Association between cagA and vacA genotypes and pathogenesis in a Helicobacter pylori infected population from South-eastern Sweden
© Karlsson et al.; licensee BioMed Central Ltd. 2012
Received: 30 January 2012
Accepted: 21 June 2012
Published: 2 July 2012
Chronic gastritis, peptic ulcer disease, and gastric cancer have been shown to be related to infection with Helicobacter pylori (H. pylori). Two major virulence factors of H. pylori, CagA and VacA, have been associated with these sequelae of the infection. In this study, total DNA was isolated from gastric biopsy specimens to assess the cagA and vacA genotypes.
Variations in H. pylori cagA EPIYA motifs and the mosaic structure of vacA s/m/i/d regions were analysed in 155 H. pylori-positive gastric biopsies from 71 individuals using PCR and sequencing. Analysis of a possible association between cagA and vacA genotypes and gastroduodenal pathogenesis was made by logistic regression analysis. We found that H. pylori strains with variation in the number of cagA EPIYA motif variants present in the same biopsy correlated with peptic ulcer, while occurrence of two or more EPIYA-C motifs was associated with atrophy in the gastric mucosa. No statistically significant relation between vacA genotypes and gastroduodenal pathogenesis was observed.
The results of this study indicate that cagA genotypes may be important determinants in the development of gastroduodenal sequelae of H. pylori infection. In contrast to other studies, vacA genotypes were not related to disease progression or outcome. In order to fully understand the relations between cagA, vacA and gastroduodenal pathogenesis, the mechanisms by which CagA and VacA act and interact need to be further investigated.
H. pylori is a microaerophilic, spiral shaped Gram-negative bacterium that chronically infects the gastric mucosa. It is recognised as a human pathogen associated with chronic gastritis, peptic ulcer and gastric cancer, the development of which are related to the virulence factors cytotoxin associated antigen (CagA)[4, 5] and vacuolating cytotoxin A (vacA)[6, 7]. It has been reported that CagA and VacA polymorphisms are associated with distinct pathological features in H. pylori infected adults with gastrointestinal diseases[8–14].
H. pylori cagA and vacA gene polymorphisms are well studied and it is assumed that these polymorphisms, alone or in concert, are associated in H. pylori associated pathogenesis[9, 10, 13, 42, 43]. However, some studies have reported a lack of association between H. pylori cagA and vacA gene polymorphisms and the severity or progression of H. pylori associated diseases[25, 44].
Statistical outcome is dependent on the population studied. We aimed to analyse a randomly selected population in South-eastern Sweden with regard to H. pylori cagA and vacA genotypes and sequelae using logistic regression analysis. By means of a previously described PCR-based strategy[45, 46] we assessed variations of cagA EPIYA and vacA s/m/i/d mosaic structure present in H. pylori DNA isolated from 155 fresh frozen (−80°C) gastric biopsy specimens.
Presence of H. pylori DNA in the gastric biopsy specimens
Using MDA-DNA and 16S rDNA variables V3 region pyrosequencing analysis, the presence of H. pylori-DNA in all 155 biopsy specimens was confirmed.
Analysis of cagA EPIYA motifs
Statistical analysis revealed that H. pylori strains with different number of cagA EPIYA motif variants present in the same biopsy was correlated to peptic ulcer development, OR = 2.77 (1.10-7.00). In the present study, peptic ulcer included four cases of duodenal ulcers, three pre-pyloric ulcers, two gastric ulcers and five cases of previously diagnosed ulcers of undefined origin (no data available).
Two or more cagA EPIYA-C motifs were associated with development of gastric atrophy, OR = 1.86 (1.05-3.30). In biopsies with mixed amplicons, the number of EPIYA-C was determined from the amplicon with the highest number of repeats. Gastritis was histologically classified according to the Sydney system, and atrophy of the gastric mucosa was graded from 1–3 (1 = mild, 2 = moderate, 3 = severe). For the purpose of the present study, moderate to severe atrophy of the gastric mucosa was classified as atrophic gastritis.
Statistical calculations were performed also in subgroups based on the location in the stomach (corpus, antrum). No differences were observed between the groups regarding their respective disease progression.
Analysis of cagE and cag-PAI empty-site
To detect deletions of cagA within cagPAI, a region surrounding cagA (cag-PAI empty site) was amplified, as well as the cagE gene (also located within the cag-PAI). Amplification of cagE was successful in 114 of the biopsies. Of the remaining 41 biopsies, only 19 successfully amplified the cag-PAI empty site region, indicating the presence of mutated primer target sites or absence of cagE.
Analysis of vacA s/i/d/m-region
Four regions of the vacA gene (s, m, i and d regions) were genotyped. PCR amplification and DNA sequence analysis in 155 H. pylori positive biopsy specimens revealed full information from all regions of vacA in 146 samples. Of the samples genotyped in the s region, the majority were of s1a (130) or s1b (19) genotype, while only three samples were s2 genotype. In the m region the distribution was more even, with 87 samples of m1 genotype and 64 samples of m2 genotype. DNA from 32 of the biopsies displayed a deletion of the d region (d2), while 115 isolates showed wild-type sequence (d1) in this region. The intermediate region is classified according to two different sequences, and may be of i1, i2, i1-i2 or i2-i1 genotype. In this material, 94 isolates were of i1 genotype, 24 isolates of i2 genotype and 31 isolates of i2-i1 genotype. None were of i1-i2 type.
In group 3, there were two isolates (6%) derived from peptic ulcer patients, while in group 1 and 2 there were 20 isolates (24%) and eight isolates (27%), respectively, originating from ulcer patients. The lower frequency of peptic ulcer observed in vacA s1d1m1 genotype compared to other genotypes was not statistically significant. Eight biopsies from group 1 (10%) and two biopsies from group 2 (7%) were derived from patients with atrophic gastritis, while in group three there was no subject with atrophic gastritis (not statistically significant).
Intraindividual variations of cagA EPIYA and vacA genotypes in corpus, antrum and duodenal bulb
In 51 of 71 individuals, biopsy specimens from all three locations of the stomach (corpus, antrum and duodenal bulb) were available for analysis. In 26 of these 51 subjects, the cagA and vacA genotypes were identical in all locations. Considering the remaining 25 subjects, 22 subjects differed with respect to the cagA EPIYA genotype, two with regard to the vacA (i) genotype, two considering the vacA (d) genotype and one with respect to the vacA (m) genotype, when comparing the locations for each subject (Additional file1).
The results of several studies have indicated that there is an association between the cagA gene and gastric cancer[14, 27, 28, 48]. There are also reports showing an association between the vacA gene and gastroduodenal sequelae (e.g. peptic ulcer, atrophic gastritis) of H. pylori infection[36, 38–40].
Here we show that of the individuals with biopsies from all locationsns (corpus, antrum and duodenal bulb), 49% had different cagA EPIYA genotype between the three locations. There is a possibility that these individuals may have been infected with different strains on different occasions. However, it is perhaps more likely those H. pylori strains acquired genetic alterations in cagA after infection. Three recombination mechanisms have been detected in the cagA gene; homologous recombination between CM sequences, recombination between EPIYA sequences or between short similar sequences. These recombination mechanisms, as well as mutations in the gene, may serve as a driving force for generating strain diversity in H. pylori, also called microevolution. It is possible that infection with multiple H. pylori ancestral strains or alternatively, a single ancestral strain undergoing microevolution in the cagA gene giving rise to H. pylori subclones with different cagA EPIYA motif variants in the same biopsy specimen, may be more aggressive than a single ancestral strains acting alone. In an early study it has been suggested that the majority of Swedish clinical isolates of H. pylori from patients of higher age (>63 years old) represent single strain infections. However, in younger ages multiple strain infection may be more common. Furthermore, it has been discussed that different subclones of each strain, some of which might be cagA-positive or -negative, may coexist, possibly colonising different areas of the stomach during different periods of life-long H pylori infection. In this context, the aim of this study was to investigate a possible association between the presence of H. pylori cagA EPIYA motifs and disease outcome. We found an association between H. pylori DNA isolated from the same biopsy specimen and generating two or more cagA EPIYA motif variant amplicons and peptic ulcer OR = 2.77 (1.10-7.00).
Gastric atrophy was associated with two or more EPIYA-C motifs in the cagA gene of the biopsy (corpus and antrum only) H. pylori strains, OR = 1.86 (1.05-3.30). Previous studies have also found this correlation[14, 27] and it has been suggested that a higher number of EPIYA-C motifs enables a higher degree of phosphorylation, and, hence, increases the risk of gastric cancer and gastric intestinal metaplasia. One explanatory mechanism in this aspect may be the interaction of CagA with the protein ASPP2, which normally activates p53 to induce apoptosis. CagA inhibits ASPP2, leading to an increased cell survival and enhanced transformation of the cell.
Other studies have shown an association of gastric cancer and atrophy to the s1 genotype, the s1m1 genotype, or the i1 genotype[27, 37–39]. In the present study, we detected a higher frequency of atrophy among the vacA s1d1m1 genotype than among other genotypes. However, none of these results were statistically significant, which could be due to small or unevenly distributed groups of samples (type II error).
Miernyk and co-workers observed an increased risk of developing peptic ulcer disease in s1m1 compared to s1m2/s2m2 vacA genotype. Our study shows a tendency towards a similar association, although not statistically significant.
In summary, H. pylori strains with variation in the number of cagA EPIYA motif variants present in the same biopsy were associated with the occurrence of peptic ulcer. Similarly, two or more EPIYA-C motifs were associated with atrophy in the gastric mucosa. No statistically significant association between vacA genotypes and gastroduodenal pathogenesis was observed.
Study subjects and tissue collection
Number of individuals with biopsies from respective location
Individuals with different biopsy combinations1
DNA was isolated from the homogenized tissue using an automated nucleic extractor M48 and MagAttract DNA Mini M48 kit following the manufacturer’s instruction (Qiagen, Hilden, Germany). The isolated DNA was enriched by whole genome amplification by means of multiple displacement amplification (MDA), using an Illustra GenomiPhi V2 DNA kit (GE-Healthcare, Uppsala, Sweden) according to standard protocols.
Initially, the presence H. pylori DNA in the biopsy specimens were verified using 16S rDNA V3 region pyrosequencing analysis.
Primers used for PCR amplification in the study
5' > 3'1
VacA (i + d)
Empty site CagA
VacA s/i/d/m region subtyping was accomplished by three single PCR amplification assays. The signal-sequence (SS) region was amplified using primer M13-SeqS.se and SeqS.as; the intermediate and deletion region (IR and DR) using primer M13-SeqVac.se and SeqVac.as; the midregion (MR) using primer M13-SeqM.se and VAG-R (Figure 2; Table 2), respectively
Amplification conditions used were identical in all assays as described previously. Prior to sequencing, amplicons were analysed by automated capillary gel electrophoresis using a QIAxcel system and a QIAxcel DNA High Resolution kit (Qiagen, Hilden, Germany).
cagA EPIYA motif and vacA s/i/d/m-region sequence analysis
M13-tagged cagA EPIYA and vacA amplicons were sequenced using M13 uni (−21) sequencing primer and a customer sequencing service (Eurofins MWG Operon, Ebersberg, Germany). The obtained cagA and vacA sequences were aligned and compared with catalogued H. pylori 26695 [GenBank:AE000511, H. pylori J99 [GenBank:AE001439], H. pylori P12 [GeneBank:CP001217], H. pylori G27 [GenBank:CP001173], and H. pylori Shi470 [GeneBank:CP001072] sequences using the CLC DNA Workbench version 5.5. Sequences were retrieved from the NCBI nucleotide database. CagE and cag-PAI (empty-site) amplicon sizes were analysed by capillary gel electrophoresis only.
Binary logistic regression analysis of data was performed using Minitab 15 software. Statistical significance was assumed at P < 0.05. All statistical analyses presented here were significant according to Hosmer-Lemeshow (HL) goodness-of-fit test, with HL p values >0.05. In the logistic regression analysis and the GLM analysis, a 95% confidence interval including 1.0 was regarded as non-significant. Odds ratios with 95% confidence intervals (CI) were calculated to explore possible associations of individual genotypes to peptic ulcer or gastric atrophy. Age and sex were included as covariates. With regard to atrophy, data from duodenal biopsies were not included in the statistical analysis.
The study was supported by grants from the Research Council in the South-East of Sweden (FORSS, the ALF program, the committee for medical R&D, and the Molecular Biology program at Clinical Microbiology, Laboratory Medicine Centre-DC, University Hospital, Linköping, Sweden. We are grateful to Statistician Olle Eriksson, PhD, for statistical calculations and advice.
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