Determination of Helicobacter pylori virulence by simple gene analysis of the cag pathogenicity island

Nucleic acid amplification was performed for five loci in the cag pathogenicity island (PAI) of Helicobacter pylori (comprising cagA, the cagA promoter region, cagE, cagT, and the left end of cagII [LEC]), and gastric inflammation in patients was evaluated. Of 204 H. pylori isolates from Japanese patients (53 with peptic ulcer, 55 with gastric cancer, and 96 with chronic gastritis), 197 (96.6%) were positive for all five loci. Two isolates (1%) were negative for all five loci, and five isolates (2.4%) were positive for only cagA and LEC. These latter seven isolates were all from patients with mild chronic gastritis. Neutrophil infiltration in gastric mucosa was significantly milder in patients infected with partially or totally deleted-PAI strains than in those with intact-PAI strains. The cagE gene was a more accurate marker of an intact cag PAI than the cagA gene, and cagE seemed to be more useful in discriminating between H. pylori strains causing different rates of disease progression.     

implication of the structure of the Helicobacter pylori cag pathogenicity island in induction of interleukin-8 secretion

Helicobacter pylori virulence is associated with the presence of the cag pathogenicity island (PAI). The cag PAI is involved in the ability to induce interleukin-8 (IL-8) secretion by human cells, which is implicated in the inflammatory response of the gastric mucosa to H. pylori infection. The aim of this study was to determine whether the genetic structure of the cag PAI is conserved and whether it is linked to IL-8 induction ability. Detection of specific markers (cagA, picB, cag13-cag14, virD4, and IS605) by PCR and dot blot hybridization and long-distance PCR determination of the presence of cagI, cagII, and the middle region of the cag PAI were performed on 153 strains isolated from adults suffering from ulcers (n = 79) or gastritis (n = 74). IL-8 induction ability was evaluated by coculture of the strains with HEp-2 cells. Eighty-three strains (54.3%) had an entire cag PAI, 12 strains (7.8%) had the cag PAI split in two, 49 strains (32%) had no cag PAI, and 9 strains exhibited other structural combinations. The presence of an entire cag PAI was statistically correlated with the presence of IS605 (P = 0.006) and the ability to induce IL-8 secretion but not with clinical presentation of the infection. The structure of the cag PAI appears to be rather conserved and is related to the proinflammatory power of a strain. The existence of strains inducing IL-8 secretion regardless of the cag PAI structure suggests that this region is not the only requirement for IL-8 secretion.     

Genetic diversity in the Helicobacter pylori cag pathogenicity island and effect on expression of anti-CagA serum antibody in UK patients with dyspepsia

AIMS: To investigate variation within the cag pathogenicity island (PAI) of Helicobacter pylori isolated from patients with dyspepsia in mid-Essex, and to evaluate the effect on expression of anti-CagA antibody. METHODS: Sixty two isolates of H pylori cultured from gastric biopsies were screened by specific PCR assays for the presence of cagA and other gene markers (cagD and cagE, and virD4) in the cag PAI. An enzyme linked immunosorbent assay (ELISA) kit (Viva Diagnostica helicobacter p120) was used to test for anti-CagA IgG antibody in matching sera. Isolates were also genotyped by vacuolating cytotoxin polymerase chain reaction (PCR) analysis, and tested for absence of the complete cag PAI (empty site PCR assay). RESULTS: Forty one of the H pylori isolates had a cag PAI containing cagA. One strain had no cagA but other cag PAI loci were present, whereas the remaining 20 strains had no detectable cag PAI markers. Anti-CagA IgG antibody was detected in 34 sera by the ELISA assay, and when compared with the cag PAI genotype of the infecting strain, accuracy, sensitivity, and specificity were 92%, 87%, and 100%, respectively. The seven discrepant or borderline strains in the ELISA were all vacA s1 but differed in other genotypic markers. CONCLUSIONS: The cag PAI was widely distributed in H pylori from patients with dyspepsia in mid-Essex who had different gastric pathologies. Infection with a strain having an uninterrupted cag PAI was associated with the presence of anti-CagA antibody in most patients. Discrepant ELISA results, mostly for elderly patients with duodenal ulcers, were attributed to cagA associated variation, particularly to the presence of mixed cagA+/cagA- cell variants in the infecting strain population. Tests for anti-CagA serum antibody were unreliable for predicting severity of clinical disease associated with H pylori infection in this series of patients.     

Serologic detection of infection with cagA+ Helicobacter pylori strains.

Approximately 60% of Helicobacter pylori isolates possess the cagA gene and express its 120- to 140-kDa product (CagA). In this study, the cagA gene was detected in H. pylori isolates from 26 (81.3%) of 32 patients with duodenal ulcers (DU), 17 (68.0%) of 25 patients with gastric ulcers, and 23 (59.0%) of 39 patients with nonulcer dyspepsia (NUD). By Western blotting (immunoblotting) with antiserum to CagA, in vitro CagA expression was demonstrated for 95.5% of cagA+ strains compared with 0% of strains lacking cagA. Sera from patients infected with cagA+ strains (n = 66) reacted with recombinant CagA in an enzyme-linked immunosorbent assay to a significantly greater extent than either sera from patients infected with strains lacking cagA (n = 30) or sera from uninfected persons (n = 25) (P < 0.001). A strain lacking cagA was isolated from eight patients who had serum immunoglobulin G antibodies to CagA, which suggests that these patients were infected with multiple strains. Serum immunoglobulin G antibodies to CagA were present in 87.5, 76.0, and 56.4% of patients with DU, gastric ulcers, and NUD, respectively (odds ratio, 5.41; 95% confidence interval, 1.44 to 24.72; P = 0.004 [DU versus NUD]). These data demonstrate an association between infection with cagA+ H. pylori and the presence of duodenal ulceration and indicate that serologic testing is a sensitive method for detecting infection with cagA+ strains.     

Correlation between cag pathogenicity island composition and Helicobacter pylori-associated gastroduodenal disease

Helicobacter pylori infection is associated with a variety of outcomes ranging from seemingly asymptomatic coexistence to peptic ulcer disease and gastric cancer. The cag pathogenicity island (PAI) contains genes associated with a more aggressive phenotype and has been suggested to be a determinant of severe disease outcome. The cagA gene has served as a marker for the cag PAI. However, the presence of this single gene does not necessarily indicate the presence of a complete set of cag PAI genes. We have analyzed the composition of the cag PAI in 66 clinical isolates obtained from patients with duodenal ulcer, gastric cancer, and nonulcer dyspepsia. Hybridization of DNA to microarrays containing all the genes of the cag PAI showed that 76 and 9% of the strains contained all or none of the cag PAI genes, respectively. Partial deletions of the cag PAI were found in 10 isolates (15%), of which 3 were cagA negative. The ability to induce interleukin-8 (IL-8) production in AGS cells was correlated to the presence of a complete cag PAI. Strains carrying only parts of the island induced IL-8 at levels significantly lower than those induced by cag PAI-positive isolates. The presence of an intact cag PAI correlates with development of more severe pathology, and such strains were found more frequently in patients with severe gastroduodenal disease (odds ratio, 5.13; 95% confidence interval, 1.5 to 17.4). Partial deletions of the cag PAI appear to be sufficient to render the organism less pathogenic.     

Distinct diversity of the cag pathogenicity island among Helicobacter pylori strains in Japan

The severity of Helicobacter pylori-related disease is correlated with the presence of a cag pathogenicity island (PAI). Genetic diversity within the cag PAI may have a modifying effect on the pathogenic potential of the infecting strain. We analyzed the complete cag PAI sequences of 11 representative Japanese strains according to their vacA genotypes and clinical effects and examined the relationship between the diversity of the cag PAI and clinical features. The cag PAI genes were divided into two major groups, a Western and a Japanese group, by phylogenetic analysis based on the entire cag PAI sequences. The predominant Japanese strains formed a Japanese cluster which was different from the cluster formed by Western strains. The diversity of the cag PAI was associated with the vacA and cagA genotypes. All strains with the s1c vacA genotype were in the Japanese cluster. In addition, all strains with the East Asian-type cagA genotype were also in the Japanese cluster. Patients infected with the Japanese-cluster strain had high-grade gastric mucosal atrophy. These results suggest that a distinct diversity of the cag PAI of H. pylori is present among Japanese strains and that this diversity may be involved in the development of atrophic gastritis and may increase the risk for gastric cancer.     

Genomic Comparison of cag pathogenicity island (PAI)-positive and -negative Helicobacter pylori strains: identification of novel markers for cag PAI-positive strains

In an analysis of Helicobacter pylori genomic DNA by macroarray methodology, genomic DNA from a panel of cag pathogenicity island (PAI)-negative H. pylori clinical isolates failed to hybridize with 27 genes located outside the cag PAI in a cag PAI-positive reference strain. PCR analyses confirmed that HP0217 (encoding a lipopolysaccharide biosynthetic protein) and HP1079 (encoding a protein of unknown function) were present significantly more frequently in cagA-positive strains than in cagA-negative strains. A low G+C content of these two genes suggests they were acquired by horizontal transfer events.     

DNA-level characterization of Helicobacter pylori strains from patients with overt disease and with benign infections in Bangladesh

The complex relation between the genotype of Helicobacter pylori and its association with clinical outcome is not well understood. Studies in the West have showed that strains expressing certain virulence factors (vacAs1, vacAm1, and cagA) are associated with duodenal ulcer disease. However, the H. pylori genotype is known to vary with geographic region. In the present study, we compared several virulence markers (cagA, vacA, and iceA) and neutral markers (IS605, IS606, and IS608) in H. pylori strains isolated from 65 adult patients with peptic ulcer (PU) and 50 patients with nonulcer dyspepsia (NUD). PCR tests indicated that cagA is present in 75% of the strains from patients with PU compared to 55% in patients with NUD, and 80% of the isolates from patients with PU carried potentially toxigenic vacAs1 alleles of the vacuolating cytotoxin gene (vacA) compared to 60% in isolates from patients with NUD. However, no significant difference in any other virulence marker was observed in isolates from both groups. Phylogenetic analysis of the vacA middle region and the 5' end of the cagA gene indicates that Bangladeshi isolates are more closely related to H. pylori isolates from India and are different from isolates from East Asia.     

Heterogeneity of cag genotypes in Helicobacter pylori isolates from human biopsy specimens

The Helicobacter pylori chromosomal cluster of genes known as the cytotoxin-associated gene (cag) island may have different compositions in infecting strains. In this study, we analyzed 150 single colonies obtained from gastric biopsy specimens from 10 patients infected with cagA-positive H. pylori strains and sweep isolates (isolates harvested with sweep in different points of the plate) from 6 patients infected with cagA-negative strains. Three loci in the cag island (cagA, cagE, and virB11) and the conserved gene glmM (ureC) were investigated by PCR. The levels of anti-H. pylori and anti-CagA antibodies in patient sera were also measured. For subjects infected with cagA-negative strains, all sweep isolates were also negative for cagE and virB11, suggesting the complete absence of the cag island. For subjects infected with cagA-positive strains, most of the isolates were positive for all three genes studied, whereas 24.7% of the isolates had a partial or total deletion of the cag island. cagA, cagE, and virB11 were, respectively, present in 87.3, 77.3, and 90% of the colonies. The deletion of virB11 was always associated with the deletion of cagA and/or cagE. H. pylori colonies with different cag genotypes were isolated within a single gastric biopsy specimen from 3 of the 10 patients and were further characterized by random amplified polymorphic DNA (RAPD) analysis and by sequencing of an arbitrarily selected gene segment. Although the colonies had different cag genotypes, their RAPD profiles were highly similar within each patient, and the nucleotide sequences of the selected gene segment were identical. All of the patients had detectable antibodies against H. pylori, and 9 of 10 had anti-CagA antibodies. In conclusion, we show that a single infecting H. pylori strain may include variable proportions of colony subtypes with different cag genotypes. The extension of our analysis to patients with well-characterized gastric diseases may provide significant information on the relationship between cag genotypes and clinical outcomes of H. pylori infections.     

Host cell responses to genotypically similar Helicobacter pylori isolates from United States and Japan.

Associations of Helicobacter pylori genotypes with disease differ between Western countries and Asia. Therefore, we directly compared histopathological and in vitro responses to clinical isolates with similar genotypes. Sixty-three cagA(+) vacAs1/m1 H. pylori isolates (United States, n = 24; Japan, n = 39) and eight cagA-negative vacAs2/m2 strains were incubated with AGS cells, and supernatants were assayed for interleukin-8 (IL-8) and for DNA fragmentation. CagA tyrosine phosphorylation in AGS cells and the sequence of the putative HP0638 (oipA) signal sequence region were determined for 22 representative strains. HP0638 and/or cag island mutant strains were created and examined in IL-8 and CagA tyrosine phosphorylation assays. Levels of IL-8 induction and DNA fragmentation were similar in the U.S. and Japanese cagA(+) vacAs1/m1 isolates. All 10 of the isolates with the highest IL-8 induction and 8 of the 10 isolates with the lowest IL-8 induction had an in-frame oipA open reading frame, and all 10 of the isolates with the highest IL-8 induction and 7 of the 10 isolates with the lowest IL-8 induction induced CagA tyrosine phosphorylation in AGS cells. Eight isolates from gastric ulcer patients induced significantly more apoptosis in vitro, and more severe gastritis and atrophy in vivo, than other Japanese isolates. Disruption of HP0638 did not affect IL-8 induction or CagA tyrosine phosphorylation. Thus, H. pylori cagA(+) vacAs1/m1 isolates from the United States and Japan induce similar IL-8 and apoptosis levels. Inactivation of HP0638 does not alter epithelial responses mediated by the cag island in vitro. Assessment of apoptosis in vitro identified a group of H. pylori isolates that induce more severe gastric inflammation and atrophy.     

Genetic characterisation of Helicobacter pylori isolates from an Argentinean adult population based on cag pathogenicity island right-end motifs, lspA–glmM polymorphism and iceA and vacA genotypes

Isolates of Helicobacter pylori from 88 patients were characterised by cagA status, cagA pathogenicity island (PAI) right-end motifs, iceA, vacA and lspA-glmM genotypes, primarily by PCR-based analysis, to investigate whether Argentinean isolates differed from those recovered in southern Europe or other Latin American countries. PCR-based analysis of vacA alleles was confirmed by reverse hybridisation in 56 cases, while sequence analysis was performed either when iceA and vacA genotypes could not be determined by PCR, or to investigate PCR and reverse hybridisation vacA genotyping discordance. Typing by lspA-glmM restriction fragment length polymorphism was performed with HhaI and AluI. The pattern of cag PAI right-end motifs and the prevalence of type Ia were similar to those in isolates from southern European countries, with cagA(+)/iceA1/vacA-s1 m1 being the commonest genotype. Reverse hybridisation identified a vacA-s1a/s1b recombinant allele, confirmed by sequencing analysis. Analysis of lspA-glmM genotypes identified at least 73 unrelated strains. Few mixed infections were identified, but in one case, isolates from a single biopsy exhibiting two vacA alleles were shown by lspA-glmM fingerprints to be two unrelated strains. No associated effect on ulcer disease risk was demonstrated by analysis of cagA, vacA and iceA status. Overall, the isolates of H. pylori from Argentina were similar to isolates from southern Europe or Latin American countries, and infections were associated mainly with single H. pylori strains.     

Tyrosine phosphorylation of CagA from Chinese Helicobacter pylori isolates in AGS gastric epithelial cells

Helicobacter pylori strains possessing the cag pathogenicity island (PaI) are associated with the development of gastroduodenal diseases, including gastric cancer. cag PaI products induce the secretion of interleukin-8 (IL-8) from epithelial cells and facilitate the translocation of CagA into the cell cytosol. In East Asia, where the incidence of gastric cancer is high, most strains possess the cag PaI. To date, however, no cag PaI phenotypic data have been provided for strains isolated in mainland China. Here we used 31 Chinese strains to determine the genotypic and phenotypic status of the cag PaI. All strains possessed cagA and cagE, and we observed a variation in the length of cagA variable regions. Nucleotide sequencing of the cagA variable region revealed that CagA was of two types, a short "Western" form with two tyrosine phosphorylation sites and a longer "East Asian" form with three tyrosine phosphorylation sites. Coculture of strains with AGS epithelial cells showed that strains could induce IL-8 secretion from the cells and that CagA with three phosphorylation sites became more phosphorylated than that with two and could induce significantly (P < 0.001) more cells to elongate. We hypothesize that the preponderance of the more active East Asian form of cagA may underlie the high rate of gastric cancer in China.     

The cag pathogenicity island of Helicobacter pylori is disrupted in the majority of patient isolates from different human populations

The cag pathogenicity island (cag-PAI) is one of the major virulence determinants of Helicobacter pylori. The chromosomal integrity of this island or the lack thereof is speculated to play an important role in the progress of the gastroduodenal pathology caused by H. pylori. We determined the integrity of the cag-PAI by using specific flanking and internally anchored PCR primers to know the biogeographical distribution of strains carrying fully integral cag-PAI with proinflammatory behavior in vivo. Genotypes based on eight selected loci were studied in 335 isolates obtained from eight different geographic regions. The cag-PAI appeared to be disrupted in the majority of patient isolates throughout the world. Conservation of cag-PAI was highest in Japanese isolates (57.1%). However, only 18.6% of the Peruvian and 12% of the Indian isolates carried an intact cag-PAI. The integrity of cag-PAI in European and African strains was minimal. All 10 strains from Costa Rica had rearrangements. Overall, a majority of the strains of East Asian ancestry were found to have intact cag-PAI compared to strains of other descent. We also found that the cagE and cagT genes were less often rearranged (18%) than the cagA gene (27%). We attempted to relate cag-PAI rearrangement patterns to disease outcome. Deletion frequencies of cagA, cagE, and cagT genes were higher in benign cases than in isolates from severe ulcers and gastric cancer. Conversely, the cagA promoter and the left end of the cag-PAI were frequently rearranged or deleted in isolates linked to severe pathology. Analysis of the cag-PAI genotypes with a different biogeoclimatic history will contribute to our understanding of the pathogen-host interaction in health and disease.     

Topographical localisation of cagA positive and cagA negative Helicobacter pylori strains in the gastric mucosa; an in situ hybridisation study

BACKGROUND: The cagA gene is a marker for the presence of the cag pathogenicity island, and the presence of cagA positive strains of Helicobacter pylori can identify individuals with a higher risk of developing gastrointestinal diseases. AIMS: To study the interaction between H. pylori cagA(+) and cagA(-) strains and the gastric mucosa. METHODS: Patients with H. pylori associated gastritis and peptic ulcers were studied. Biopsies were obtained from the antrum, corpus, fundus, and incisura for H pylori culture, and for in situ hybridisation studies. From each biopsy, multiple single H. pylori colonies were isolated and propagated for DNA isolation, and cagA was detected by the polymerase chain reaction (PCR). For in situ detection of H. pylori an oligonucleotide specific for an H. pylori common antigen and an oligonucleotide specific for cagA were used as probes. Biotinylated probes were incubated with biopsy sections, developed with streptavidin-horseradish peroxidase, and amplified with the tyramide system. RESULTS: PCR results for cagA in isolated colonies confirmed the in situ hydridisation studies. In situ hybridisation identified cagA(+) bacteria in patients with cagA(+) isolates; cagA(-) bacteria in patients with cagA(-) isolates, and cagA(+) and cagA (-) bacteria in patients with both cagA(+) and cagA(-) isolates. CagA(-) bacteria usually colonised the mucous gel or the apical epithelial surface, whereas cagA(+) bacteria colonised the immediate vicinity of epithelial cells or the intercellular spaces. CONCLUSIONS: These results document a different in vivo interaction between H. pylori cagA(+) or cagA(-) strains and the gastric mucosa.     

The Helicobacter pylori cag pathogenicity island protein CagN is a bacterial membrane-associated protein that is processed at its C terminus

Helicobacter pylori infects nearly half the world's population and is associated with a spectrum of gastric maladies. Infections with cytotoxin-associated gene pathogenicity island (cag PAI)-containing strains are associated with an increased risk for gastric cancer. The cag PAI contains genes encoding a type IV secretion system (T4SS) and a delivered effector, CagA, that becomes tyrosine phosphorylated upon delivery into host cells and initiates changes in cell signaling. Although some cag PAI genes have been shown to be required for CagA delivery, a subset of which are homologues of T4SS genes from Agrobacterium tumefaciens, the majority have no known function or homologues. We have performed a detailed investigation of one such cag PAI protein, CagN, which is encoded by the gene HP0538. Our results show that CagN is not delivered into host cells and instead is associated with the bacterial membrane. We demonstrate that CagN is cleaved at its C terminus by a mechanism that is independent of other cag PAI proteins. Finally, we show that a delta cagN mutant is not impaired in its ability to deliver CagA to gastric epithelial cells and initiate cell elongation.     

Assessment of the cag pathogenicity island status of Helicobacter pylori infections with serology and PCR

The serological characterisation of Helicobacter pylori strains has been questioned, e.g., when the presence or absence of the cag pathogenicity island (PAI) is determined. This study compared CagA-reactive serum antibodies, assessed with immunoblot, with cag PAI status, as determined by PCR. CagA serology results were available for 101 individuals contributing 202 bacterial samples for cag PAI PCR. There was a high degree of correlation between the two methods (kappa coefficient, 0.82; 95% confidence interval, 0.68-0.97). Combined with suggested biological explanations for the discrepancies, this finding supports the application of well-evaluated serological assays in the assessment of the cag PAI status of H. pylori infections in clinical and epidemiological studies.