cagA and vacA genotype of Helicobacter pylori associated with gastric diseases in Xi＇an area

AIM: To establish stock of clinical Helicobacter pylori (H.pylon) isolates, to perform cagA and vacA typing of these isolates, to evaluate the relationship between genotypes of cagA and vacA and upper gastrointestinal diseases and to assess the association of vacA genotypes with presence of the pathogenicity marker-cagA.METHODS: Clinical H.pylori strains were isolated from the antrum of 259 patients in Clumbia agar. The isolated H.pylori strains were identified by histology, and16SrRNA PCR.CagA genotypes were detected by colony hybridization, the probe was derived from the cloned plasmid PcagA, and digested by EcoRI-HindⅢ and the isolated PcagA DNA fragment was radioactively labelled by the random priming method. vacA genes types (s,m)and subtypes (s1a, s1b,s2) were typed by PCR. Vacuolating toxin was detected with neutral red absorb test. The results were treated statistically by χ2test, ttest, and rank sum test.RESULTS: A total of 192 clinical H. pylori strains were isolated and the stock of Helicobacter pylori was established. The total positive rate of cagA was 87 % in all gastric diseases,and 95 % in gastric cancer group. There was a difference between gastric cancer group and the other groups (P＜0.05)except duodenal ulcer group. The expression of type s1 of vacA was more than type s2 (P＜0.05), and, the expression of type m1 was equal to type m2. In gastric cancer group,there was a difference between s1a and s1b (P＜0.05), and s1a was more than s1b. Vacuolating toxins were more in Xi′an area isolates.CONCLUSION: The cagA+ vacA type s1 clinical isolates are more in Xi′an area, but this can not serve as an index to predict gastric cancer.     

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 aHelicobacter py/ori ( H pylori) cagA gene fragment andestablish enzyme-linked immunosorbent assays (ELISA) fordetecting CagA and its antibody,so as to understand themanner in which the infection of CagA-expressing Hpylori(CagA~ Hpylori) isolates cause diseases.METHODS:Hpyloristrains in gastric biopsy specimens from156 patients with positive results in rapid urease test wereisolated.PCR was used to detect the frequency of cagAgene in the 109 Hpyloriisolates and to amplify a 2 148-bpfragment (cagA1) of cagA gene from a clinical strain Y06.Aprokaryotic expression system of cagA1 gene was constructed,and the expression of the target recombinant protein(rCagA1) was examined by SDS-PAGE.Western blotting andimmunodiffusion assay were employed to determine theimmunoreactivity and ant igenicity of rCagA1,respectively.Two ELISAs were established to detect CagA expression in109 Hpyloriisolates and the presence of CagA antibody inthe corresponding patients'sera,and the correlationsbetween infection with CagA Hpyloriand gastritis as wellas peptic ulcer were analyzed.RESULTS:Of all the clinical specimens obtained,80.8%(126/156) were found to have Hpyloriisolates and 97.2%of the isolates (106/109) were positive for cagA gene.Incomparison with the reported data,the cloned cagA1fragment possessed 94.83% and 93.30% homologies withthe nucleotide and putative amino acid sequences,respectively.The output of rCagA1 produced by theconstructed recombinant prokaryotic expression system wasapproximately 30% of the total bacterial protein,rCagA1was able to bind to the commercial antibody against thewhole-cells of Hpyloriand to induce the immunized rabbitsto produce antibody with an immunodiffusion titer of 1:4.Aproportion as high as 92.6% of the Hpyloriisolates (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 biopsyspecimens of peptic ulcer appeared to be higher than thatfrom gastritis(88.5%),but the difference was not statisticallysignificant(x~2=3.48,P>0.05).CONCLUSION:rCagAl produced by the prokaryoticexpression system constructed in this study possesses goodimmunoreactivity and antigenicity,and the establishedELISAs can be used to detect CagA of Hpyloriand its antibody.H pylori isolates show high frequencies of cagA gene andCagA expression,but the infections by CagA~ H pylori strainsare not the most decisive factors to cause gastric diseases.     

Expression of cytokeratins in Helicobacter pylori -associated chronic gastritis of adult patients infected with cagA ＋ strains： An immunohistochemical study

AIM:To investigate the expression of differentcytokeratins(CKs)in gastric epithelium of adult patientswith chronic gastritis infected with Helicobacter pylori(Hpylori)cagA strains.METHODS:The expression of CK 7,8,18,19 and 20was studied immunohistochemically in antral gastricbiopsies of 84 patients.All the CKs were immunostainedin cagA H pylori gastritis(57 cases),non-H pylori gastritis(17 cases)and normal gastric mucosa(10 cases).RESULTS:In cagA H pylori gastritis,CK8 wasexpressed comparably to the normal antral mucosafrom surface epithelium to deep glands.Distributionof CK18 and CK 19 was unchanged,i.e.transmucosal,but intensity of the expression was different in foveolarregion in comparison to normal gastric mucosa.Cytokeratin 18 immunoreactivity was significantly higherin the foveolar epithelium of H pylori-positive gastritiscompared to both Hpylori-negative gastritis and controls.On the contrary,decrease in CK19 immunoreactivityoccurred in foveolar epithelium of H pylori-positive gastritis.In both normal and inflamed antral mucosawithout Hpylori infection,CK20 was expressed strongly/moderately and homogenously in surface epithelium andupper foveolar region,but in H pylori-induced gastritissignificant decrease of expression in foveolar regionwas noted.Generally,in both normal antral mucosa andH pylori-negative gastritis,expression of CK7 was notobserved,while in about half cagA H pylori-infectedpatients,moderate focal CK7 immunoreactivity of theneck and coiled gland areas was registered,especially inareas with more severe inflammatory infiltrate.CONCLUSION:Alterations in expression of CK 7,18,19 and 20 together with normal expression of CK8 occurin antral mucosa of H pylori-associated chronic gastritisin adult patients infected with cagA strains.Alterationsin different cytokeratins expression might contribute toweakening of epithelial tight junctions observed in Hpylori-infected gastric mucosa.     

Expression of nuclear factor-kappa B and target genes in gastric precancerous lesions and adenocarcinoma:Association with Helicobactor pylori cagA ( ) infection

AIM:To examine the expression of nuclear factor kappaB(NF-xB) and its target genes in intestinal metaplasia (IM),dysplasia (DYS) and gastric carcinoma (GC) infected withHelicobacter pylori (H pylori) and to investigate themechanism underlying Hpyloricytotoxin associated gene A(cag A) infection leading to gastric adenocarcinoma.METHODS:Expressions of NF-kB/p65 and its target genes:c-myc,cyclinD1 and bcl-xl were immunohistochemicallyexamined in 289 cases of gastric biopsy and resectionspecimens from patients with IM,DYS and GC infected withH pylori.H pylori in the above mentioned tissues wasdetected by Warthin-Starry stain and rapid urease tests.IgG antibody to cagA in sera of the patients was measuredby ELISA.RESULTS:The positive rates of NF-kB/p65 were significantlyhigher in groups with cagA of IMⅠ-Ⅱ(28/33),IMⅢ(48/52),DYSI(27/31),DYS Ⅱ-Ⅲ(28/32),GC(35/40) than in groupswithout cagA of IMⅠ-Ⅱ(4/17),IMⅢ(3/20),DYSI(3/20),DYSⅡ-Ⅲ(6/21),GC(10/23).The expressions of c-myc,cyclinD1,and bcl-xl were significantly higher in groups withcagA of IM Ⅲ(47/52,49/52,46/52),DYSⅡ-Ⅲ(29/32,26/32,25/32) than in groups without cagA of IM Ⅲ(8/20,7/20,5/20),DYSⅡ-Ⅲ(10/21,8/21,3/21),which were in conformitywith the expression of NF-kB in IM Ⅲ,and DYSⅡ-Ⅲ.Asignificantly higher expression level of NF-kB/p65,c-myc,cyclinD1 and bcl-xl was detected in intestinal type GC(27/28,18/28,22/28,24/28) than in diffuse type GC(8/12,3/12,3/12,6/12),respectively.CONCLUSION:There may be two different molecularmechanisms in the occurrence of intestinal and diffuse typegastric carcinomas,intestinal type gastric carcinoma isstrongly associated with high expression of c-myc,cyclinD1and bcl-xl through NF-kB/p65 activated by Hpylori cagA.inhibiting the activity of NF-kB is an effective and promisingway to prevent intestinal type gastric carcinoma.     

Association between Helicobacter pylori,Epstein-Barr virus,human papillomavirus and gastric adenocarcinomas

AIM To correlate Helicobacter pylori(H. pylori), Epstein-Barr virus(EBV) and human papillomavirus(HPV) with gastric cancer(GC) cases in Pará State, Brazil.METHODS Tissue samples were obtained from 302 gastric adenocarcinomas. A rapid urease test was used to detect the presence of H. pylori, and the presence of the cagA gene in the HP-positive samples was confirmed by PCR. An RNA in situ hybridization test designed to complement Eber1 RNA was used to detect the presence of EBV in the samples, and the L1 region of HPV was detected using nested PCR. Positive HPV samples were genotyped and analyzed for E6 and E7 viral gene expression. Infections were also correlated with the clinical and pathological characteristics of the patients.RESULTS The majority of the 302 samples analyzed were obtained from men(65%) aged 55 years or older(67%) and were classified as the intestinal subtype(55%). All three pathogens were found in the samples analyzed in the present study(H. pylori : 87%, EBV: 20%, HPV: 3%). Overall, 78% of the H. pylori-positive(H. pylori+) samples were cag A +(H. pylori-cag A+), and there was an association between the cytotoxic product of this gene and EBV. Coinfections of H. pylori-cagA+ and EBV were correlated with the most advanced tumor stages. Although only 20% of the tumors were positive for EBV, infection with this virus was associated with distant metastasis. Only the HPV 16 and 18 strains were found in the samples, although no expression of the E6 and E7 oncoproteins was detected. The fundus of the stomach was the region least affected by the pathogens.CONCLUSION HPV was not involved in gastric tumorigenesis. Prophylactic and therapeutic measures against H. pylori and EBV may prevent the development of GC, especially the more aggressive forms.     

Mutation of cytotoxin-associated gene A affects expressions of antioxidant proteins of Helicobacter pylori

AIM： To determine if disruption of the cagA gene of Helicobacter pylori （H pylori） has an effect on the expression of other proteins at proteome level. METHODS： Construction of a cagA knock out mutant Hp27_△cagA （cagA） via homologous recombination with the wild-type strain Hp27 （cagA＋） as a recipient was performed. The method of sonication-urea-CHAPS-DTT was employed to extract bacterial proteins from both strains. Soluble proteins were analyzed by two-dimensional electrophoresis （2-DE）. Images of 2-DE gels were digitalized and analyzed. Only spots that had a statistical significance in differential expression were selected and analyzed by matrix-assisted laser desorption/ionization- time of flight mass spectrometry （MALDI-TOF-MS）. Biological information was used to search protein database and identify the biological function of proteins. RESULTS： The proteome expressions between wild-type strain and isogenic mutant with the cagA gene knocked-out were compared. Five protein spots with high abundance in bacteria proteins of wild-type strains, down-regulated or absently expressed in bacteria proteins of mutants, were identified and analyzed. From a quantitative point of view, the identified proteins are related to the cagA gene and important antioxidant proteins of Hpylori, including alkyl hydroperoxide reductase （Ahp）, superoxide dismutase （SOD） and modulator of drug activity （Mda66）, respectively, suggesting that cagA is important to maintain the normal activity of antioxidative stress and ensure H pylori persistent colonization in the host. CONCLUSION： cagA gene is relevant to the expressions of antioxidant proteins of H pylori, which may be a novel mechanism involved in H pylori cagA pathogenesis.     

Gene distribution of cagⅡ in Helicobacter pylori-infected patients of Zhejiang Province

AIM: To determine the prevalence of genotypes of cagⅡ in Helicobacter pylori( H pylon)-infected patients in Zhejiang Province and investigate the relationship between these genotypes and the types of gastroduodenal diseases.METHODS: One hundred and seventy one clinical isolates were collected from 70 chronic superficial gastritis, 31 chronic atrophic gastritis, 41 gastric ulcer, 21 duodenal ulcer, 3 gastric and duodenal ulcer, and 5 gastric adenocarcinoma patients. Polymerase chain reaction assays were performed for analysis of cagT, ORF13 and ORF10 genes in the cagⅡ region.RESULTS: Of 171 Hpyloriisolates from Zhejiang patients,159(93.0%) were positive for all the three loci. One isolate (0.6%) was negative for all the three loci, and 11(6.4%) were partially deleted in cagⅡ. The positive rates of cagT,ORF13 and ORF10 genes were 97.1%, 94.7% and 99.4%,respectively. In the strains isolated from the patients with diseases including chronic superficial gastritis, chronic atrophic gastritis, gastric ulcer and duodenal ulcer, the sitive rates of cagT were 95.7%, 100.0%, 95.1% and 100.0%, respectively. The positive rates of ORF13 were 94.3%, 93.5%, 95.1% and 100.0%, respectively. The sitive rates of ORF10 were 98.6%,100.0%,100.0% and 100.0%, respectively. The three genes were all positive in the three H pylori strains isolated from the patients with both gastric and duodenal ulcer. In the five strains isolated from the patients with gastric adenocarcinoma,only one isolate was negative for ORF13. There were no significant differences of the cagT, ORF13 and ORF10 genes among the different gastroduodenal diseases including chronic superficial gastritis, chronic atrophic gastritis,gastric ulcer, duodenal ulcer, both gastric and duodenal ulcer and gastric adenocarcinoma (χ^2=3.098, P＞0.05 for cagT;χ^2=3.935, P＞0.05 for ORF13 and χ^2=6.328,P＞0.05 for ORF10).CONCLUSION: The cagⅡ is not a uniform and conserved entity. Although the genes in cagⅡ are highly associated with the gastroduodenal diseases, the clinical outcome of Hpyloriinfection is not reliably predicted by the three genes in cagⅡ in patients from Zhejiang Province.     

Helicobacter pylori and other Helicobacter species DNA in human bile samples from patients with various hepato-biliary diseases

AIM: To investigate the presence of Helicobacter species by nested PCR of 16S rRNA genes followed by the presence of Helicobacter pylori (H pylori) 16S rRNA, ureA, cagA genes in bile obtained at endoscopic retrograde cholangio-pancreatography (ERCP) from 60 Indian subjects. METHODS: Sixty bile samples were obtained from patients diagnosed with various hepato-biliary diseases and control subjects at ERCP. PCR analysis was carried out using primers for Helicobacter genus 16S rRNA gene and H pylori (16S rRNA, ureA and cagA) genes. Gastric H pylori status was also assessed from biopsies obtained at endoscopy from patients with various hepato-biliary diseases and controls. The control group mainly consisted of subjects with gastric disorders. Sequencing analysis was performed to confirm that PCR products with 16S rRNA and cagA primers were derived from H pylori. RESULTS No Helicobacters were grown in culture from the bile samples. Helicobacter DNA was detected in bile of 96.7% and 6.6% of groups I and II respectively. Ten from group I were positive for 16S rRNA and ureA and 9 were positive for cagA gene. In contrast of the 2 from the control, 1 amplified with 16S rRNA, ureA and cagA primers used. The sequences of the 16S rRNA genes and cagA were 99% similar to Helicobacter pylori. CONCLUSION: Helicobacters are associated with the pathogenesis of various hepato-biliary disorders.     

Seroepidemiology of Helicobacter pylori infection in elderly people in the Beijing region,China

AIM:To investigate seroepidemiology of cagA+and vacA+strains of Helicobacter pylori(H.pylori)in an elderly population in Beijing and to determine risk factors for seropositivity.METHODS:A total of 2006 elderly persons(>60years)were selected using a random cluster sampling method in different parts of the Beijing area(urban,suburban and mountainous districts).Structured questionnaires were completed during home visits,including history of H.pylori infection,history of gastrointestinal diseases,diet types,hygiene habits,occupation and economic status.Blood samples(2 mL)were collected from each participant,and serum IgG antibodies to cagA,vacA and H.pylori urease antigens were measured by immunodetection.RESULTS:The prevalence of H.pylori infection in elderly subjects was 83.4%and the typeⅠH.pylori strain infection rate was 56%.The seroprevalence for typeⅠH.pylori strain infection in urban and suburbandistricts was higher than that in the mountainous areas(P<0.001).Elderly subjects who had previously performed manual labor or were in the young-old age group(age<75 years)had a higher seroprevalence of H.pylori infection than those who had previously performed mental labor or were in the oldest-old age group(age≥75 year)(P<0.05).The typeⅠH.pylori strain infection rate in the elderly with vegetarian diets was higher than in those eating high-protein foods(P<0.001).There was no significant difference in the prevalence of H.pylori strains between male and female elderly participants(P>0.05).CONCLUSION:TypeⅠH.pylori seroprevalence is higher in elderly people.The distribution of strains of H.pylori is significantly affected by age,area and dietary habits.     

H pylori iceA alleles are disease-specific virulence factors

AIM: To characterize and compare genotype profiles of H pylori strains isolated from patients with chronic gastritis and duodenal ulcer in western part of Turkey. METHODS: A total of 46 patients [30 chronic gastritis (CG) and 16 duodenal ulcer (DU)] who had undergone endoscopy because of dyspeptic complaints were studied. The antral biopsy specimens were evaluated for the presence of H pylori by rapid urease test and culture, and the genotype profiles were determined by real-time PCR. RESULTS: The cagA gene was observed in 43 (93.5%) isolates. The vacA s1m2 genotype was the predominant subtype, found in 63.3% and 68.7% of isolates in patients with CG and DU, respectively. Twenty (66.6%) isolates from patients with CG were iceA2 positive while the iceA1 was predominant in those with DU (68.8%). In terms of the association of the iceA alleles to other genes, both alleles were significantly associated with the cagA vacA s1m2 genotype. CONCLUSION: The prevalent circulating genotypes in CG and DU were cagA vacA s1m2 iceA2 and cagA vacA s1m2 iceA1 genotype, respectively. It was found that cagA vacA s1m2 genotype seems to be common virulence factors in both CG and DU while iceA alleles show specificity for gastroduodenal pathologies in this study.     

幽门螺杆菌中国株cagA全长基因克隆及其分子特征分析

目的克隆幽门螺杆菌中国株MEL-Hp 27cagA全长基因,并进行分子特征分析。方法参考Hp26695基因组序列,分别针对cagA基因编码区保守序列和位于cagA基因上、下游的基因序列设计两对PCR引物,交叉分段扩增包括cagA基因编码区、5′、3′非编码区在内的全长基因序列,并对cagA基因调控序列、编码序列及源自中国的CagA分子EPIYA基序分布特点进行分析。结果Hp27cagA基因编码区长3510bp,编码1169个氨基酸。5′端非编码区长649bp,-10区、-35区分别位于起始密码子ATG上游89bp和154bp处,3′端非编码区长476bp。Hp27cagA基因编码区核苷酸序列与东亚菌株同源性为96%,与西方菌株的同源性仅为86%左右。Hp27CagA分子存在典型的EPIYA基序,属于ABD型,与国际参考株NCTC11637的AB-CCC型不同。比较源自中国的HpCagA序列的EPIYA基序分布特点,未发现中国Hp分离株之间存在CagAEPIYA基序与疾病结局(胃癌、慢性胃炎和十二指肠溃疡)的聚类关系。结论成功克隆幽门螺杆菌全长ca-gA基因;cagA基因编码区及非编码区序列存在东西方差异;未发现中国Hp分离株之间存在CagA EPIYA基序与疾病结局的聚类关系。     

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.     

幽门螺杆菌不同基因型和基因亚型与甲硝唑耐药性的研究

目的研究Helicobacter pylori菌株本身的毒力差异是否与H.pylori菌株对甲硝唑的敏感性有关。方法 用E-test方法检测109株H.pylori菌株对甲硝唑的敏感性;PCR检测H.pylori菌株不同的vacA基因亚型、cagA、iceA和babA2基因型。结果 云南地区甲硝唑耐药率为67.89％;vacA、cagA、iceAl、babA2基因的各种基因亚型和基因型在H.pylori菌株甲硝唑耐药率上无显著性差异。结论 H.pylori菌株本身的毒力差异与H.pylori菌株对甲硝唑的敏感性无关。     

上海地区幽门螺杆菌cagA基因3’区和vacA基因的多态性分析及其临床意义

探讨上海地区人群中幽门螺杆菌（H．pylori）cagA基因3’区和vacA基因的多态性及其临床意义。方法：99株H．pylori菌株分离自17例慢性浅表性胃炎（CSG）、21例慢性萎缩性胃炎（CAG）、19例胃溃疡（GU）、23例十二指肠溃疡（DU）和19例胃癌（GC）患者。用聚合酶链反应（PCR）技术对H．pylori菌株的cagA基因3’区和vacA基因信号序列及中间区等位基因进行扩增和检测。结果：99株H．pylori菌株中84株（84．8％）cagA基因阳性,其3’区产物大小均约650bp,属A型。vacA基因信号序列仅检出sla型,见于从94．1％（16／17）的CSG、952％（20／21）的CAG、89．5％（17／19）的GU、87．00（20／23）的DU和89．5％（17／19）的GC患者中分离的菌株（P＝0．87）;中间区等位基因仅检出m2型,见于从70．6％（12／17）的CSG、71．4％（15／21）的CAG、63．20（12／19）的GU、73．9％（17／23）的DU和57．9％（11／19）的GC患者中分局的菌株（P＝0．72）。结论：上海地区人群中H．pylori菌株的cagA基因3’区相对保守;绝大多数vacA基因属sla／m2型。本研究结果不支持这些基因的多态性与H．pylori感染临床结局相关的观点。     

VacA genotyping directly from gastric biopsy specimens and estimation of mixed Helicobacter pylori infections in patients with duodenal ulcer and gastritis.

BACKGROUND: The vacA genotypes and the cagA gene status were investigated in 80 Helicobacter pylori-infected patients with duodenal ulcer (DU) and 49 with gastritis only. METHODS: Lysates of gastric biopsy specimens were used directly for polymerase chain reaction-based detection. RESULTS: The ml subtype was found in 36% and 31% and the m2 in 36% and 46% of specimens from patients with DU and gastritis, respectively (P > 0.05). In 15% of samples the midregion remained unclassified. The prevalence rate of s1 subtypes was higher in cases of DU (69%) than in gastritis (43%) (P < 0.0001); the opposite correlation was observed for s2. The cagA gene was detected in 80% of patients with DU and in 52% of those with gastritis (P < 0.0001). Infections with multiple H. pylori strains exceeded 50% in both groups. CONCLUSIONS: These results suggest that vacA s1 genotype and cagA+ status are associated with higher DU prevalence and that mixed H. pylori infections are very common in our geographic region.     

Significance of cagA status and vacA subtypes of Helicobacter pylori in determining gastric histopathology: virulence markers of H. pylori and histopathology

BACKGROUND: It has been suggested that Helicobacter pylori strains containing the cytotoxin-associated gene A (cagA), and s1m1 genotype of vacuolating cytotoxin gene A (vacA) may have been associated with peptic ulcer disease. The aim of the present study was to analyze such an association of cagA presence and vacA subtypes of H. pylori with histopathological findings in patients with gastritis. METHODS: Sixty-five independent H. pylori strains isolated from Turkish patients with gastritis were analyzed. The antral biopsy specimens were processed for culture and histopathology. Histopathological features were recorded and graded according to updated Sydney system. The vacA subtypes and cagA gene were tested by polymerase chain reaction. RESULTS: Mild degree of antral density was associated with mild degree of gastric neutrophil infiltration (P = 0.010). Positive cagA status correlated significantly with the presence of atrophy (P = 0.035) and neutrophil infiltration (P < 0.001), but not with H. pylori density (P = 0.754) nor the degree of mononuclear cell infiltration (P = 0.945). The vacA subtypes were independent of gastric histopathology. The odds ratios for atrophy and neutrophil infiltration of cagA+ versus cagA- strains were 3.62 (95% confidence interval [CI]: 1.04-12.66) and 53.18 (95%CI: 11.08-255.23), respectively. CONCLUSION: The presence of the cagA gene is strongly associated with atrophic and active gastritis. Distinct vacA subtypes of H. pylori appear to have no association with histopathological findings of gastritis.     

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.     

中国三个地区幽门螺杆菌CagA羧基端多态性分析

目的分析我国不同地区幽门螺杆菌(H.pylori)的CagA羧基端可变区特征,比较CagA可变区序列差异。方法选取分离自西安、浙江、云南地区的41株H.pylori,PCR扩增CagA羧基端可变区。测序后使用Primer Premier 5软件将核苷酸序列翻译为氨基酸序列,使用Vector NTI Suit6软件将所有菌株的CagA羧基端可变区氨基酸序列进行比较分析。结果41株菌的CagA氨基酸序列可分为东亚型和西方型两类:38株分离株为东亚型模式,其中有31株为典型东亚型,7株为变异东亚型;1株分离株为典型的西方型模式,2株表现为片段缺失的变异西方型。结论中国不同地区H.pylori的CagA蛋白序列以东亚型模式为主,占92.7%(38/41);但在至少2个地区的菌株中发现有2株和1株菌CagA羧基端可变区为西方型模式。     

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.     

Helicobacter pylori vacA genotypes and cagA status and their relationship to associated diseases

Helicobacter pylori (H. pylori ) is a major causativebacterium of chronic gastritis, peptic ulcer and mucosaassociated lymphoid tissue lymphoma in humans, and associated with an increased risk of gastric cancer[1 -8]. An important virulant factor of H. pylori is the vacuolating cytotoxin ( VacA ) encoded by vacA that induces cytoplasmic vacuolation in target cells both in vitro and in vivo[9-11]. VacA is produced as a 140 kDa precursor which contains an N-terminal signal peptide and an approximately 33 kDa C-terminal outer membrance exporter. The precursor is cleaved at both N-terminal and C-terminal and secreted into the extracellular milieu as a 95 kDa mature protein. The mature protein futher undergoes specific cleavage to yield 37 kDa and 58 kDa subunits[12-14] Although vacA is present in all H. pylori strains, only about 50% to 60% of strains can induce vacuolation of epithelial cells as assessed by the HeLa cell assay. vacA shows considerable genetic variation in H. pylori isolated from all over the world and contains at least two variable regions. The s region exists as sl or s2 allelic types. Among type sl strains, subtypes sla and slb have been identified. The m region occurs as ml or m2 allelic types. Specific vacA genotype of H. pylori strains are associated with the production of the cytotoxin in vitro, epithelial damage in vivo, and clinical consequences[15-27]. The other virulant factor is the cytotoxin-associated protein (CagA) encoded by the cytotoxin-associated gene (cagA). The cagA gene is present in about 60% to 70% of strains and all of these strains express the cagA. The presence of cagA is also associated with the production of the cytotoxin in vitro, and clinical outcome[24-30]. The aim of this study was (i) to identify vacA genotypes and cagA status of H. pylori isolated from Chinese patients; (ii) to evaluation the relatioship beween vacA genotypes, cagA status and related gastroenterological disorders.