cagA, vacA and iceA Virulence Genes of Helicobacter pylori Isolates of Children in Finland

 cagA, vacA s and m genotypes and iceA alleles were analyzed from Helicobacter pylori strains isolated from 17 Finnish children and 32 children of non-Finnish origin living in Finland. Twelve children in the latter group were eastern European and 15 were of African origin. Only three children of non-Finnish origin were born in Finland. The vacA s1a subtype was more prevalent in the isolates from Finnish children than African children (76% vs. 7%, P<0.001);vacA s1b frequencies were 5% and 67%, respectively (P<0.001). The iceA1 allele was significantly more prevalent in African than Finnish isolates (93% vs. 35%, P<0.01). Considerable variation was noted in the frequency of vacA s1 subtypes and iceA alleles in children originating from different geographic regions, but the geographic variation of s1 subtypes resembled that described in other reports.     

Helicobacter pylori cagA and vacA cytotoxin genes in Changsha,China

Cytotoxin-associated protein (cagA) and the vacuolating cytotoxin (vacA) encoded by cagA and vacA genes are virulence determinants of Helicobacter pylori. In earlier studies among Chinese patients, all H. pylori strains were cagA-positive and vacAs1a/m2 type. Here, we determine the cagA, vacA and allele status of H. pylori strains isolated from patients with upper gastrointestinal symptoms in Changsha, China. Forty strains of H. pylori isolated from patients with peptic ulcer disease between March 1997 and August 1999 were recovered from storage at -80 degrees C and studied by the polymerase chain reaction (PCR) for cagA and vacA genotypes. cagA was positive in 75% of H. pylori isolates. Patients with peptic ulcer demonstrated cagA in 83% (15/18), compared with 68% (15/22) patients with superficial gastritis. vacAs1 allele was carried in 82.5% (33/40) isolates, of which 52.5% (21/40) were subtype vacAs1a/m2 and 17.5% (7/40) were subtype vacAs1b/m2.     

Association of cagA and vacA Genotypes of Helicobacter pylori with Gastric Diseases in Estonia

Gastric biopsy specimens from 156 adult patients from southern Estonia suffering from chronic gastritis, peptic ulcer disease, and perforated peptic ulcer were analyzed by PCR. The cagA gene was evenly distributed throughout 87% of the specimens from the patients with the different gastric diseases. The presence of the cagA gene correlated with that of vacA signal sequence type s1a (99%). However, no clear differences were found in the distribution of cagA and vacA genotypes among patients in Estonia with severe perforated peptic ulcer, uncomplicated peptic ulcer, or chronic gastritis.     

Expanding Allelic Diversity of Helicobacter pylori vacA

The diversity of the gene encoding the vacuolating cytotoxin (vacA) of Helicobacter pylori was analyzed in 98 isolates obtained from different geographic locations. The studies focused on variation in the previously defined s and m regions of vacA, as determined by PCR and direct sequencing. Phylogenetic analysis revealed the existence of four distinct types of s-region alleles: aside from the previously described s1a, s1b, and s2 allelic types, a novel subtype, designated s1c, was found. Subtype s1c was observed exclusively in isolates from East Asia and appears to be the major s1 allele in that part of the world. Three different allelic forms (m1, m2a, and m2b) were detected in the m region. On the basis of sequence alignments, universal PCR primers that allow effective amplification of the s and m regions from H. pylori isolates from all over the world were defined. Amplimers were subsequently analyzed by reverse hybridization onto a line probe assay (LiPA) that allows the simultaneous and highly specific hybridization of the different vacA s- and m-region alleles and tests for the presence of the cytotoxin-associated gene (cagA). This PCR-LiPA method permits rapid analysis of the vacA and cagA status of H. pylori strains for clinical and epidemiological studies and will facilitate identification of any further variations.     

Assessment of Helicobacter pylori vacA and cagA genotypes and host serological response

Helicobacter pylori strains can be distinguished by genotyping of virulence-associated genes, such as vacA and cagA. Because serological discrimination between strain types would reduce the need for endoscopy, 61 patients carrying H. pylori were studied by vacA and cagA genotyping of H. pylori in gastric biopsy specimens and by detection of specific serum antibodies. Serological responses to H. pylori were determined by Helicoblot (versions 2.0 and 2.1). Antibodies to CagA also were determined by a rapid anti-CagA assay (Pyloriset screen CagA) as well as by two noncommercially developed enzyme immunoassays, each using a recombinant CagA protein. Assessment of performance of the Helicoblot assays indicated substantial interobserver variation, with kappa values between 0.20 and 0.93. There was no relationship between the serological profiles on the Helicoblot and the genotypes from the same patients, except for strong associations between the presence of anti-CagA and the cagA-positive and vacA s1 H. pylori genotypes. Detection of anti-CagA by the five different assays varied considerably, with kappa values ranging from 0.21 to 0.78. Using the cagA genotype as the "gold standard," the sensitivity and specificity of the anti-CagA assays varied from 71.4 to 85.7% and from 54.2 to 100%, respectively. Thus, serological profiles of antibodies to H. pylori are heterogeneous and, with the exception of anti-CagA antibodies, show no relation to the H. pylori vacA and cagA genotypes. Detection of anti-CagA antibodies is strongly dependent on the test used.     

Frequency of vacA Genotypes and Cytotoxin Activity in Helicobacter pylori Associated with Low-Grade Gastric Mucosa-Associated Lymphoid Tissue Lymphoma

The vacA genotypes s1,m1 and s1,m2 were detected in 44 and 30% of Helicobacter pylori isolates, respectively, from patients with gastric mucosa-associated lymphoid tissue lymphoma, compared to 26 and 56% of isolates, respectively, from individuals with gastritis. The vacA s1 genotype was significantly associated with, but not predictive of, the presence of vacuolating cytotoxin activity.     

Prevalence of Helicobacter pylori vacA and cagA genotypes in Ethiopian dyspeptic patients

A total of 300 gastric biopsy samples and 50 Helicobacter pylori isolates were collected from Ethiopian adult dyspeptic patients. The vacA and cagA genes were detected in 90 and 79% of biopsy specimens, respectively, and in 100 and 87% of clinical isolates, respectively. Both genes were detected in 84% of the gastric biopsy samples and in 87% of the clinical isolates. Among vacA genotypes, the s1/m1 genotype was the most common in gastric biopsy samples (48%). The vacA and cagA positive H. pylori strains were detected to a higher degree in patients with chronic active gastritis (71%) than patients with other histopathological findings (29%) (P < 0.05).     

Diversity of Helicobacter pylori vacA gene and cytotoxin production

Objective: To determine the frequency of different alleles of the vacA gene among the strains of Helicobacter pylori isolated from children and adults, and to compare vacuolating cytotoxin production with different combinations of vacA alleles.
Methods: H. pylori strains were isolated from the gastric biopsy samples of 100 patients with duodenal ulcer (32) and chronic gastritis (68). Bacterial DNA was extracted and the vacA alleles typed using PCR. Cytotoxin activity was assessed by an Intestine 407 cell vacuolation assay.
Results: Alleles s1 and s2 of vacA were identified in 72% and 28% of the tested strains, respectively. Of the 72 type s1 strains, 71 were classified as type s1a and only one as s1b. The predominant sequence observed in the vacA middle region was the m2 type (65%). Combinations of vacA genotypes s1a m1, s1a m2 and s2 m2 were found in 35%, 36% and 28% of strains, respectively. Vacuolating cytotoxin was produced by vacA s1a m1 strains isolated from patients with duodenal ulcer (84.6%) and chronic gastritis (68.2%). Subtype s1a m2 strains expressed vacuolating cytotoxin less frequently in both duodenal ulcer (47.1%) and chronic gastritis (21.1%) patients (   p <0.01  ). Antibodies to VacA (89 kDa) were found in 71.9% of patients with duodenal ulcer and in 42.6% of patients with chronic gastritis.
Conclusions: Most H. pylori vacA s1 m1 strains are toxic, whereas s2 m2 forms are essentially non-toxic.     

Distinct variants of Helicobacter pylori cagA are associated with vacA subtypes.

The diversity of the cytotoxin-associated gene (cagA) of Helicobacter pylori was analyzed in 45 isolates obtained from nine countries. We examined variation in the 5' end of the cagA open reading frame as determined by PCR and sequencing. Phylogenetic analysis revealed the existence of at least two distinct types of cagA. One variant (cagA1) was found exclusively in strains from Europe, the United States, and Australia, whereas a novel variant (cagA2) was found in strains from East Asia. The greatest diversity between cagA1 and cagA2 was found in the first 20 amino acids of the cagA open reading frame, where several consistent insertions or deletions were observed. Additional cagA sequence variants that could be classified as separate subtypes were found in two of three Peruvian and in five of seven U.S. strains tested. The calculated isoelectric point of the first 154 amino acids of the cagA1 variants (7.52 +/- 1.54) was significantly higher than that of the first 154 amino acids of the cagA2 variants (5.61 +/- 0.94; P < 0.001). Most cagA2 strains contained vacA subtype s1c (P < 0.001), and in vacA m1 strains cagA1 was more frequently observed than cagA2. These results show the epidemiological relationship between cagA and vacA at the subtype level and indicate the existence of distinct H. pylori lineages that are not uniformly distributed over the globe.     

Relationship between Helicobacter pylori iceA, cagA, and vacA Status and Clinical Outcome: Studies in Four Different Countries

There is continuing interest in identifying Helicobacter pylori virulence factors that might predict the risk for symptomatic clinical outcomes. It has been proposed that iceA and cagA genes are such markers and can identify patients with peptic ulcers. We compared H. pylori isolates from four countries, looking at the cagA and vacA genotypes, iceA alleles, and presentation of the infection. We used PCR to examine iceA, vacA, and cagA status of 424 H. pylori isolates obtained from patients with different clinical presentations (peptic ulcer, gastric cancer, and atrophic gastritis). The H. pylori isolates examined included 107 strains from Bogota, Colombia, 70 from Houston, Tex., 135 from Seoul, Korea, and 112 from Kyoto, Japan. The predominant genotype differed among countries: the cagA-positive iceA1 vacA s1c-m1 genotype was predominant in Japan and Korea, the cagA-positive iceA2 vacA s1b-m1 genotype was predominant in the United States, and the cagA-positive iceA2 vacA s1a-m1 genotype was predominant in Colombia. There was no association between the iceA, vacA, or cagA status and clinical outcome in patients in the countries studied. iceA status shows considerable geographic differences, and neither iceA nor combinations of iceA, vacA, and cagA were helpful in predicting the clinical presentation of an H. pylori infection.     

Prevalence of serum antibodies to Helicobacter pylori VacA and CagA and gastric diseases in Chile

The objective of this study was to evaluate the prevalence of antibodies to Helicobacter pylori CagA and VacA proteins and correlate this prevalence with gastric diseases in colonised Chileans. The study was performed in 418 adults colonised with H. pylori: 316 with gastroduodenal pathology (152 duodenal ulcer, 14 gastric cancer and 150 gastritis patients) and 102 asymptomatic subjects. Serum IgG antibodies to H. pylori were determined by enzyme immunoassay (EIA). Antibodies to VacA and CagA proteins were detected by Western blotting. In a subgroup of the patients, the vacuolating activity was determined by HeLa cell assay and the CagA product was confirmed by PCR assay. IgG antibodies to both VacA and CagA proteins of H. pylori were found in 270 (85%) of 316 colonised gastric patients and in 72 (71%) of 102 asymptomatic subjects. Colonisation with virulent strains was significantly higher among duodenal ulcer and gastric cancer patients than in gastritis patients or asymptomatic subjects. Infections with VacA+/ CagA+ H. pylori strains is common in Chile but, in contrast to some Asian countries, this phenotype was more prevalent in isolates from patients with more severe gastric pathologies.     

Clinical and histological associations of cagA and vacA genotypes in Helicobacter pylori gastritis.

AIMS: To determine the relation among the cytotoxin associated gene (cagA) and vacuolating cytotoxin gene (vacA) status of Helicobacter pylori isolates, the associated clinical diseases, and the severity and pattern of chronic gastritis. METHODS: Helicobacter pylori was cultured from gastric biopsies obtained from dyspeptic patients. DNA was extracted from the isolates and the cagA and vacA status determined by the polymerase chain reaction (PCR). The prevalence of the different cagA and vacA genotypes in three clinical groups, duodenal ulcer, gastric ulcer, and non-ulcer dyspepsia was compared. The histological features in sections from two antral and two corpus biopsies were graded by one blinded observer. The grades were compared with age and sex matched groups with different cagA and vacA genotypes, and with duodenal ulcers, or non-ulcer dyspepsia. RESULTS: Isolates from 161 patients were included. One hundred and nine (68%) harboured a cagA+ strain and 143 (89%) harboured a vacA s1 strain. The prevalence of cagA+ strains in duodenal ulcer patients (94%) was highly significantly greater than in those with non-ulcer dyspepsia (56%). However, of the patients infected with a cagA+ strain, almost equal numbers had non-ulcer dyspepsia or peptic ulceration. Chronic inflammation, polymorph activity, surface epithelial degeneration, atrophy, and intestinal metaplasia were all significantly more severe in the cagA+ than in the cagA- group, whereas only corpus epithelial degeneration was significantly more severe in the vacA s1 group compared with the vacA s2 group. Patients infected with cagA+ strains were almost four times more likely to have antral intestinal metaplasia than cagA- patients. An antral predominant gastritis was present in duodenal ulcer patients compared with matched non-ulcer dyspepsia patients, but this was not attributable to cagA or vacA status. CONCLUSIONS: Helicobacter pylori strains showing cagA positively and the vacA s1 genotype are associated with more severe gastritis but these virulence factors do not appear to determine the overall pattern. The pattern is closely linked to clinical disease. Therefore, it is likely that the nature of the disease complicating chronic infection is determined by host and environmental factors, while bacterial factors determine the magnitude of the risk of developing such disease.     

幽门螺杆菌的CagA和VacA的致病机制及其临床意义

幽门螺杆菌(Helicobacterpylori,H.pylori)是消化道的主要致病因素之一,随着对H.pylori认识的深入,人们发现H.pylori的两个标志性毒素--细胞毒素相关蛋白(cytotoxin-associatedprotein,cagA)和空泡毒素(vacuolatingcytotoxin,VacA)不仅关系紧密,而且在致病过程中扮演了重要角色.本文主要阐述CagA和VacA的致病机制和它们的临床意义.     

Antibody against Helicobacter pylori CagA and VacA and the risk for gastric cancer.

AIM: Helicobacter pylori is associated with gastric cancer. Our aim was to investigate whether CagA or VacA seropositivity provides additional risk for gastric cancer. METHODS: Sera from 110 gastric cancer patients were sex and aged matched with asymptomatic controls. H pylori status was determined by IgG enzyme immunoassay (HM-CAP EIA); CagA status was assessed by enzyme linked immunosorbent assay (ELISA) (OraVax) and immunoblotting (Chiron), and VacA status by immunoblotting using recombinant proteins as antigens. RESULTS: H pylori infection was associated with an increased risk of gastric cancer (odds ratio (OR) = 2.19, 95% confidence interval 1.17 to 4.1). Subgroup analysis showed a significant association with intestinal type (OR = 2.94, 1.35 to 6.41), distal type (OR = 2.97, 1.39 to 6.33), early gastric cancer (OR = 3.74, 1.54 to 9.06), and age < or = 55 years (OR = 8.33, 2.04 to 34.08), but not with diffuse type (OR = 0.83), proximal type (OR = 1.0), advanced gastric cancer (OR = 1.13), or age > 55 years (OR = 1.40). Serum CagA IgG and VacA antibody positivity was present in similar proportions in patients with and without cancer, with no significant differences in histological classification, clinical stage, or location (p > 0.3). CONCLUSIONS: H pylori infection causes chronic gastritis and is associated with the development of gastric cancer. Neither CagA nor VacA seropositivity added additional information or stratification.     

vacA Genotypes and Genetic Diversity in Clinical Isolates of Helicobacter pylori

Genetic diversity in Helicobacter pylori strains may affect the function and antigenicity of virulence factors associated with bacterial infection and, ultimately, disease outcome. In this study, DNA diversity of H. pylori isolates was examined by analysis of vacA genotypes and by restriction fragment length polymorphism (RFLP) analysis of H. pylori-associated genes (vacA, cagA, flaA, ureAB, and ureCD). Thirty-seven H. pylori isolates from 26 patients were successfully classified into distinct vacA allelic genotypes. The signal sequence allele s1 (31 of 37) predominated over the s2 allele (6 of 37) and was significantly associated with the occurrence (past or present) of gastric ulcers. A novel midregion allele, designated as m3, has been identified in two H. pylori isolates which could not be typed with midregion allele m1- or m2-specific primers. Additionally, significant nucleotide diversity yielding different amino acid sequences was demonstrated by DNA sequencing of vacA fragments from clinical isolates of H. pylori. Furthermore, RFLP analysis of 45 H. pylori isolates (including 15 paired isolates) obtained from antrum and corpus biopsy specimens from 30 individual patients showed remarkably high interhost diversity (one patient, one H. pylori strain) and intrahost identity in gene sequences coding for VacA, CagA, flagellin, and urease. Only in a single patient was a minor genotypic variation at different anatomic sites within the stomach identified. These data warrant the detailed analysis of the effect of genetic diversity on the function and antigenicity of H. pylori-associated virulence factors.

Helicobacter pylori is one of the most prevalent causes of infection in human beings worldwide. H. pylori persists in the human gastric mucus layer for decades and possibly for life, even in the face of a brisk humoral antibody response (3, 26). The extent of mucosal lesions induced by H. pylori varies, with only a small number of patients developing peptic ulceration and gastric cancer (3, 19). With the increasing emergence of antibiotic-resistant H. pylori strains (27), development of an effective vaccine may represent an alternative means of controlling or even preventing H. pylori infection (5, 21).Individual H. pylori isolates demonstrate a high level of genomic diversity as defined by different techniques, including conventional or pulsed-field gel electrophoresis and restriction endonuclease digestion of genomic DNA, PCR-amplified genomic DNA, individual genes, or ribosomal DNA (1, 9, 10, 14, 24). Genomic differences may affect virulence factors, altering their function and antigenicity. Antigenic variation of certain gene products may represent an immune escape mechanism for H. pylori strains in the host organism.Vacuolating cytotoxin (VacA) is a major H. pylori-associated virulence factor (25, 29), but it may also serve as a target for induction of a strong, long-lived, and effective anti-H. pylori-directed immune response (12, 18, 25). Although almost all H. pylori strains bear the vacuolating cytotoxin gene (vacA) and through expression of this gene produce an immunoreactive protein (2), only 50 to 60% exhibit detectable cytotoxin activity (6, 24). Hence, nonfunctional but immunogenic variants of VacA exist. It was recently reported that the 95-kDa protein isolated from culture supernatants of Tox⁻ strains (the Tox protein being a homolog of VacA) is recognized by an antiserum raised against the H. pylori cytotoxin from Tox⁺ strains but is not capable of inducing cell vacuolation (2). The humoral anti-VacA-directed immune response is predominantly directed against conformational epitopes of the cytotoxin (17). Therefore, the nucleotide or amino acid sequences of cytotoxin and its homolog provide the basis for the design of a genetically “detoxified” molecule which retains the structure and immunogenicity of the native protein.The level of in vitro cytotoxin activity appears to correlate with the clinical consequences of H. pylori infection. VacA functionality in turn correlates with specific vacA genotypes, defined by certain combinations of vacA signal sequences (s1a, s1b, and s2) and vacA midregion alleles (m1 and m2), including s1a-m1, s1b-m1, s1a-m2, s1b-m2, and s2-m2 (2). It is unknown whether these structural vacA differences impact on anticytotoxin-directed immunity.In this study, we employed a PCR-based method to define the vacA genotypes of 37 individual H. pylori isolates from antrum and corpus biopsy specimens obtained from 26 patients undergoing endoscopy. Genetic diversity was characterized in detail by restriction fragment length polymorphism (RFLP) analysis of H. pylori-associated genes (i.e., vacA, cagA, flaA, ureAB, and ureCD) coding for virulence factors (VacA, CagA, flagellin, and urease). This analysis was complemented by sequence analysis of individual vacA and flaA genes.