Detection of CagA EPIYA motifs in H.pylori DNA extracted from recently collected, frozen, or deparaffinized biopsies and clinical samples

Abstract

CagA is a major virulence factor of Helicobacter pylori that, once injected into the epithelial cells and phosphorylated on specific bacterial tyrosine residues within repeating EPIYA-A,-B,-C, and -D motifs, localizes to the plasma membrane and interacts with a number of intracellular effectors suggested to play an important role in Helicobacter pylori pathogenesis. EPIYA-D (in East Asian CagA) and EPIYA-C motifs (in Western CagA) are the main sites of CagA phosphorylation and the presence both of EPIYA-D or an increasing number of EPIYA-C motifs, rather than the general CagA positivity, has been associated with more severe gastroduodenal disease. With the aim to analyze EPIYA motifs in 24 cagA+ H.pylori isolates and in a number of recently collected, frozen, or, in particular, deparaffinized biopsies and clinical sample, all obtained from 62 patients with different H.pylori pathology, we comparatively evaluated EPIYA profiles by polymerase chain reaction amplification using single sets of primers flanking the variable EPIYA coding region, or a single forward primer and multiple reverse primers specific for the individual EPIYA motifs. Only the primers originally employed by Rudi et al. (1988)to amplify the variable 3'' region of the cagA gene allowed identification of EPIYA motifs in all biopsies and clinical samples. Multiple infections and EPIYA profiles with more than one EPIYA-C motif, in some cases confirmed by DNA sequencing, were observed in 12 and 22 patients, respectively. As expected, the increasing numbers of EPIYA-C motifs were associated with more severe gastric pathologies.