Distinct cagA EPIYA Motifs are Associated with Ethnic Diversity in Malaysia and Singapore

Background:  In vitro studies have shown that the biologic activity of CagA is influenced by the number and class of EPIYA motifs present in its variable region as these motifs correspond to the CagA phosphorylation sites. It has been hypothesized that strains possessing specific combinations of these motifs may be responsible for gastric cancer development. This study investigated the prevalence of cagA and the EPIYA motifs with regard to number, class, and patterns in strains from the three major ethnic groups within the Malaysian and Singaporean populations in relation to disease development.
Materials and methods:  Helicobacter pylori isolates from 49 Chinese, 43 Indian, and 14 Malay patients with functional dyspepsia (FD) and 21 gastric cancer (GC) cases were analyzed using polymerase chain reaction for the presence of cagA and the number, type, and pattern of EPIYA motifs. Additionally, the EPIYA motifs of 47 isolates were sequenced.
Results:  All 126 isolates possessed cagA , with the majority encoding EPIYA-A (97.6%) and all encoding EPIYA-B. However, while the cagA of 93.0% of Indian FD isolates encoded EPIYA-C as the third motif, 91.8% of Chinese FD isolates and 81.7% of Chinese GC isolates encoded EPIYA-D ( p  < .001). Of Malay FD isolates, 61.5% and 38.5% possessed EPIYA-C and EPIYA-D, respectively. The majority of isolates possessed three EPIYA motifs; however, Indian isolates were significantly more likely to have four or more ( p  < .05).
Conclusion:  Although, H. pylori strains with distinct cagA -types are circulating within the primary ethnic groups resident in Malaysia and Singapore, these genotypes appear unassociated with the development of GC in the ethnic Chinese population. The phenomenon of distinct strains circulating within different ethnic groups, in combination with host and certain environmental factors, may help to explain the rates of GC development in Malaysia.     

幽门螺杆菌cagA克隆表达

克隆表达4株幽门螺杆菌的cagA基因,以方便地获得大鼠CagA蛋白和重组表达质粒,为临床诊断CagA阳性幽门螺杆菌感染,以及进一步研究不同类型CagA功能及其与疾病关系提供材料。PCR扩增幽门螺杆菌的cagA基因,克隆至PinPoint^TMXa-1T载体,酶切鉴定连接方向,IPTG诱导正向连接克隆表达CagA融合蛋白并进行SDS－PAGE和Western blots鉴定。结果显示PCR扩增得到3.5-3.8kb的CagA基因,PCR及酶切鉴定得到正向连接的重组克隆,SDS－PAGE及Western blots证实正向连接的重组克隆表达CagA融合蛋白。构建了4种cagA的重组表达质粒,通过转化同一宿主菌可研究不同CagA的功能和致病性差异;通过亲和层析纯化融合蛋白可获大量CagA蛋白,用于血清学诊断CagA阳性幽门螺杆菌感染,及不同抗原性CagA与疾病之间的关系。     

Helicobacter pylori cagA Gene Polymorphism Affects the Total Antioxidant Capacity of Human Saliva

Background:  We aimed to evaluate the total antioxidant capacity (TAC) of saliva in healthy Helicobacter pylori -positive and negative saliva individuals.
Materials and Methods:  A total of 102 human saliva samples were checked for the presence of H. pylori DNA ( ureA and cagA gene fragments).
TAC of saliva was estimated by ABTS radical cation (ABTS^{• +} ) decolorization assay.
Results:  PCR analysis revealed that 36 subjects were ureA-/cagA- , 24 were ureA+/cagA- and 42 were ureA+/cagA+ . Smoking habits had no evident effect on H. pylori infection.
We found that TAC of the ureA-/cagA- material, after 10 seconds reaction reflecting fast-reacting antioxidants, was significantly higher than of ureA+/cagA- and ureA+/cagA+ samples (p < .01 and p < .001, respectively). Similar results were obtained for reaction time of 3 minutes measuring slow-reacting antioxidants (p < .001). We also estimated ureA+/cagA- and ureA+/cagA+ samples alone and reported a statistically significant decrease in the TAC_3min value of ureA+/cagA+ compared with ureA+/cagA- samples (p < .05).
Conclusions:  Our data demonstrated that altered redox equilibrium may be associated with more frequent occurrence of H. pylori in the saliva samples.     

Prevalence of Helicobacter pylori pathogenicity-associated cagA and vacA genotypes among Pakistani dyspeptic patients

The cytotoxin-associated gene A ( cagA ), and the vacuolating cytotoxin gene A ( vacA ) products are considered the most important pathogenic determinants of Helicobacter pylori , a gram-negative bacterium causing gastrointestinal disorders such as duodenal ulcers, gastritis and mucosa-associated lymphoid tissue disease. A higher prevalence of H. pylori has been reported in various regions in the Pakistani population; however, no data are available about the virulence-associated genetic determinants. The objective of this study was to determine the prevalence of virulence-associated genes, cagA, vacA and particularly vacA allelic variants among dyspeptic patients from Pakistan. Gastric biopsy samples were obtained from 78 adult patients presenting dyspepsia symptoms. DNA was isolated and analyzed for the presence of H. pylori and its genotypes by PCR. Genus-specific PCR involving 16S rRNA gene revealed that 66 of the 78 patients were positive for H. pylori , an overall prevalence of 84.6% for this particular study. The most common vacA genotype was s1b/m2 (54.5%) followed by s1a/m1 (19.7%). cagA was positive in 24.2% of the cases and strongly associated with s1a/m1, vacA . The prevalence of virulent cagA , and vacA allelic form s1a/m1 was lower than that reported from neighboring countries.     

The cagA status of Helicobacter pylori isolates from dyspeptic children in Turkey

There are inconsistent reports regarding cytotoxin-associated gene A (cagA) status of Helicobacter pylori isolates and the severity of the mucosal lesions in children. The aim of this study was to determine the prevalence of cagA(+) strains and to evaluate its correlation with clinic and endoscopic findings. We examined 45 H. pylori strains that were grown on brain-heart infusion agar supplemented with 7% horse blood. Following 72 h of incubation colonies were harvested and bacterial DNA was extracted. Polymerase chain reaction primers F1 and B1 were used to amplify a 348-bp internal fragment of cagA. The prevalence of cagA in Turkish pediatric patients was 55.6%. No association was found between cagA status and the severity of gastro-duodenal lesions.     

The diversity of vacA and cagA genes of Helicobacter pylori in East Asia

It has been reported that Helicobacter pylori infection with the type I strain, which expresses the VacA and CagA antigens, is associated with duodenal ulcer. We examined the diversity of vacA and cagA genes in 143 isolates obtained from patients with duodenal ulcer or chronic gastritis in East Asia (two different areas of Japan, Fukui and Okinawa, and also in Hangzhou, China) by polymerase chain reaction (PCR) and sequence analysis. Diversities of cagA and vacA genes were detected in East Asia. The prevalence of cagA-positive H. pylori was significantly different between Fukui and Okinawa (P=0.0032). The prevalence of Western type CagA was significantly higher in Okinawa than in Fukui (P<0.0001). However, there was no significant association between the genotype of cagA and clinical outcome. In Japan, the predominant vacA genotype was s1c/m1b. In contrast, in Hangzhou, the predominant vacA genotype was s1c/m2, and they were all East Asian CagA-positive. These findings suggest that a distinct distribution of the vacA and cagA genotypes is present in East Asia, regardless of clinical outcome.     

利用杆状病毒表达幽门螺杆菌cagA基因

幽门螺杆菌cagA基因克隆到杆状病毒表达系统的pBlueBacHis2A转移载体中,将重组质粒pBlueBacHis2A-CagA与亲本病毒Bac-N-blue DNA共转染Sf9细胞,以空斑法纯化获得的重组杆状病毒.经PCR法鉴定后进行扩增培养,SDS-PAGE和Western bolt检测结果证实所表达的蛋白为CagA蛋白,间接ELISA分析表明,表达产物可与Hp感染者血清发生特异性的免疫反应.     

Identification of a new segment involved in cagA 3' region variation of Helicobacter pylori

The cagA 3' region shows marked variation among Helicobacter pylori strains. Two segments of 102 bp and 57 bp are reportedly responsible for this variation. We analysed the cagA 3' region in 70 H. pylori strains using polymerase chain reaction and sequencing. We found that another segment, namely beta segment, was also involved in the variation of this region. The beta segment was 105 bp long and located between the aforementioned two segments. Six genotypes were identified based on the structure of the cagA 3' region. No relationship was found between these genotypes and the clinical outcomes or vacA genotypes. The numbers of tyrosine phosphorylation sites within the cagA 3' region varied among strains, but this was not related to the cagA genotypes. Our data suggest that the cagA 3' region is significantly variable. It appears that the variation of the cagA 3' region might contribute to the modification of virulence.     

The effect of Helicobacter pylori on neutrophil chemotaxis is independent of cagA

Sixteen Helicobacter pylori strains were studied in order to determine their neutrophil chemotactic activity and the association with the presence cagA gene. Neutrophil chemotactic activity was detected by a modified Boyden chamber method and the results were expressed in terms of chemotactic index (CI). The presence of cagA was determined by PCR. Of the 16 strains, eight were cagA+ and eight were cagA-. All of the isolated strains showed chemotactic activity. The mean value of CI of the patient group was significantly higher than the negative control (P < 0.01). The mean value of CI of zymosan-activated serum (P < 0.05) and the reference strain H. pylori NCTC 11637 (HP11637) (P < 0.01) was significantly higher than the patient group's mean value of CI. There were no statistical significance in the CI between cagA+ and cagA- strains (P > 0.05). It is concluded that H. pylori attracts neutrophils by chemotaxis, however, there is no association with cagA.     

利用杆状病毒表达幽门螺杆菌cagA基因

幽门螺杆菌cagA基因克隆到杆状病毒表达系统的 pBlueBacHis2A转移载体中 ,将重组质粒 pBlueBacHis2A CagA与亲本病毒Bac N blueDNA共转染Sf9细胞 ,以空斑法纯化获得的重组杆状病毒。经PCR法鉴定后进行扩增培养 ,SDS PAGE和Westernbolt检测结果证实所表达的蛋白为CagA蛋白 ,间接ELISA分析表明 ,表达产物可与Hp感染者血清发生特异性的免疫反应     

Identification of Helicobacter pylori and the cagA genotype in gastric biopsies using highly sensitive real-time PCR as a new diagnostic tool

The CagA protein is one of the virulence factors of Helicobacter pylori, and two major subtypes of CagA have been observed, the Western and East Asian type. CagA is injected from the bacteria into gastric epithelial cells, undergoes tyrosine phosphorylation, and binds to Src homology 2 domain-containing protein-tyrosine phosphatase SHP-2. The East Asian type CagA binds to SHP-2 more strongly than the Western type CagA. Here, we tried to distinguish the CagA type by highly sensitive real-time PCR with the objective of establishing a system to detect H. pylori and CagA subtypes from gastric biopsies. We designed primers and probe sets for Western or East Asian-cagA at Western-specific or East Asian-specific sequence regions, respectively, and H. pylori 16S rRNA. We could detect the H. pylori 16S rRNA gene, Western and East Asian-cagA gene from DNA of gastric biopsies. The sensitivity and specificity for H. pylori infection was 100% in this system. In Thai patients, 87.8% (36/41) were cagA-positive; 26.8% (11/41) were Western-cagA positive and 53.7% (22/41) were East Asian-cagA positive, while 7.3% (3/41) reacted with both types of cagA. These results suggest that this real-time PCR system provides a highly sensitive assessment of CagA type as a new diagnostic tool for the pathogenicity of H. pylori infection.     

cagA gene variants in Malaysian Helicobacter pylori strains isolated from patients of different ethnic groups

Helicobacter pylori infection of a distinct subtype of cagA may lead to different pathological manifestation. The aim of this study is to determine the presence of cagA gene and its variants in H. pylori infection among different ethnic groups and its effect on gastroduodenal diseases. Overall detection of cagA among the 205 clinical isolates of H. pylori was 94%. Variations in size of the 3' region of cagA gene were examined among 192 Malaysian H. pylori cagA-positive strains. Results showed that three cagA variants differing in fragment length of PCR products were detected and designated as type A (621-651bp), type B (732-735bp) and type C (525 bp). Although there was no association between any of the cagA subtypes with peptic ulcer disease (p>0.05), an association between cagA subtypes with a specific ethnic group was observed. Specific-cagA subtype A strains were predominantly isolated from Chinese compared to Malays and Indians (p<0.0005), and cagA subtype B strains were predominantly isolated from Malays and Indians compared to Chinese (p<0.05). The cagA type A strains of H. pylori is commonly found in the Chinese patients who have a higher risk of peptic ulcer disease, thus indicating that it could be used as an important clinical biomarker for a more severe infection.     

cagA gene and vacA alleles in Spanish Helicobacter pylori clinical isolates from patients of different ages

The prevalence of the cagA gene and vacA alleles in 124 Spanish Helicobacter pylori clinical isolates from patients of different ages ranging from 3 to 78 years was studied (21 patients < or = 10 years, 30 patients 11-20 years, 17 patients 21-40 years, 31 patients 41-60 years and 25 patients 61-80 years). The cagA gene and vacA s1 or vacA s2 alleles were identified by PCR from the strain. 66.9% of the isolates were cagA+ and 33.1% cagA-. vacA s1 was detected in 48.4% of the isolates and vacA s2 in 51.6%. 44.4% of patients were cagA+/vacA s1, 22.5% were cagA+/vacA s2, 4% were cagA-/vacA s1 and 29% were cagA-/vacA s2. The percentage of cagA+ isolates and the vacA s1 alleles in the different groups were as follows: 23.8% and 28.6% in 0-10 years, 40% and 30% in 11-20 years, 88.2% and 70.6% in 21-40 years, 90.3% and 70.9% in 41-60 years and 92% and 44% in the 61-78 years group. 93% (54/58) of isolates found in ulcer patients and 90.9% (10/11) of isolates from gastritis patients older than 20 years were cagA+. In patients younger than 20 years ulcer disease was rare with 60% of isolates being cagA+ (3/5) compared with 31.6% cagA+ isolates (12/38) in patients suffering from gastritis in the younger group. The prevalence of the cagA gene and vacA s1 allele increased with age, being more frequent in older patients than in younger.     

幽门螺杆菌vacA和cagA基因全长分子系统发育分析

文章从GenBank中下载所有含有vacA和cagA基因的H.pylori菌株的VacA和CagA全长氨基酸序列,利用ClastalX 2.0和MEGA 5.05软件构建VacA和CagA分子系统发育树,探讨两基因之间的分子系统发育关系和不同聚类群的临床感染结果与基因型特征。结果显示,VacA和CagA具有高度相似的分子系统发育树,并且所有H.pylori菌株在系统发育树中具有相同的分布特点,分别聚类为东亚株群1、2和西方株群3个聚类群。其中东亚株群1患萎缩性胃炎比例较高,vacA基因型以s1c/m1b和s1a/m1b为主,cagA基因型以EPIYA-ABD为主;东亚株群2患十二指肠溃疡的比例较高,vacA基因型以s1c/m2和s1a/m2为主,cagA基因型以EPIYA-AB’C为主;西方株群患十二指肠溃疡和胃炎的比例相当,萎缩性胃炎比例较低,vacA基因型以s1a/m1a和s1b/m1a为主,cagA基因型以EPIYA-AB/B’CC为主。这些结果说明,vacA和cagA基因可能具有共进化的遗传关系;东亚株群1、2和西方株群分别具有不同的vacA和cagA基因亚型,这可能与其临床感染结果密切相关,因此,在进行H.pylori相关性疾病分析时,有必要结合vacA和cagA基因型的亚型做深入分析。     

Identification of the repeated number of C and D regions of tyrosine phosphorylation motifs in Helicobacter pylori cagA using multiplex PCR

Various tyrosine phosphorylation motif regions of H. pylori cagA exist. The number of these regions was found to have some influence on cell signaling, which was found to be more pronounced when in D (ESS) region than in C (WSS) region. A molecular biological method with multiplex PCR was developed to distinguish C and D regions, and to identify the repetition number of tyrosine phosphorylation of the cagA gene. Multiplex PCR using novel primer sets was performed on 73 strains of H. pylori isolated from Korean patients with upper gastrointestinal diseases. The Western cagA was identified in only 3 strains (4.1%) whereas East Asia cagA was identified in 69 strains (94.5%). These results were reconfirmed through a sequencing analysis. The method developed in this study would be useful for monitoring the repeated number of C and D regions of tyrosine phosphorylation motifs in H. pylori cagA.     

Comparison of genotyping of Helicobacter pylori cagA and vacA virulence genes from gastric biopsies and stool specimens

Background. We compared results of genotyping of Helicobacter pylori cagA and vacA virulence genes in DNA from gastric biopsies, both paraffin‐embedded and frozen, and from stool samples, in order to evaluate the comparative sensitivity of the stool assay. Methods. Genomic DNA from paraffin‐embedded biopsies, unfixed frozen biopsies, and stool samples of the same 20 patients was amplified for the cagA gene, an empty site (which provides a positive signal for cagA negative strains) and for the s and m alleles of the vacA gene. Composite genotypes were determined by combining data from analysis of all three materials. Results. Analysis of none of the materials taken singly showed all of the genotypes revealed by all three materials taken together, probably because of sampling error. Analysis of paraffin biopsies revealed 83.5%, that of frozen biopsies revealed 74.7% and that of stools revealed 75.9% of the genotypes. There was no significant difference in the percentage of the H. pylori genotypes identified from the three materials. Analysis of combinations of frozen biopsies and stools revealed 89.9% of the composite genotypes, and that of paraffin biopsies and stools revealed 96.2% of the composite genotypes. Evidence of multiple genotypes was found in 10 of 20 (50%) of the cases. Conclusions. Any one of the investigated biological materials can be used for detection of cagA and vacA genes, but no single assay provided a complete genotype. The use of a combination of two materials may generate a more accurate representation of H. pylori genotypes in each individual.     

Experimental colonization of mice by fresh clinical isolates of Helicobacter pylori is not influenced by the cagA status and the vacA genotype

Developing murine models of infection by Helicobacter pylori is quite useful but not all the strains are able to colonize the mouse. In order to study the influence of the two main virulence factors, CagA and VacA, on the establishment of H. pylori in mice, we have inoculated C57BL/6 mice with 15 strains randomly chosen among clinical strains freshly isolated from biopsy specimens of infected patients and five reference strains. Only six of the clinical strains and two of the reference strains could infect the animals regardless of the cagA status and the vacA genotype. We concluded that 40% of the H. pylori strains are able to infect mice and that the capacity of colonization is not influenced by the cagA status and the vacA genotype. These factors cannot be used to predict the success of an experimental infection.     

No correlation of babA2 with vacA and cagA genotypes of Helicobacter pylori and grading of gastritis from peptic ulcer disease patients in Brazil

BACKGROUND: The babA2 gene, which encodes a blood-group antigen-binding adhesin that mediates attachment of Helicobacter pylori to human Lewis(b) antigens on gastric epithelial cells, has been associated with a higher risk of peptic ulcer and gastric cancer. The purpose of this study was to ascertain the frequency of babA2 genotype in H. pylori strains of patients with peptic ulcer and to correlate with other virulence factors. MATERIALS AND METHODS: vacA, cagA, and babA2 genotypes of H. pylori were determined by using polymerase chain reaction (PCR). DNA was extracted from positive urease test gastric samples of 150 patients with peptic ulcer. Antrum and corpus biopsies were taken for histologic examination according to the updated Sydney system classification. RESULTS: babA2 genotype was present in 104 (69.3%) and cagA in 113 (75.3%) gastric samples. No significant correlation was observed between babA2 and vacAs1 genotype or between babA2 and cagA status. The correlation of vacAs1 genotype with positive cagA was statistically significant ( p < .001). The babA2-positive strain was more frequently found from the gastric samples of men, than of women (p = .01). Strains harboring cagA, vacAs1, and babA2 genotypes had no association to the grading of gastritis, presence of glandular atrophy, or intestinal metaplasia. The simultaneous presence of cagA, vacAs1, and babA2 was found in 32.6% of the H. pylori strains. CONCLUSIONS: babA2 genotype is frequently found in H. pylori strains from peptic ulcer disease in Brazil, although it has no significant correlation to the worsening of the gastritis and to other virulence markers such as vacAs1 and cagA.     

Helicobacter pylori and the birth cohort effect: evidence for stabilized colonization rates in childhood

Background: The prevalence of Helicobacter pylori has declined over recent decades in developed countries. The increasing prevalence with age is largely because of a birth cohort effect. We previously observed a decline in H. pylori prevalence in 6‐ to 8‐year‐old Dutch children from 19% in 1978 to 9% in 1993. Knowledge about birth‐cohort‐related H. pylori prevalence is relevant as a predictor for the future incidence of H. pylori‐associated conditions. Aim: The aim of this study was to investigate whether the birth cohort effect of H. pylori observed between 1978 and 1993 continued in subsequent years. Methods: Anti‐H. pylori IgG antibodies and anti‐CagA IgG antibodies were determined in serum samples obtained in 2005/2006 from 545 Dutch children aged 7–9 years who participated in the Prevention and Incidence of Asthma and Mite Allergy birth cohort. The H. pylori and CagA antibodies were determined by enzyme‐linked immunosorbent assays that have been extensively validated in children, with a 94% sensitivity for H. pylori colonization and a 92.5% sensitivity for colonization with a cagA‐positive strain. Results: Of the 545 children (M/F 300/245), most (91.5%) were of Dutch descent. The H. pylori positivity rate was 9% (95% CI 6.6–11.4%). The prevalence of CagA antibodies was 0.9% (95% CI 0.1–1.6%). No significant differences were demonstrated in H. pylori and cagA prevalence in relation to gender or ethnicity. Conclusion: The prevalence of H. pylori in childhood has remained stable in the Netherlands from 1993 to 2005, suggesting a stabilization of the previously decreasing trend in subsequent birth cohorts. This finding may reflect stabilization in determinants such as family size, housing, and hygienic conditions (or offset by day care). If confirmed in other populations in developed countries, it implies that colonization with H. pylori will remain common in the coming decades. Remarkably however, the rate of colonization with cagA⁺H. pylori strains has become very low, consistent with prior observations that cagA⁺ strains are disappearing in Western countries.