hundred children were enrolled in the study. H. pylori infection was assessed using an enzyme-linked immunosorbent assay, and CagA status was assessed using immunoblotting. Serum gastrin and pepsinogen concentrations were measured by radioimmunoassay. H. pylori seroprevalence was 12.3% (37/300) and CagA status was identified in 28/37 H. pylori-seropositive children (75.7%). Serum pepsinogen I and II levels were significantly higher in CagA-seropositive than CagA-seronegative children with H. pylori infection. There was no significant relationship between CagA seropositivity and serum gastrin levels. In conclusion, CagA status has a significant impact on serum pepsinogen levels, possibly through enhanced gastric mucosal inflammation.     

幽门螺杆菌VacA蛋白体外诱导胃上皮AGS细胞内HMGB1的表达

摘要：目的：探讨幽门螺杆菌（Hp）感染的胃上皮AGS细胞内高迁移率族蛋白B1(high mobility group box 1, HMGB1)的表达。 方法：Hp11638（CagA⁺,VacA⁺）和Hp11638突变株（Hp11638M,CagA⁺,VacA^-）的提取液与AGS细胞共同温育后,收集细胞及培养上清液。裂解AGS细胞,western blot分析AGS细胞内HMGB1的表达,ELISA法检测培养上清液中HMGB1的水平。 结果:Hp11638提取液刺激AGS细胞后HMGB1表达量为(123.33±25.2) μg/mL,明显高于Hp11638M提取液刺激后的(46.67±7.23) μg/mL（q=8.49,P<0.01）。Hp11638和Hp11638M提取液刺激的AGS细胞培养上清液中HMGB1的水平分别为（115.59±16.62）和（48.32±6.30） ng/mL,差异有统计学意义（q=12.25,P<0.01）。 结论:在胃炎发生、发展过程中,VacA蛋白是刺激细胞中HMGB1高表达的主要因子。     

Association of CagA-positive infection with Helicobacter pylori antibodies of IgA class

cagA gene, the best known virulence factor of Helicobacter pylori, codes for an immunodominant CagA protein. In this study, CagA antibodies of the IgG class were measured by immunoblot or enzyme immunoassay in subjects with positive H. pylori serology, and the presence of CagA antibodies was compared with that of H. pylori antibodies of IgA and IgG classes. Serum samples were available for a total of 1,481 subjects, including gastroscopied patients with biopsy-verified H. pylori infection, smoking men with a normal or low serum pepsinogen I level indicating atrophic corpus gastritis, and subjects who later developed gastric cancer and their matched controls. CagA antibodies were significantly more prevalent among individuals with elevated H. pylori antibody titres of the IgA class than in those with IgG antibodies only, with the exception of a small subgroup of individuals who later developed gastric cancer. CagA-positive H. pylori strains seem to induce an immune response with a markedly higher frequency of IgA than what is found in inflammation caused by CagA-negative strains. The presence of serum IgA antibodies to H. pylori seems to indicate a higher risk for CagA-positive H. pylori infection and possibly more severe late sequelae of the disease.     

High prevalence of VacA, CagA protein expression and presence of cag pathogenicity island in Japanese Helicobacter pylori isolates

VacA, CagA proteins and cag pathogenicity island (PAI) were reported to be major virulence factors of Helicobacter pylori. By using specific antibodies, the expression of VacA and CagA was evaluated in Japanese isolates, together with vacuolating assay. To characterize the status of not only cagA, but entire cag PAI, H. pylori isolates were evaluated for cagA and 13 other cagPAI genes by Southern blot. VacA and CagA proteins were expressed in 87% and in 90%, respectively. Vacuolating assay was positive in 84% isolates. Most strains had all cag PAI genes and the only 6% were cag PAI deleted despite of retaining cagA gene. The majority of Japanese isolates were positive for VacA, CagA proteins and cag PAI, and the high prevalence of infection with virulence strains may contribute to the characteristics of H. pylori infection in Japan.     

Multistep activation of the Helicobacter pylori effector CagA

Chronic infection with the Gram-negative bacterium Helicobacter pylori is a major risk factor for the development of gastric cancer. Accumulating evidence indicates that the H. pylori virulence determinant cytotoxin-associated gene A (CagA) has a key oncogenic role in the process. Certain biological activities of CagA require its tyrosine phosphorylation by host cell kinases. In this issue of the JCI, Mueller and colleagues report their detailed kinetic and functional analysis of CagA phosphorylation, which indicates that c-Src and c-Abl kinases sequentially phosphorylate CagA. Interestingly, the two phosphorylation events need not occur on the same CagA molecule but are both required for the biological effects of CagA. The results provide a clinically relevant example of how a successful bacterial pathogen has evolved to exploit the tightly coordinated, sequential activity of host cell kinases for virulence factor activation and induction of pathology.     

Frequencies of serum antibodies to Helicobacter pylori CagA and VacA in a Turkish population with various gastroduodenal diseases

Infection with Helicobacter pylori (H. pylori) strains secreting cytotoxin-associated gene A (CagA) and vacuolating cytotoxin A (VacA) proteins is associated with more severe gastroduodenal pathologies. However, this association varies among geographical regions and ethnic groups. We investigated the frequencies of antibodies to CagA and VacA proteins in 131 H. pylori-infected dyspeptic patients [40 duodenal ulcer (DU), 19 gastric ulcer (GU), 28 gastric cancer (GC), and 44 non-ulcer dyspepsia (NUD)] across 30 H. pylori-infected and endoscopically normal asymptomatic subjects (AS). Anti-CagA and anti-VacA antibodies were detected by Western blotting. The positivity rates of anti-CagA and anti-VacA antibodies were higher in patients with DU (92.5 and 75%), GU (89.5 and 84.2%) and GC (96.4 and 85.7%) than patients with NUD (70.5 and 50%) and AS (50 and 23.3%) (p < 0.05). CagA+ VacA+ phenotype was more frequent in patients with DU, GU and GC than patients with NUD and AS (75, 84.2, 85.7 vs. 47.7 and 20%, respectively) (p < 0.01). Our results showed that there is a significantly positive association between the presence of anti-CagA and anti-VacA antibodies and DU, GU and GC in our region.     

诱导胃上皮细胞白细胞介素-8分泌的幽门螺杆菌基因型

目的：分析临床分离的幽门螺杆菌的cag致病岛的差异和不同激素抑制剂对幽门螺杆菌诱导人胃上皮细胞白细胞介素－8（IL－8）分泌的影响。方法：分别用临床分离的不同基因型的cagA^ cagE^ 、cagA^ cagE^-、cagA^-cagE^ 、cagE^-幽门螺杆菌与人胃上皮细胞MGC－803共同培养，IL－8分泌用酶联免疫吸附试验进行检测，比较蛋白激酶A、C、G和蛋白酪氨酸激酶的抑制剂对幽门螺杆菌诱导胃上皮细胞IL－8分泌的影响。结果：cagA^ cagE^ 基因型幽门螺杆菌不能增加胃上皮细胞IL-8的分泌。蛋白激酶A、C、G的抑制剂不能阻断幽门螺杆菌增加胃上皮细胞IL－8的分泌，而蛋白酪氨酸激酶的抑制剂阻断了幽门螺杆菌增加胃上皮细胞IL－8的分泌。结论：cagA^ cagE^ 基因型幽门螺杆菌显著增加了胃上皮细胞IL－8的分泌并且依赖于蛋白酪氨酸激酶的磷酸化。     

Gastric mucosal immunity induced by H. pylori infection

H. pylori infection induces various humoral and cellular immunities in gastric mucosa. Some reports indicate predominant CD4+ cells infiltrate in H. pylori infected gastric mucosa, and these cells express the T helper 1 phenotype. Local humoral immunity is also induced. Gastric plasma cells produce anti-H. pylori antibodies, however, their protective immunity is not enough to eradicate bacteria in human. We found heat shock protein 60 kDa (hsp60) may be closely associated with pathogenesis in MALT lymphoma. IgG1 antibodies to hsp60 were significantly correlated with the antibodies to H. pylori whole cell in patients with MALT lymphoma. CD40-CD40L dependent B cell proliferation was induced by cytokine and/or hsp60 stimulations in those patients. Cytotoxicity of gastric epithelial cells which is associated with host immunity induced by H. pylori infection is still unclear. We found that lymphocytes from patients with peptic ulcer showed cytotoxicity to gastric cell line HGC-27 in vitro. Cytotoxicity was enhanced by cytokine stimulus to T-lymphocytes and by heat stress and/or patients' antibodies treatment of HGC-27 cells. The pathogenicity of H. pylori may involve not only bacterial virulence factor but also host immunity. Studies of mucosal local immunity will help explain the mechanisms of H. pylori induced gastrodoudenal diseases.     

Anti-inflammatory effect of activated protein C in gastric epithelial cells

It has been previously demonstrated that activated protein C (APC) plays an important role in the inhibition of inflammation in the gastric mucosa from patients with Helicobacter pylori infection. However, the role of gastric epithelial cells in the anti-inflammatory activity of APC remains unknown. In the present study, we evaluated the anti-inflammatory activity of APC and the expression of thrombomodulin (TM) and endothelial protein C receptor (EPCR) in gastric epithelial cells. The gastric epithelial cell lines, MKN-1 and AGS, and gastric biopsy samples from patients with and without H. pylori infection were used in the experiments. Polymerase chain reaction showed that gastric epithelial cell lines express EPCR and TM. Flow cytometry analysis also showed EPCR expression in both cells. H. pylori infection significantly increased EPCR expression compared with non-infected cells. Similar results were observed in vivo when samples from patients with and without H. pylori infection were analyzed for EPCR protein expression. Significant TM activity was found on AGS and MKN-1 cells stimulated with LPS from Escherichia coli and VacA antigen. APC was able to significantly inhibit the secretion of MCP-1 and IL-1beta induced by H. pylori homogenate in AGS cells. APC also remarkably suppressed the mRNA expression and secretion of MCP-1 from AGS cells infected with H. pylori. These results demonstrated the expression of components of the protein C pathway on gastric epithelial cells and that APC may play a critical role in the protection against gastric mucosal inflammation.     

Helicobacter pylori is associated with modified lipid profile: impact on Lipoprotein(a)

OBJECTIVES: Helicobacter pylori is a controversial risk factor for atherosclerosis. We investigated whether the bacterium persistent inflammation or the expression of the cytotoxin-associated gene A (CagA) may affect serum lipids as well as Lipoprotein(a). DESIGN AND METHODS: Two hundred-eleven healthy volunteers were evaluated for lipids and Lipoprotein(a). Helicobacter pylori was characterized by Urea Breath Test and IgG-anti-CagA. apo(a) Kringle-IV polymorphism was genotyped. RESULTS: Prevalence of the infection was 72%; 43% of subjects expressed CagA reactivity. Infected subjects showed increased levels of cholesterol, LDL-cholesterol, and cholesterol/HDL-cholesterol atherogenic index. Association with the Helicobacter pylori CagA(-) strains persisted after the adjustment for covariates. Significant difference between infected and uninfected subjects was found in Lipoprotein(a) levels. This difference did not arise from the Kringle-IV genotype. CONCLUSIONS: The infection per se significantly modified serum lipid and Lipoprotein(a) concentrations. CagA does not seem to be a reliable marker of pathogenicity for the atherogenic complications of H. pylori infection.     

幽门螺旋杆菌感染与子痫前期的关系

幽门螺旋杆菌是最常见的消化道致病菌,参与急性胃肠炎、消化性溃疡、胃肠肿瘤等胃肠道疾病的发生发展, 与胃肠外疾病也有密切联系。研究发现,幽门螺旋杆菌感染与子痫前期存在明确的流行病学因果关系,特别是细胞毒素相关基因A(cytotoxin-associated gene A, cagA)阳性的幽门螺旋杆菌菌株诱导产生的抗cagA抗体可与滋养层细胞发生免疫反应导致滋养细胞浸润异常。此外,幽门螺旋杆菌感染引起的炎症反应、脂质代谢异常可造成血管内皮功能异常,导致子痫前期。本文将对幽门螺旋杆菌感染与子痫前期的关系作一综述。     

High seroprevalence of Helicobacter pylori in chronic bronchitis among Chinese population

An increased seroprevalence of Helicobacter pylori (H. pylori), especially high virulent cytotoxin-associated gene-A (CagA) positive strains, has been found in many extragastrointestinal disorders. Moreover, it has been reported that the risk of chronic bronchitis may be increased in H. pylori infected patients. However, until now there are no data regarding the relationship between H. pylori infection and chronic bronchitis among Chinese population. Therefore the aim of the present study was to assess the seroprevalence of H. pylori and in particular of CagA positive virulent strains in patients with chronic bronchitis among Chinese population. We evaluated 46 patients with chronic bronchitis, 48 age- and sex-matched patients with peptic ulcer and 48 healthy control subjects. All enrolled subjects underwent a serologic test for H. pylori IgG and CagA by enzyme linked-immunosorbent assay (ELISA). There was no significant difference in the seropositivity for these parameters between chronic bronchitis and peptic ulcer groups (86.9% vs 89.6% for anti-H. pylori IgG and 67.4% vs 72.9% for anti-H. pylori-CagA IgG). However, these serological parameters were significantly higher in the patients with chronic bronchitis or peptic ulcer than those in control group, who showed 60.4% for anti-H. pylori IgG seropositivity and 20.8% for anti-H. pylori-CagA IgG seropositivity. Among the patients with chronic bronchitis, no significant difference was found in these serological parameters between the current cigarette smokers and never smokers. This is the first report of a high seroprevalence of H. pylori infection in chronic bronchitis among Chinese population.     

CagA+幽门螺杆菌与胃黏膜上皮细胞凋亡的分子研究

目的：观察CagA^ Hp对胃黏膜上皮细胞凋亡的影响，进而探讨CagA^ Hp增加胃癌发生危险性的机制。方法：以TUNEL染色法研究30例CagA^ Hp阳性患者Hp根除前后胃黏膜上皮细胞凋亡的情况；通过免疫组织化学法和RT－PCR检测凋亡相关基因bcl-2和bax的表达。结果：CagA^ Hp阳性患者胃黏膜上皮细胞凋亡指数为13．42％，Hp根除后，胃黏膜上皮细胞凋亡指数降为4．8％，较治疗前明显减少（P＜0．01），而CagA^ Hp仍为阳性患者胃黏膜上皮细胞凋亡指数无明显减少（P＞0．05）；bcl-2蛋白表达阳性的CagA^ Hp阳性患者Hp根除后，胃黏膜上皮细胞bcl-2蛋白表达阳性明显增多（P＜0．01），且胃黏膜上皮细胞bcl-2的mRNA表达明显增强（P＜0．01），而CagA^ Hp仍为阳性患者胃黏膜上皮细胞bcl-2蛋白表达和mRNA表达无明显增强（P＞0．05）；bax蛋白表达阳性的CagA^ Hp阳性患者Hp根除后，胃黏膜上皮细胞bax蛋白表达阳性明显减少（P＜0．01），且胃黏膜上皮细胞bax的mRNA表达明显减少（P＜0．01）；而CagA^ Hp仍为阳性患者胃黏膜上皮细胞bax蛋白表达和mRNA表达无明显减少（P＞0．05）。结论：诱导胃黏膜上皮细胞凋亡是CagA^ Hp参与胃癌发生的机制之一，CagA^ Hp通过下调bcl-2的表达、促进bax的表达诱导胃黏膜上皮细胞凋亡。     

胃癌组织环氧合酶-2表达与幽门螺杆菌cagA+菌株感染的关系

目的探讨胃癌组织H pylori cagA感染对环氧合酶2(COX2)表达的影响。方法采用原位聚合酶链反应(PCR)及免疫组织化学技术检测60例慢性浅表性胃炎6、0例慢性萎缩性胃炎、64例肠上皮化生6、4例不典型增生、64例胃癌组织中cagA基因及COX2蛋白表达情况。结果COX2在胃癌、异型增生的阳性表达率升高,分别为70.3%、64.1%与肠上皮化生、萎缩性胃炎、浅表性胃炎相比有显著性差异(P<0.05)。在H pyloricagA阳性胃癌组、异型增生组COX2表达率明显高于H pyloricagA阴性组,两组之间有显著性差异(P<0.05);COX2阳性表达与患者性别、年龄、肿瘤大小无关,与患者浸润深度、淋巴转移、H pyloricagA感染有关。结论H pylori可通过诱导COX2表达参与胃癌病变,COX2参与胃癌的发生发展,CagA是H pylori的重要致病因素,与COX2表达上调及胃癌的发生密切相关。     

不同毒力亚株幽门螺杆菌感染在特发性血小板减少性紫癜发病中的意义

目的分析幽门螺杆菌（H.pylori）不同毒力亚株感染与特发性血小板减少性紫癜（ITP）发病的相关性。方法应用免疫印记法检测64例ITP患者和59例对照者的血清H.pylori抗体：细胞毒素相关基因A（CagA）、空泡毒素蛋白A（VacA）、尿素酶A（UreA）、尿素酶B（UreB）,并对CagA＋和/或VacA＋阳性患者随机分组,一组抗H.pylori治疗,另一组、CagA-和VacA-以及H.pylori阴性患者组未作抗H.pylori治疗作为对照组,比较治疗前后血小板计数。结果ITP组患者H.pylori阳性率高于对照组,差异有统计学意义（P〈0.05）;ITP组H.pylori阳性患者CagA＋和/或VacA＋型者所占比例高于对照组,差异有统计学意义（P〈0.001）。CagA＋和/或VacA＋阳性患者经H.pylori根治后血小板计数较CagA＋和/或VacA＋阳性未根治组升高明显,P〉0.05有统计学意义。结论ITP患者幽门螺杆菌的感染率高,而且不同毒力亚株感染与其发病相关。     

Genetic diagnosis of H. pylori strains

Helicobacter pylori strains are divided into two broad families, type I and type II, based on whether or not they possess the cag pathogenicity island (cagPAI). It has been suggested that cagPAI is inherited by horizontal transfer from an unknown microorganism, and the genes of cag are thought to be encoded by a putative type IV secretory system. In addition, CagA may be delivered from attached H. pylori into the host crytoplasm by this system and is tyrosine phosphorylated in gastric epithelial cells. The phosphorylated CagA may play a crucial role in promoting the inflammatory responses of gastric mucosa. These findings suggest that type I H. pylori is a pathogenic H. pylori.     

Heterogeneity of cag genotypes and clinical outcome of Helicobacter pylori infection

Helicobacter pylori infecting strains may include colony subtypes with different cytotoxin-associated gene (cag) genotypes. We sought to determine whether the cag heterogeneity of infecting strains is related to the clinical outcome of infection. Gastric biopsies for culture and histologic study were taken from 19 patients infected with cagA-positive strains (6 with duodenal ulcer, 8 with atrophic gastritis, and 5 with nonatrophic gastritis). For each biopsy, DNA was extracted from 10 single colonies and from a sweep of colonies. Polymerase chain reaction (PCR) for cagA and cagE (both located in the right half of cag) and virB11 (located in the left half of cag) was performed. Random amplified polymorphic DNA PCR (RAPD-PCR) and sequencing of glmM PCR product were performed to verify strain identity of colonies with different cag genotypes. In all patients, PCR from sweeps were positive for cagA, showing that all specimens contained cagA-positive H. pylori subtypes. In 11 patients, PCR products from all colonies were positive for cagA, cagE, and virB11, but in 8 patients, PCR products from varying numbers of colonies were negative for 1 or more cag genes. RAPD-PCR and sequencing of glmM PCR product confirmed the strain identities of colonies with different cag genotypes. We detected cag deletions in 6 of 8, 2 of 5, and 0 of 6 patients with atrophic gastritis, nonatrophic gastritis, and duodenal ulcer, respectively (P = .02). In conclusion, changes in cag genotype in single colony isolates from subjects infected with cagA-positive H. pylori strains are more common in atrophic than in nonatrophic gastritis or duodenal ulcer. These findings are consistent with host-induced (acid secretion?) adaptive changes in cag genotype during infection.     

Polymorphonuclear oxidative burst after Helicobacter pylori water extract stimulation is not influenced by the cytotoxic genotype but indicates infection and gastritis grade.

H. pylori-associated gastric mucosal inflammation is characterized by the presence of polymorphonuclear (PMN) leukocyte infiltrate, which is more severe when the infecting strain is cagA positive. After appropriate stimuli, such as bacterial products, PMN release large amounts of oxygen derived free radicals and proteases, to kill the bacterium. H. pylori seems to be particularly resistant to the oxidative machinery of PMN, which can in turn damage the host gastric mucosa. We evaluated peripheral PMN oxidative burst response after stimulation with water extracts from cagA positive (WEcagA+) or negative (WEcagA-) H. pylori strains in infected (n=31) and non-infected patients (n=32) in comparison with healthy controls (n=16); the influence of gastric mucosal inflammatory infiltrate and activity grade on PMN oxidative burst were also assessed. PMN oxidative burst was measured by FACS analysis. H. pylori water extracts were obtained from bacterial culture. H. pylori genotype was determined by means of the polymerase chain reaction. The PMN oxidative burst in H. pylori infected patients was significantly higher than that in H. pylori negative or healthy controls, no differences being found when the results following WEcagA+ and WEcagA- stimulation were compared. The difference in PMN oxidative burst obtained after WEcagA- and E. coli (standard stimulus for PMN oxidative burst) stimulation discriminated H. pylori infected from non-infected patients with a sensitivity of 90% and a specificity of 97%. The grade of PMN oxidative burst correlated with PMN infiltration grade of the gastric mucosa. Our findings allow to conclude that PMN oxidative burst activation by H. pyloriWE is species- but not strain-correlated. PMN priming, probably consequent to the action of soluble mediators released by mononuclear cells, makes PMN hyper-responsive to H. pylori products, thus favoring the release in the gastric mucosa of infected patients of large amounts of oxygen-derived free radicals, which are not enough to eliminate the infection, but may contribute to damaging the gastric mucosa itself. Peripheral PMN oxidative burst response to H. pyloriWE might furthermore be of help in diagnosing H. pylori infection.