Identification of cagA tyrosine phosphorylation DNA motifs in Helicobacter pylori isolates from peptic ulcer patients by novel PCR-restriction fragment length polymorphism and real-time fluorescence PCR assays

Abstract

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.

FIG. 1.

Schematic representation of H. pylori cagA showing locations of TPMs A, B, and C; primer and probe positions for each LC-PCR assay; and primer sites and motif-determining restriction enzyme cut sites for TPM-A and TPM-C RFLP assays. The diagram is not drawn to scale.

FIG. 2.

Examples of PCR-RFLP analysis of cagA TPM regions to detect the presence of motifs A and C in H. pylori isolates from mid-Essex. (A) HinfI digestion of the 356-bp amplicon separated by agarose gel electrophoresis to detect the presence of TPM-A. Lanes: 1 and 8, X174 HaeIII ladder; 2, NCTC 11637 (TPM-A negative, RFLP type H5); 3, NCTC 11638 (TPM-A positive, RFLP type H3); 4, ATCC 43526 (TPM-A negative, RFLP type H5); 5, J99 (TPM-A negative, RFLP type H4); 6, strain 26695 (TPM-A positive, RFLP type H3); 7, negative control. (B) DdeI digestion of the 365-bp amplicon to detect the presence of TPM-A. Lanes: 1, 7, and 8, as for panel A; 2, NCTC 11637 (TPM-A negative, RFLP type D7); 3, NCTC 11638 (TPM-A positive, RFLP type D2); 4, ATCC 43526 (TPM-A negative, RFLP type D7); 5, J99 (TPM-A negative, RFLP type D1); 6, strain 26695 (TPM-A positive, RFLP type D2). (C) Tsp509I digestion of the 179-bp amplicon to detect the presence of TPM-C. Lanes: 1, 7, and 8, as for panel A; 2, NCTC 11637 (TPM-C positive, RFLP type T3); 3, NCTC 11638 (TPM-C negative, RFLP type T1); 4, ATCC 43526 (TPM-C positive, RFLP type T3); 5, J99 (TPM-C negative, RFLP type T1); 6, strain 26695 (TPM-C negative, RFLP type T1).

FIG. 3.

Example of melting curve analysis performed in the LC instrument to detect the presence of TPM-C in H. pylori cagA. Amplification was monitored by single fluorescent acquisition (F1 channel gains set at 20) at the end of each extension. Probe hybridization melting point analysis was then performed after denaturation (94°C for 10 s) by continuous measurement of Cy5 fluorescence (F3 channel gains set at 50) over a temperature gradient of 43 to 90°C (temperature transition rate, 0.2°C/s).