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.     

Virulence gene of H. pylori

H. pylori is a well-recognized pathogen that infects up to 50% of humans in the world. H. pylori lives for decades in the hostile environment of the human stomach. H. pylori is closely associated with histologic gastritis, gastric ulceration, duodenal ulceration, gastric cancer and MALT lymphoma. These various clinical outcomes are considered by 1) different virulence, 2) host response, 3) other environmental factors, and their interactions. Since the whole genome was sequenced in 1997, the virulence genes have been investigated in molecular genetic aspects. The cag pathogenicity island (cagPAI) is a complex of virulence genes, which code approximately 30 proteins. The cagPAI acquired by horizontal transfer and is coding for type 4 secretion machinery system. Via this system, many virulence gene products or other interactive proteins are transferred into the host cells.     

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.     

c-Src and c-Abl kinases control hierarchic phosphorylation and function of the CagA effector protein in Western and East Asian Helicobacter pylori strains

Many bacterial pathogens inject into host cells effector proteins that are substrates for host tyrosine kinases such as Src and Abl family kinases. Phosphorylated effectors eventually subvert host cell signaling, aiding disease development. In the case of the gastric pathogen Helicobacter pylori, which is a major risk factor for the development of gastric cancer, the only known effector protein injected into host cells is the oncoprotein CagA. Here, we followed the hierarchic tyrosine phosphorylation of H. pylori CagA as a model system to study early effector phosphorylation processes. Translocated CagA is phosphorylated on Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs EPIYA-A, EPIYA-B, and EPIYA-C in Western strains of H. pylori and EPIYA-A, EPIYA-B, and EPIYA-D in East Asian strains. We found that c-Src only phosphorylated EPIYA-C and EPIYA-D, whereas c-Abl phosphorylated EPIYA-A, EPIYA-B, EPIYA-C, and EPIYA-D. Further analysis revealed that CagA molecules were phosphorylated on 1 or 2 EPIYA motifs, but never simultaneously on 3 motifs. Furthermore, none of the phosphorylated EPIYA motifs alone was sufficient for inducing AGS cell scattering and elongation. The preferred combination of phosphorylated EPIYA motifs in Western strains was EPIYA-A and EPIYA-C, either across 2 CagA molecules or simultaneously on 1. Our study thus identifies a tightly regulated hierarchic phosphorylation model for CagA starting at EPIYA-C/D, followed by phosphorylation of EPIYA-A or EPIYA-B. These results provide insight for clinical H. pylori typing and clarify the role of phosphorylated bacterial effector proteins in pathogenesis.     

Helicobacter pylori CagA protein can be tyrosine phosphorylated in gastric epithelial cells

Attachment of Helicobacter pylori to gastric epithelial cells induces various cellular responses, including the tyrosine phosphorylation of an unknown 145-kD protein and interleukin 8 production. Here we show that this 145-kD protein is the cagA product of H. pylori, an immunodominant, cytotoxin-associated antigen. Epithelial cells infected with various H. pylori clinical isolates resulted in generation of tyrosine-phosphorylated proteins ranging from 130 to 145 kD in size that were also induced in vitro by mixing host cell lysate with bacterial lysate. When epithelial cells were infected with [(35)S]methionine-labeled H. pylori, a radioactive 145-kD protein was detected in the immunoprecipitates with antiphosphotyrosine antibody or anti-CagA (cytotoxin-associated gene A) antibody. Consistently, the 145-kD protein recognized by the anti-CagA and antiphosphotyrosine antibodies was induced in epithelial cells after infection of wild-type H. pylori but not the cagA::Km mutant. Furthermore, the amino acid sequence of the phosphorylated 145-kD protein induced by H. pylori infection was identical to the H. pylori CagA sequence. These results reveal that the tyrosine-phosphorylated 145-kD protein is H. pylori CagA protein, which may be delivered from attached bacteria into the host cytoplasm. The identification of the tyrosine-phosphorylated protein will thus provide further insights into understanding the precise roles of CagA protein in H. pylori pathogenesis.     

湖南地区幽门螺杆菌CagA基因3’端多态性与胃小二指肠疾病的关系

目的 探讨湖南地区幽门螺杆菌（Helifcobacter Hpylori）细胞毒素相关基因（CagA基因）3＇端可变区序列特征及其与胃十二指肠疾病的关系.方法 本地区有明显上消化道症状患者235例,其中慢性胃炎（CG）57例,胃溃疡（GU）62例,十二指肠溃疡（DU）70例,胃癌（GC）46例.于胃镜检查时用灭菌活检钳取胃窦组织1块,分离培养出H.pylori 89株,用PCR法对上述菌株的CagA基因扩增及测序,并通过生物信息学软件进行多重序列比对和相似性分析.结果 H.pylori培养阳性率为37.9%（89/235）,其中H.pylori CagA阳性者占91.7%（77/84）,GU组、DU组和GC组CagA阳性率高于CG组,其差异有统计学意义（P〈0.05）.77株H.pylori CagA基因3＇端均具有3个EPIYA重复序列,其中第2个EPIYA序列存在3种突变型,占18.2%（14/77）.H.pylori CagA基因3＇端序列特征以东亚型为主,占88.3%（68/77）,东亚型的CagA阳性菌株在GU组、DU组及GC组高于CG组（P〈0.05）.所有东亚型CagA阳性菌株CagA序列特征类似于Yamaoka报道的A型.结论 湖南地区H.pylori CagA阳性菌株以东亚型为主,均具有3个EPIYA重复序列,其中第2个序列存在3种突变型,其与消化性溃疡和胃癌发生有关.     

幽门螺杆菌感染及其毒力因子和胃十二指肠疾病关系的研究

目的 探讨幽门螺杆菌（Hp)感染病人中Hp细胞毒相关蛋白（CagA)、细胞空泡毒素（VacA)抗体在胃十二指肠疾病的比例，并评估不同类型幽门螺杆菌感染对胃窦粘膜炎症程度的影响。方法 应用免疫印迹法测定Hp感染者病人体内的CagA、VacA抗体。结果 73％的Hp感染者病人中出现CagA抗体，61％Hp感染者病人中出现VacA抗体。CagA抗体和VacA抗体在各疾病之间差异无显著性（P＞0．05）。在胃溃疡、十二指肠溃疡和复合性溃疡三类病人中，具CagA和VacA双阳性的比例较CagA和VacA双阴性的比例差异有显著性（P＜0．05）。胃窦萎缩病变在CagA和VacA双阳性的病人中比CagA和VacA双阴性的病人严重（P＜0．05）。结论 具有高毒力因子（CagA和VacA)的Hp是胃肠疾病病人感染的常见菌株，具有CagA和VacA的Hp可能在诱导胃粘膜发展到萎缩病变过程中起重要作用。但不能作为特异性指标来判断胃十二指肠疾病。     

Virulent strains of Helicobacter pylori demonstrate delayed phagocytosis and stimulate homotypic phagosome fusion in macrophages

Helicobacter pylori colonizes the gastric epithelium of approximately 50% of the world's population and plays a causative role in the development of gastric and duodenal ulcers. H. pylori is phagocytosed by mononuclear phagocytes, but the internalized bacteria are not killed and the reasons for this host defense defect are unclear. We now show using immunofluorescence and electron microscopy that H. pylori employs an unusual mechanism to avoid phagocytic killing: delayed entry followed by homotypic phagosome fusion. Unopsonized type I H. pylori bound readily to macrophages and were internalized into actin-rich phagosomes after a lag of approximately 4 min. Although early (10 min) phagosomes contained single bacilli, H. pylori phagosomes coalesced over the next approximately 2 h. The resulting "megasomes" contained multiple viable organisms and were stable for 24 h. Phagosome-phagosome fusion required bacterial protein synthesis and intact host microtubules, and both chloramphenicol and nocodazole increased killing of intracellular H. pylori. Type II strains of H. pylori are less virulent and lack the cag pathogenicity island. In contrast to type I strains, type II H. pylori were rapidly ingested and killed by macrophages and did not stimulate megasome formation. Collectively, our data suggest that megasome formation is an important feature of H. pylori pathogenesis.     

Helicobacter pylori induces beta3GnT5 in human gastric cell lines, modulating expression of the SabA ligand sialyl-Lewis x

Chronic Helicobacter pylori infection is recognized as a cause of gastric cancer. H. pylori adhesion to gastric cells is mediated by bacterial adhesins such as sialic acid-binding adhesin (SabA), which binds the carbohydrate structure sialyl-Lewis x. Sialyl-Lewis x expression in the gastric epithelium is induced during persistent H. pylori infection, suggesting that H. pylori modulates host cell glycosylation patterns for enhanced adhesion. Here, we evaluate changes in the glycosylation-related gene expression profile of a human gastric carcinoma cell line following H. pylori infection. We observed that H. pylori significantly altered expression of 168 of the 1,031 human genes tested by microarray, and the extent of these alterations was associated with the pathogenicity of the H. pylori strain. A highly pathogenic strain altered expression of several genes involved in glycan biosynthesis, in particular that encoding beta3 GlcNAc T5 (beta3GnT5), a GlcNAc transferase essential for the biosynthesis of Lewis antigens. beta3GnT5 induction was specific to infection with highly pathogenic strains of H. pylori carrying a cluster of genes known as the cag pathogenicity island, and was dependent on CagA and CagE. Further, beta3GnT5 overexpression in human gastric carcinoma cell lines led to increased sialyl-Lewis x expression and H. pylori adhesion. This study identifies what we believe to be a novel mechanism by which H. pylori modulates the biosynthesis of the SabA ligand in gastric cells, thereby strengthening the epithelial attachment necessary to achieve successful colonization.     

Relationship of Helicobacter pylori CagA(+) status to gastric juice vitamin C levels

BACKGROUND: To date it is not known whether gastric juice vitamin C levels are influenced by Helicobacter pylori CagA(+) strains. The aim of the present study, therefore, was to study the impact of H. pylori CagA status on gastric juice vitamin C levels. MATERIALS AND METHODS: We studied 30 H. pylori(+) patients, and the results were compared with 10 endoscopically and histologically normal H. pylori(-) subjects (control group) who were similar to the H. pylori(+) group in terms of age and sex. In all patients, gastric juice vitamin C levels were determined and the severity of gastritis was graded on a scale of 0 (absent) to 3 (severe). CagA was determined by immunoblotting the sera from patients against H. pylori antigens. RESULTS: Among 30 H. pylori(+) patients, 20 were CagA(+) and 10 CagA(-). In the entire group of H. pylori(+) patients, the median gastric juice vitamin C levels (mg L-1) were 16.35 (range 3.5-33.6) and were significantly lower (P < 0.001) than in the control group of H. pylori(-) patients [35.5 (23.1-50.2)]. In addition, in the entire group of H. pylori(+) patients there was a highly significant (P < 0.0001) inverse correlation between the gastritis activity score and the gastric juice vitamin C levels. In the group of H. pylori CagA(+) patients, the median levels of gastric juice vitamin C were 13.8 (3.5-31.2) and were significantly lower than the corresponding levels in both the H. pylori CagA(-) group [24.8 (22-33.6), P < 0.01] and the H. pylori(-) control group [35.5 (23.1-50.2), P < 0.001], the last groups being similar. Furthermore, the gastritis activity median score in the H. pylori CagA(+) group [2 (1-3)] was significantly higher (P < 0.05) than in the H. pylori CagA(-) group [1 (1-2)]. CONCLUSION: These data indicate that infection with CagA(+) H. pylori strains significantly lowers the gastric juice vitamin C levels in comparison with CagA(-) H. pylori strains, which might have a significant impact on gastric carcinogenesis.     

cag PAI and gastric carcinogenesis-association with p53 gene mutation

It is widely accepted that carcinogenesis is a multistep process in which regulation of both cell proliferation and apoptosis is disturbed. p53, which is considered the cellular gatekeeper for growth and division, induces apoptosis. Helicobacter pylori(Hp) infection is an accepted risk factor for the development of gastric cancer, but not all infected individuals develop gastric cancer. Because CagA+ Hp induces increased cell proliferation, the CagA+ strain is believed to play an important role in the pathogenesis of gastric cancer. We have reported that p53 alteration were more frequently found in the CagA+ Hp infection in gastric cancer patients. In this chapter, we summarized recent findings of the relation among p53, CagA and cag PAI.     

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.     

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.     

Virulence factors of Helicobacter pylori responsible for gastric diseases in Mongolian gerbil

Helicobacter pylori infection induces various gastroduodenal diseases. We examined the role of two genes, vacA and cagE, in the gastric pathogenesis induced by H. pylori using a long-term (62 wk) animal model. Reportedly, both genes are associated with the virulence of H. pylori: vacA encodes vacuolating cytotoxin, and cagE, with other genes in the cag pathogenicity islands, encodes a type IV secretion system. Mongolian gerbils were challenged in this study by a wild-type TN2 strain and its isogenic mutants of cagE or vacA. The wild-type and vacA mutants induced severe gastritis, whereas cagE mutants induced far milder changes. Gastric ulcer was induced at the highest rate (22/23) by the wild-type TN2, followed by the vacA mutant (19/28). No ulcer was found in the gerbils infected with the cagE mutant (0/27) or in controls (0/27). Intestinal metaplasia was also found in the gerbils infected with the wild-type (14/23) or vacA mutant (15/28). Gastric cancer developed in one gerbil with wild-type infection and in one with vacA mutant infection. In conclusion, the knocking out of the cagE gene deprived wild-type H. pylori of the pathogenicity for gastritis and gastric ulcer, suggesting that the secretion system encoded by cag pathogenicity island genes plays an essential role.     

Helicobacter pylori strain-specific differences in genetic content, identified by microarray, influence host inflammatory responses

Helicobacter pylori enhances the risk for ulcer disease and gastric cancer, yet only a minority of H. pylori-colonized individuals develop disease. We examined the ability of two H. pylori isolates to induce differential host responses in vivo or in vitro, and then used an H. pylori whole genome microarray to identify bacterial determinants related to pathogenesis. Gastric ulcer strain B128 induced more severe gastritis, proliferation, and apoptosis in gerbil mucosa than did duodenal ulcer strain G1.1, and gastric ulceration and atrophy occurred only in B128+ gerbils. In vitro, gerbil-passaged B128 derivatives significantly increased IL-8 secretion and apoptosis compared with G1.1 strains. DNA hybridization to the microarray identified several strain-specific differences in gene composition including a large deletion of the cag pathogenicity island in strain G1.1. Partial and complete disruption of the cag island in strain B128 attenuated induction of IL-8 in vitro and significantly decreased gastric inflammation in vivo. These results indicate that the ability of H. pylori to regulate epithelial cell responses related to inflammation depends on the presence of an intact cag pathogenicity island. Use of an H pylori whole genome microarray is an effective method to identify differences in gene content between H. pylori strains that induce distinct pathological outcomes in a rodent model of H. pylori infection.     

Association between Helicobacter pylori cytotoxic type I CagA-positive strains and migraine with aura

Recent studies have suggested an association between Helicobacter pylori infection and migraine. However, various strains of the bacterium are present, some endowed with greater pathogenicity. In particular, H. pylori type I CagA-positive strains induce a higher release of proinflammatory substances by the gastric mucosa that could trigger systemic vasospasms. The aim of the present study was to assess the prevalence of H. pylori CagA-positive strains in subjects with migraine. One hundred and seventy-five patients affected by migraine (49 with aura, 126 without aura) were consecutively enrolled and matched for sex, age, social background and geographical origin with 152 controls. Helicobacter pylori infection was assessed through 13C-urea breath test. Specific serological IgG against CagA were detected through ELISA. The prevalence of H. pylori infection was similar in migraine patients and in controls (40% vs. 39%, respectively). Among migraine patients, prevalence of infection was not related to presence or absence of aura (45% vs. 37%, respectively). However, among infected subjects, a significantly higher prevalence of CagA-positive strains was observed in patients affected by migraine with aura when compared with those affected by migraine without aura (41% vs. 19%, P < 0.01) and with controls (41% vs. 17%, P < 0.01). CagA-positive H. pylori strains were found to be strongly associated with migraine with aura. A higher inflammatory response of the gastric mucosa to more virulent strains could release substances that may act as triggers of vasospasm in peculiar cerebral arterial districts, probably implicated in the 'aura' phenomenon.     

Helicobacter pylori-induced apoptosis in T cells is mediated by the mitochondrial pathway independent of death receptors

BACKGROUND: Chronic infection with Helicobacter pylori is related to the pathogenesis of the noncardia carcinoma of the stomach. In this study we investigated the mechanisms of H. pylori-induced apoptosis in T lymphocytes, which could explain a mechanism of immune evasion facilitating chronic inflammation of the mucosa and gastric carcinogenesis. MATERIALS AND METHODS: The supernatant of H. pylori culture was used to study the mechanism of apoptosis induction in human leukaemia T cell lines Jurkat and CEM and in primary T cells. The cytotoxin associated gene A (CagA) and vacuolating cytotoxin A (Vac A) positive bacterial strain H. pylori 60190 (CagA(+), VacA(+)) and as a control the less toxic H. pylori strain Tx30a (CagA(-), VacA(-)) were used to produce the supernatant. Cell death was determined by DNA fragmentation and protein expression by Western blot. RESULTS: H. pylori 60190-induced apoptosis was neither blocked by inhibition of the death ligands TRAIL (TNF-related apoptosis-inducing ligand), CD95L/FasL and TNF-alpha (tumour necrosis factor-a) in wild type Jurkat cells nor in FADD(def) (Fas-associated death domain protein) and caspase-8(def) subclones of the Jurkat cell line. Yet, the pancaspase inhibitor zVAD-fmk could inhibit up to 90% of H. pylori-induced apoptosis. Stable transfection of Jurkat wild type cells with Bcl-x(L and) Bcl-2 resulted in marked reduction of H. pylori-induced apoptosis, showing that the mitochondrial pathway is the key regulator. This is supported by the finding that surviving primary human lymphocytes upregulate Bcl-2 when exposed to H. pylori supernatant. CONCLUSIONS: H. pylori-induced apoptosis of T cells is mediated by the mitochondrial pathway and could create a local environment that facilitates life-long infection by immune evasion.     

幽门螺杆菌血清分型与上消化道疾病的关系

目的 探讨幽门螺杆菌(helicobacter pylori,Hp or H.pylori)分型与消化道不同疾病的关系。方法 入选198例Hp阳性的胃镜检查患者，采用免疫印迹法进行Hp的血清学分型，并取胃窦黏膜经HE染色观察胃窦黏膜病理组织学变化。结果 198例患者中检出HpI型菌株173例(87．4％)，Ⅱ型菌株25例(12．6％)。I型较II型Hp感染者胃镜下消化性溃疡、胃癌的比例更高，P=0.012；与胃炎组比较，十二指肠球部溃疡组、胃癌组的I型感染者更高（P值分别为0.026、0.048），而与胃溃疡组无显著差别(P=0.125)。病理组织学改变I型较II型Hp感染者的结果更为严重（P=0.038）。结论 临床上消化道疾病患者Hp感染以I型菌株最为多见。Hp感染的分型诊断有助于对胃、十二指肠疾病类型及病情的判断，I型菌株感染者需要更为积极的治疗。     

消化性溃疡患者CagA-Hp感染与血清IL-8、IL-10的相关性研究

目的：探讨消化性溃疡（PU）患者伴有CagA-Hp感染与其血清IL-8、IL-10之间的关系。方法：取H.pylori阳性PU患者100例，其中CagA-Hp抗体阳性患者50例，CagA-Hp抗体阴性患者50例，采用ELISA法分别测定其血清IL-8和IL-10水平。结果：消化性溃疡患者中CagA-Hp抗体阳性组血清IL-8和IL-10显著高于CagA-Hp抗体阴性组。CagA-HpIgG抗体阳性患者中，胃溃疡与十二指肠球溃疡患者血清中IL-8和IL-10水平与溃疡发生的部位无关。