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Short nucleotide sequence inserts within the signal (s) and mid (m) regions of the vacuolating cytotoxin gene (vacA) of Helicobacter pylori provide the basis for defining the allelic forms widely used for strain typing and as markers for toxin functionality and severity of interactions with host gastric epithelial cells. Here 484 signal region and 411 mid-region sequences (new and from public databases) from 32 countries were analysed to determine the effect of geographical location on insert diversity, which is currently undefined. Short (27 bp) inserts of 52 mol% G+C from 201 sequences (98 %) of the s2 allelic family encoded a highly conserved nine amino acid sequence irrespective of geographical origin. The longer (75 bp) mid-region insert of 38 mol% G+C in 255 sequences of the m2 allelic family was more diverse and represented by 23 peptide variants, with one predominant sequence (MRI type 4) representing 62 % of inserts. Mid-region inserts were widespread throughout European/North American (Western) sequences in the dataset whereas a lower insert frequency was a geographical feature of East Asian sequences. Each insert was preceded by an associated conserved motif that provided a marker of the insertion sites within vacA, and facilitated identification of the Chinese m2b genotype. It is concluded that the observed sequence conservation supports the continued global use of vacA genotyping, and that inserts could have a functional significance in the mature protein, particularly the s2 form of the toxin, as the same combination of signal and mid-region insert type and preinsert motif was highly conserved.

Background & Aims: The Helicobacter pylori cag pathogenicity island encodes a secretory system that translocates CagA into epithelial cells, where it becomes tyrosine phosphorylated and induces cytoskeletal rearrangements. Strains with more CagA tyrosine phosphorylation motifs are most closely associated with gastric cancer. Here we assess whether clinical strains can deliver CagA, whether strains with different numbers of CagA phosphorylation motifs have CagA phosphorylated to different degrees, and whether this induces different amounts of epithelial cytoskeletal change., Methods: Forty-four H. pylori strains from South African patients, all cagA gene positive, were cocultured with the gastric adenocarcinoma cell line AGS. CagA expression and phosphorylation were determined by Western blot and interleukin-8 secretion by enzyme-linked immunosorbent assay. The cagA 3' variable regions of 22 strains were sequenced and shown to possess 3-6 phosphorylation motifs. These strains were used to quantify CagA phosphorylation and cytoskeletal rearrangements., Results: cagA genotype and typing of cag pathogenicity island genes were poorly predictive of phenotype. Thirty-four of 44 strains expressed CagA protein that could be delivered to and phosphorylated within AGS cells. Only these 34 strains induced interleukin-8 secretion from AGS cells. Among those strains, the number of CagA tyrosine phosphorylation motifs determined the degree of CagA phosphorylation and the level of biologic activity in terms of degree and extent of AGS cell elongation., Conclusions: H. pylori strains that deliver CagA with more phosphorylation motifs induce higher levels of CagA phosphorylation in epithelial cells, induce more cytoskeletal changes, and are more likely to be associated with gastric cancer.

Cag pathogenicity island-containing Helicobacter pylori (type I) induces signal transduction pathways resulting in tyrosine phosphorylation of proteins adjacent to the site of bacterial adhesion on host gastric epithelial cells. Conventional block PCR-restriction fragment length polymorphism (RFLP) and real-time LightCycler (LC) PCR hybridization assays, validated by direct sequencing, were designed to test for the presence of three nucleotide sequences corresponding to tyrosine phosphorylation motifs (TPMs) A, B, and C in 84 isolates of H. pylori type I from patients in England. Overall, the PCR assays demonstrated that one or more TPMs were present in 62 strains (75%). Motif A was common (71% of strains), whereas motifs B and C were rarer (8% of strains). Strains lacking a TPM were typically vacuolating cytotoxin genotype vacA m2. Motif A was widely distributed in relation to disease severity and was more commonly (but not significantly [P = 0.071]) associated with gastric ulcer than with duodenal ulcer (86 versus 56%). The LC hybridization assay provided a rapid means of detecting all three motifs, but RFLP analysis was more specific for TPM-A. TPMs provide novel additional strain markers for defining cagA variation, including identification of RFLP types within TPM-A. The presence of a particular TPM was not of direct diagnostic value, either singly or in combination, but the higher proportion of TPM-A strains in gastric ulcer patients merits further investigation.

Isolates of Helicobacter pylori from dyspeptic patients in England and South Africa were tested for ability to induce interleukin-8 (IL-8) in gastric cells. All isolates were cagA-positive, which was used as a marker for the presence of the cag pathogenicity island. The aims were to determine if activities were related to diversity within cagE (HP0544), a locus encoding a key component in the Type IV secretion system, and if disease severity might be linked to a combination of strain features. We found that isolates were heterogeneous in ability to induce IL-8 activity with the 23 positive isolates (59%) showing activities ranging from 260 to 3200 pg ml(-1). The cagE locus was detected in most isolates and RFLP analysis of a 1.52-kb internal fragment showed interstrain diversity with 12 combined (MboI/NlaIII) types. Most cagE genotypes were not associated with IL-8 induction, however two genotypes were found only in IL-8-inducing strains and one genotype was associated with lack of IL-8 induction. IL-8 activity was not associated with either the number or composition of cagA tyrosine phosphorylation motifs and vacA m-type. Although we found a weak association between cagE type and the ability to induce IL-8, our results imply that gastric cell factors or bacterial factors other than vacA, cagA and cagE are involved in the induction of IL-8 and the development of severe gastric disease.