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. 2005 Feb;43(2):791-5.
doi: 10.1128/JCM.43.2.791-795.2005.

Simple method for determination of the number of Helicobacter pylori CagA variable-region EPIYA tyrosine phosphorylation motifs by PCR

Richard H Argent  1 , Youli Zhang, John C Atherton
Affiliations

Simple method for determination of the number of Helicobacter pylori CagA variable-region EPIYA tyrosine phosphorylation motifs by PCR

Richard H Argent et al. J Clin Microbiol. 2005 Feb.

Abstract

Helicobacter pylori strains possessing the cag pathogenicity island are associated with the development of gastric cancer. The CagA protein is translocated into epithelial cells and becomes phosphorylated on tyrosine residues within EPIYA motifs, which may be repeated within the variable region of the protein. Strains possessing CagA with greater numbers of these repeats have been more closely associated with gastric carcinogenesis. Phosphorylated CagA leads to epithelial cell elongation, which is dependent on the number of variable-region EPIYA motifs. Thus, determination of the degree of CagA phosphorylation and the number of EPIYA motifs appears to be more important than detection of cagA alone. Determination of the number of EPIYA motifs by nucleotide sequencing, however, is a laborious and expensive process. We describe here a novel and rapid PCR method for determination of the pattern of repeats containing the EPIYA motif. This will aid in the identification of those strains that may be more likely to cause disease.

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Figures

FIG. 1.
FIG. 1.
PCR amplification of cagA variable-region TPMs from South African H. pylori strains. (A and C) Genomic DNA from H. pylori strains HP508, GC102, and GC78 (A) and GC77 (C) were used to PCR amplify the cagA variable-region EPIYA motifs, using the forward primer cagA28F and the reverse primers cagA-P1C (P1), cagA-P2CG and cagA-P2TA (equimolar mixture; P2), or cagA-P3E (P3). M, size markers (in base pairs). (B) Schematic representation of the cagA variable region from H. pylori strain GC77 showing the EPIYA motifs (gray), the annealing positions of the cagA28F (A28F) and cag2 (2) forward primers and the cagA-P1C (P1C), cagA-P2TA (P2TA), cagA-P2CG (P2CG), and cagA-P3E (P3E) reverse primers, and the expected sizes of the amplified products.
FIG. 2.
FIG. 2.
PCR amplification of cagA variable-region TPMs from H. pylori strains. Genomic DNA from H. pylori strains J99, AB36, AB5, AB29, Z11, and Z28 were used to amplify the cagA variable-region EPIYA motifs, using the forward primers cagA28F (J99, AB36, AB5, and AB29) or cag2 (Z11 and Z28) and the reverse primers cagA-P1C (P1), cagA-P2CG and cagA-P2TA (equimolar mixture; P2), or cagA-P3E (P3). H. pylori strain J99 lacks the P1 motif, and strain AB36 does not possess cagA. M, size markers (in base pairs).
See this image and copyright information in PMC

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