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.
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.
Owen
Robert J
RJ
Laboratory of Enteric Pathogens, Specialist and Reference Microbiology Division, Health Protection Agency, London, United Kingdom. rowen@phls.nhs.uk
Sharp
Sally I
SI
Chisholm
Stephanie A
SA
Rijpkema
Sjoerd
S
D000942:Antigens, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D005456:Fluorescent Dyes; D016481:Helicobacter Infections / Q000382:microbiology; D016480:Helicobacter pylori / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D010437:Peptic Ulcer / Q000382:microbiology*; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D014443:Tyrosine / Q000378:metabolism*
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D005456:Fluorescent Dyes; C080866:cagA protein, Helicobacter pylori; D014443:Tyrosine