幽门螺杆菌cagA基因3''''端可变区和CagA蛋白EPIYA基序研究进展

细胞毒素相关基因A（cagA）编码CagA蛋白，是研究最多的幽门螺杆菌（H．pylori）基因之一。CagA蛋白经由Ⅳ型分泌系统进入Hpylori黏附的胃上皮细胞，其C-端发生酪氨酸磷酸化，磷酸化的CagA可引起细胞骨架重排、诱导细胞发生形态学变化、增强细胞动力，造成细胞异常运动和增殖．最终导致癌变。因此cagA阳性H．pylori菌株较cagA阴性菌株毒力更强。cagA基因3’端可变区重复序列数目的差异造成了cogA基因结构的多态性以及CagA蛋白大小、功能和细菌毒力（致病性）的差异。因此，cagA基因3’端可变区及其蛋白的EPIYA基序与H．pylori感染临床结果的相关性，成为目前H.pylori研究的热点之一。     

CagA蛋白序列可变区多态性分析

背景:幽门螺杆菌(H.pylori)与胃十二指肠疾病关系密切。由于菌株之间CagAEPIYA基序数目及其间隔的氨基酸序列的不同,导致H.pylori主要毒力因子CagA蛋白的多态性和毒力差异。目前关于CagA蛋白多态性的研究缺乏系统性。目的:系统了解H.pyloriCagA蛋白序列多态性及其特征。方法:采用生物信息学软件序列比对分析与统计学软件数据加工整理技术相结合的方法,对NCBI、swiss_prot/tremble、DDBJ三大蛋白数据库CagA蛋白的97条全长序列和442条3’端部分序列进行多态性分析。结果:CagA蛋白氨基酸序列长短不等,主要是由可变区的变化引起的,可变区平均大小为(114.2±24.9)个氨基酸。539株H.pylori菌株CagA蛋白可变区EPIYA有14种突变型,占7.8%;EPIYA基序平均重复(3.3±0.7)次,最少1次,最多7次。两个EPIYA基序间的间隔序列主要有7种,其中R3C和R4C中的“FPLKRHDKVDELIKVG”以及R4C中的“TIDDLGGP”是西方株特征序列,R3D中的“KIASAGKGVGGFSGAG”和R4D中的“FPLRRSAAVNDLSKVG”、“TIDFDEAN”是东亚株特征序列。EPIYA及其间隔序列的不同组合构成了CagA可变区的17种不同类型。东亚株EPIYA基序重复次数显著少于西方株,而EPIYA-A、EPIYA-B位点数显著多于西方株。结论:CagA蛋白可变区呈现明显多态性,但有其内在规律;进一步研究CagA蛋白多态性与细菌毒力的关系可能揭示出更多H.pylori致病的分子生物学机制。     

幽门螺杆菌CagA C-端功能域特征及其生物学功能研究

目的　克隆源自临床分离的幽门螺杆菌 (Hp)菌株CagADNA序列 ,比较其C 端氨基酸序列与国外菌株的差异 ,并分析其磷酸化能力和转染胃上皮细胞后的生物学功能。方法　从 39株临床分离的HpDNA基因组中用PCR方法扩增cagAC 端DNA ,经琼脂糖凝胶电泳后割胶回收DNA片段进行测序。分别构建cagA的原核细胞和真核细胞表达载体并进行CagAC 端氨基酸磷酸化能力测定。 结果　有 38株菌株检测到CagADNA片段 ,CagA阳性率为 97.4 % (38/ 39)。测序后发现 ,克隆的 38株阳性菌株CagA蛋白均仅含 1个重复序列和 2～ 4个串联的谷氨酸 脯氨酸 异亮氨酸 酪氨酸 丙氨酸(EPIYA)序列 ,且在重复序列的D2区氨基酸序列均为天冬氨酸 苯丙氨酸 天冬氨酸 (DFD)。来自胃癌和非胃癌患者的CagA蛋白串联的EPIYA序列数目差异无显著性。用原核细胞表达的CagA蛋白进行体外磷酸化试验 ,重复序列中的酪氨酸能被磷酸化 ,其余的在EPIYA中的酪氨酸也能被磷酸化 ,但转染的AGS细胞株中仅重复序列中的酪氨酸能被磷酸化。在转染AGS细胞株后少数细胞 (<10 % )因骨架重构而出现典型的“蜂鸟”样改变 ,与直接用Hp感染AGS细胞时类似 ,且对转染的AGS细胞再用Hp感染后 ,其“蜂鸟”样细胞的百分比也无明显增加。结论　源自国人的HpCagA蛋白结构不同于欧美国家     

胃癌相关幽门螺杆菌毒力和粘附基因进化分析

目的分析幽门螺杆菌(Hp)细胞毒素相关基因羧基末端(cagA 3')可变区和血型抗原结合粘附基因(babA)序列变异及进化特征,探寻致癌相关Hp亚群。方法对150株Hp菌株采用聚合酶链反应(PCR)方法扩增cagA、vacA和babA基因并进行毒力基因分型,比较毒力基因型在胃炎(91株)组和胃癌组(59株)中的分布差异;分别对Hp cagA 3'可变区和babA~+ PCR产物进行一代测序并构建系统进化树。结果在150株Hp菌株中,胃癌组cagA~+基因型为100.00%,胃炎组为93.41%,且大部分(92.14%)为携带东亚型CagA EPIYA-ABD基序的菌株;胃癌组中vacA s1/m1、vacA s1/m2和babA~+基因型Hp菌株分别占44.07%、55.93%和98.31%,胃炎组分别占48.35%、50.55%和98.90%,差异无统计学意义(P均0.05)。基于Hp cagA 3'可变区及babA基因序列构建Neighbour-Joining系统进化树,在cagA 3'Cluster I亚群和babA Cluster I亚群中胃癌组Hp菌株,分别占84.48%和80.36%,胃炎组分别为29.27%和39.47%,差异均有统计学意义(P均0.05)。结论胃癌相关Hp菌株分别在cagA 3'可变区和babA序列构建的系统进化树上明显聚集成亚群,具有更相似的遗传进化特征。     

威海地区幽门螺杆菌cagA基因3′端多态性分析

目的研究威海地区幽门螺杆菌（Helicobacter pylori,Hp）临床分离株cagA基因的3′端多态性。方法采用PCR法扩增Hp临床分离株的cagA基因3′端并测序,用DNAStar 5.0软件对其进行编码氨基酸序列的比对分析。结果 63株Hp临床分离株均cagA阳性（cagA＋）,其中93.7%（59/63）为东亚型（EPIYYA-ABD）,6.3%（4/63）为西方型（EPIYYA-ABC）。3.4%（2/59）的东亚型菌株EPIYA-B突变为ESIYA-B,4株西方型型均突变为EPIYT-B。胃癌患者分离株中88.9%（16/18）为东亚型,11.1%（2/18）为西方型;非胃癌分离株中95.6%（43/45）为东亚型,4.4%（2/45）为西方型。2株分离自胃癌患者的西方型Hp EPIYA-A后的第一个氨基酸残基由赖氨酸（K）突变为谷氨酸（E）。结论威海地区Hp cagA基因以东亚型为主,但东、西方亚型Hp的致病性无显著差别,两型都存在cagA基因3′端编码氨基酸序列的改变。     

幽门螺杆菌EPIYA多态性与胃癌关系的研究进展

胃癌是世界第二位致死性肿瘤。大量研究表明,胃癌的发生与幽门螺杆菌(Hp)合成分泌的CagA蛋白密切相关。CagA蛋白羧基末端EPIYA重复序列是酪氨酸磷酸化的主要位点,EPIYA的不同基序片段磷酸化后会引起细胞信号转导通路改变,进而影响细胞有丝分裂,引起细胞形态发生改变,最终导致胃黏膜细胞癌变。因此EPIYA基序多态性可能与胃癌发生密切相关,但目前有关这一关系的前瞻性流行病学研究结果存在争议。此文从EPIYA结构的多态性出发,对比不同方法、不同地域的研究,就EPIYA多态性与胃癌发生的关系作一综述。     

不同地区幽门螺杆菌cagA基因羧基端可变区及其蛋白序列差异分析

目的:比较不同地区H pylori cagA 3'端可变区序列差异及序列的地域特征,分析差异与疾病的相关性.方法:选择本实验室所收集的20例CagA 的PCR产物进行测序,并通过ExPASy-Translation软件推测其氨基酸序列,搜索并收集GenBank中已公布的不同地区3-6个H pyloricagA 3'端可变区序列及其氨基酸序列进行比较分析.结果:cagA蛋白的氨基酸序列可分为具有明显地域特征的东亚型和西方型两类;部分东亚型菌株表现出西方型变异.87.4%的菌株具有3个串联排列的EPIYA基序,8.2%的菌株具有4个或以LEPIYA基序.新发现1条具有区域特征的含有3个氨基酸的序列.cagA蛋白氨基酸序列中的EPIYA数目与临床结果没有相关性.结论:Hpylori cagA基因及其cagA蛋白序列具有明显多态性,可以根据Hpylori cagA 3'端可变区的主要序列差异进行地域分型,cagA 3'端可变区的EPIYA基序的数目与临床结果没有相关性.     

镇江地区H pylori的cagA基因及其3'可变区结构的变化

目的:评估镇江地区H pylori临床株的cagA基因阳性率、了解菌株CagA蛋白的可能分型、其羧端可变区EPIYA的数目以及与临床结果之间有无关联.方法:培养分离来自临床胃十二指肠疾病患者的H pylori,提取基因组DNA,通过PCR方法检测H pylori cagA基因的状况.随机选择不同病种的cagA基因阳性(cagA )的PCR产物,通过转化质粒,进行cagA PCR产物测序,通过ExPASY-Tranlation软件获得cagA基因相应的CagA蛋白的氨基酸序列.国际标准株NCTC11637的cagA基因以及CagA蛋白序列通过搜索NCBI(www.ncbi.nlm.nih.gov)数据库获得.结果:60株H pylori临床株中,56例为cagA ,阳性率达93.3%.20例测序的结果表明,CagA蛋白结构可分为2种类型,19株东亚型和1株西方型.所有19株东亚型均为Yamaoka分型的A型,所有20例菌株的EPIYA的数目均为3个,与临床结果无关联.结论:cagA基因阳性率在不同病种之间无差异.镇江地区H pylori临床株的CagA蛋白的一级结构,与日本、韩国菌株一样,主要为东亚型,其CagA蛋白羧端可变区EPIYA数目均为3个,与临床结果无关联.     

用噬菌体展示技术筛选幽门螺杆菌细胞毒素相关蛋白基因结合蛋白的研究

目的筛选幽门螺杆菌细胞毒素相关蛋白（CagA）基因结合蛋白，探索CagA致病机理。方法应用噬菌体展示技术，以幽门螺杆菌CagA基因片段作为固相筛选分子，对噬菌体人胃细胞cDNA文库进行4轮“吸附-洗脱-扩增”富集，噬斑裂解液PCR扩增后，进行DNA序列分析和同源性生物信息学搜索，确定编码结合蛋白的种类。结果噬菌体经富集后，筛选出36个阳性克隆，构建了克隆载体。序列测定后经过同源性搜索，确定幽门螺杆菌CagA基因共编码核纤层蛋白B1和胰岛素样生长因子结合蛋白2两种已知蛋白。结论用噬菌体人胃cDNA文库筛选得到幽门螺杆菌CagA基因DNA结合蛋白，为研究CagA致病机理，幽门螺杆菌疫苗的研制和治疗等提供依据。     

镇江地区H pylori的cagA基因及其3′可变区结构的变化

目的:评估镇江地区H pylori临床株的cagA基因阳性率、了解菌株CagA蛋白的可能分型、其羧端可变区EPIYA的数目以及与临床结果之间有无关联.方法:培养分离来自临床胃十二指肠疾病患者的H pvlori,提取基因组DNA,通过PCR方法检测H pylori cagA基因的状况.随机选择不同病种的cagA基因阳性(cagA~ )的PCR产物.通过转化质粒,进行cagA~ PCR产物测序,通过ExPASY-Tranlation软件获得cagA基因相应的CagA蛋白的氨基酸序列.国际标准株NCTC11637的cagA基因以及CagA蛋白序列通过搜索NCBI(www.ncbi.nlm.nih.gov)数据库获得.结果:60株H pylori临床株中,56例为cagA~ ,阳性率达93.3%.20例测序的结果表明,CagA蛋白结构可分为2种类型,19株东亚型和1株西方型.所有19株东亚型均为Yamaoka分型的A型,所有20例菌株的EPIYA的数目均为3个,与临床结果无关联.结论:cagA基因阳性率在不同病种之间无差异.镇江地区H pylori临床株的CagA蛋白的一级结构,与日本、韩国菌株一样,主要为东亚型,其CagA蛋白羧端可变区EPIYA数目均为3个,与临床结果无关联.     

Analysis of 3'-end variable region of the cagA gene in Helicobacter pylori isolated from Iranian population

Background and Aims:  The 3' region of the cagA gene, the most well-known virulence factor of Helicobacter pylori , contains Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs. Four segments flanking the EPIYA motifs, EPIYA-A, -B, -C, or -D, were reported to play important roles in H. pylori -related gastroduodenal pathogenesis. The aim was to determine the roles of EPIYA segments in gastroduodenal pathogenesis in an Iranian population.
Methods:  A total of 92 cagA -positive Iranian strains isolated from dyspepsia patients with non-ulcer dyspepsia ( n  = 77), peptic ulcer ( n  = 11) and gastric cancer ( n  = 4) were studied. The EPIYA motif genotyping was determined by polymerase chain reaction and sequencing.
Results:  A total of 86 (93.5%) strains had three copies of EPIYA (ABC type), three (3.3%) had four copies (ABCC type) and three (3.3%) had two copies (AB type). The alignment of the deduced protein sequences confirmed that there were no East Asian type EPIYA-D sequences (EPIYATIDFDEANQAG) in Iranian strains. When the prevalence of strains with multiple EPIYA-C segments in Iran was compared with previously published data, it was much lower than that in Colombia and Italy, but was higher than that of Iraq, and the patterns were parallel to the incidence of gastric cancer in these countries.
Conclusion:  The structure of the 3' region of the cagA gene in Iranian strains was Western type. Although we could not find differences between EPIYA types and clinical outcomes, low prevalence of strains with multiple EPIYA-C segments might be reasons for low incidence of gastric cancer in Iran.     

Characterization of CagA variable region of Helicobacter pylori isolates from Chinese patients

AIM: To characterize the CagA variable region of Helicobacter pylori isolates from Chinese patients. METHODS: DNA fragments in CagA variable region were amplified and sequenced respectively from genomic DNA of 19 isolates from patients with gastric cancer and 20 isolates from patients with chronic gastritis. The tendency of phosphorylation in tyrosine(s) of CagA proteins was evaluated subsequently by phosphorylation assay in vivo and in vitro respectively. RESULTS: About 97.44% (38/39) H pylori isolates possessed CagA gene. CagA+ strains contained 2-4 tandem five-amino-acid motifs EPIYA but only one EPIYA had repeated sequence in CagA variable region in different isolates. There was no significant difference between the number of EPIYA motifs in H pylori from patients with different diseases. However, only tyrosine site in EPIYA within repeated sequence could be phosphorylated by AGS cells in vivo although all tyrosine sites in EPIYA could be phosphorylated in vitro. CONCLUSION: CagA in Chinese has no functional difference in perturbing cellular signal pathway among different H pylori isolates.     

Biological activity of the Helicobacter pylori virulence factor CagA is determined by variation in the tyrosine phosphorylation sites

Helicobacter pylori is a causative agent of gastritis and peptic ulcer. cagA(+) H. pylori strains are more virulent than cagA(-) strains and are associated with gastric carcinoma. The cagA gene product, CagA, is injected by the bacterium into gastric epithelial cells and subsequently undergoes tyrosine phosphorylation. The phosphorylated CagA specifically binds SHP-2 phosphatase, activates the phosphatase activity, and thereby induces morphological transformation of cells. CagA proteins of most Western H. pylori isolates have a 34-amino acid sequence that variably repeats among different strains. Here, we show that the repeat sequence contains a tyrosine phosphorylation site. CagA proteins having more repeats were found to undergo greater tyrosine phosphorylation, to exhibit increased SHP-2 binding, and to induce greater morphological changes. In contrast, predominant CagA proteins specified by H. pylori strains isolated in East Asia, where gastric carcinoma is prevalent, had a distinct tyrosine phosphorylation sequence at the region corresponding to the repeat sequence of Western CagA. This East Asian-specific sequence conferred stronger SHP-2 binding and morphologically transforming activities to Western CagA. Finally, a critical amino acid residue that determines SHP-2 binding activity among different CagA proteins was identified. Our results indicate that the potential of individual CagA to perturb host-cell functions is determined by the degree of SHP-2 binding activity, which depends in turn on the number and sequences of tyrosine phosphorylation sites. The presence of distinctly structured CagA proteins in Western and East Asian H. pylori isolates may underlie the strikingly different incidences of gastric carcinoma in these two geographic areas.     

Association between diversity in the Src homology 2 domain--containing tyrosine phosphatase binding site of Helicobacter pylori CagA protein and gastric atrophy and cancer

We investigated the relationship between the diversity of Helicobacter pylori CagA protein and clinical outcome. The cagA gene was sequenced in 115 clinical isolates. The binding affinity of CagA to Src homology 2 domain-containing tyrosine phosphatase (SHP-2) was examined by in vitro infection. Two major CagA subtypes were observed--the East Asian and the Western type. The grades of inflammation, activity of gastritis, and atrophy were significantly higher in patients with gastritis infected with the East Asian CagA-positive strain than in patients with gastritis infected with cagA-negative or Western CagA-positive strains. All strains isolated from patients with gastric cancer were East Asian CagA positive. East Asian CagA exhibited stronger SHP-2-binding activity than did Western CagA. These findings suggest that infection with East Asian CagA-positive H. pylori is associated with atrophic gastritis and gastric cancer and that persistent active inflammation induced by the East Asian CagA-positive strain may play a role in the pathogenesis of disease.     

Influence of EPIYA-repeat polymorphism on the phosphorylation-dependent biological activity of Helicobacter pylori CagA

BACKGROUND & AIMS: Helicobacter pylori CagA-positive strain is associated with gastric adenocarcinoma. CagA is delivered into gastric epithelial cells, where it undergoes tyrosine phosphorylation at the EPIYA sites by Src family kinases (SFKs). Owing to homologous recombination within the 3'-region of the cagA gene, 4 distinct EPIYA sites, each of which is defined by surrounding sequences, are variably assembled in both number and order among CagA proteins from different clinical H pylori isolates. Tyrosine-phosphorylated CagA specifically binds and deregulates SHP-2 via the Western CagA-specific EPIYA-C or East Asian CagA-specific EPIYA-D site, and C-terminal Src kinase (Csk) via the EPIYA-A or EPIYA-B site. Here we investigated the influence of EPIYA-repeat polymorphism on the CagA activity. METHODS: A series of EPIYA-repeat variants of CagA were expressed in AGS gastric epithelial cells and the ability of individual CagA to bind SHP-2 or Csk was determined by the sequential immunoprecipitation and immunoblotting method. RESULTS: CagA proteins carrying multiple EPIYA-C or EPIYA-D sites bound and deregulated SHP-2 more strongly than those having a single EPIYA-C or EPIYA-D. Furthermore, the ability of CagA to bind Csk was correlated with the number of EPIYA-A and EPIYA-B sites. Because Csk inhibits SFK, CagA with greater Csk-binding activity more strongly inhibited Src-dependent CagA phosphorylation and more effectively attenuated induction of cell elongation caused by CagA-SHP-2 interaction. CONCLUSIONS: EPIYA-repeat polymorphism of CagA greatly influences the magnitude and duration of phosphorylation-dependent CagA activity, which may determine the potential of individual CagA as a bacterial virulence factor that directs gastric carcinogenesis.     

Relationship between the cagA 3' repeat region of Helicobacter pylori, gastric histology, and susceptibility to low pH.

BACKGROUND & AIMS: The variation in size of Helicobacter pylori CagA is related to repeat sequences in the 3' region of the cagA gene. We investigated whether structural subtypes of the cagA 3' region are associated with presentation of the infection or to susceptibility to acid. METHODS: We examined 319 cagA-positive H. pylori isolates: 84 isolates from Bogota, Colombia; 83 from Houston, Texas; 24 from Siena, Italy; and 128 from Seoul, Korea. The cagA 3' region was amplified by polymerase chain reaction. Gastric histology and susceptibility to pH 3 were evaluated in relation to the number of cagA repeat regions. RESULTS: Strains with more than three repeat regions were associated with significantly higher scores for gastric mucosal atrophy and intestinal metaplasia than those with fewer repeat regions. H. pylori strains with three repeat regions were also significantly more susceptible to pH 3 than isolates with fewer repeat regions. CONCLUSIONS: H. pylori strains with more than three repeat regions in the 3' region of the cagA gene are associated with enhanced histological injury and with reduced survival in acidic conditions. It is hypothesized that these variants arise within the stomach.     

Construction of prokaryotic expression system of 2 148-bp fragment from cagA gene and detection of cagA gene, CagA protein in Helicobacter pylori isolates and its antibody in sera of patients

AIM: To construct a prokaryotic expression system of a Helicobacter pylori (H pylori) cagA gene fragment and establish enzyme-linked immunosorbent assays (ELISA) for detecting CagA and its antibody, so as to understand the manner in which the infection of CagA-expressing H pylori (CagA(+) H pylori) isolates cause diseases. METHODS: H pylori strains in gastric biopsy specimens from 156 patients with positive results in rapid urease test were isolated. PCR was used to detect the frequency of cagA gene in the 109 H pylori isolates and to amplify a 2 148-bp fragment (cagA1) of cagA gene from a clinical strain Y06. A prokaryotic expression system of cagA1 gene was constructed, and the expression of the target recombinant protein (rCagA1) was examined by SDS-PAGE. Western blotting and immunodiffusion assay were employed to determine the immunoreactivity and antigenicity of rCagA1, respectively. Two ELISAs were established to detect CagA expression in 109 H pylori isolates and the presence of CagA antibody in the corresponding patients' sera, and the correlations between infection with CagA(+) H pylori and gastritis as well as peptic ulcer were analyzed. RESULTS: Of all the clinical specimens obtained, 80.8% (126/156) were found to have H pylori isolates and 97.2% of the isolates (106/109) were positive for cagA gene. In comparison with the reported data, the cloned cagA1 fragment possessed 94.83% and 93.30% homologies with the nucleotide and putative amino acid sequences, respectively. The output of rCagA1 produced by the constructed recombinant prokaryotic expression system was approximately 30% of the total bacterial protein. rCagA1 was able to bind to the commercial antibody against the whole-cells of H pylori and to induce the immunized rabbits to produce antibody with an immunodiffusion titer of 1:4. A proportion as high as 92.6% of the H pylori isolates (101/109) expressed CagA and 88.1% of the patients' serum samples (96/109) were CagA antibody-positive. The percentage of CagA(+) H pylori strains (97.9%) isolated from the biopsy specimens of peptic ulcer appeared to be higher than that from gastritis (88.5%), but the difference was not statistically significant (chi (2)=3.48, P>0.05). CONCLUSION: rCagA1 produced by the prokaryotic expression system constructed in this study possesses good immunoreactivity and antigenicity, and the established ELISAs can be used to detect CagA of H pylori and its antibody. H pylori isolates show high frequencies of cagA gene and CagA expression, but the infections by CagA(+) H pylori strains are not the most decisive factors to cause gastric diseases.