TITLE

THE IMPORTANCE AND GENETIC DETERMINANTS OF DIFFERENCES IN VACA TRANSCRIPTION BETWEEN HELICOBACTER PYLORI STRAINS

AUTHOR(S)
Narayanan, G.L.; Letley, D.P.; Aviles, F.; Bebb, J.R.; Hardie, K.R.; Atherton, J.C.
PUB. DATE
April 2003
SOURCE
Gut;Apr2003 Supplement 1, Vol. 52, pA53
SOURCE TYPE
Academic Journal
DOC. TYPE
Article
ABSTRACT
Introduction: Levels of vacuolating cytotoxin gene (vacA) transcription vary among laboratory strains of H pylori. We aimed to study the importance of vacA transcriptional differences among wild type strains and explore whether specific genetic determinants within vacA explained these differences. Methods: H pylori single colonies were isolated from gastric biopsy specimens from 8 patients and their vacA type determined by allele-specific PCR. Total RNA was prepared for each isolate and for the control strains 60190 (Tox[sup +]; vacA s1/m1) and Tx30a (Tox; s2/m2). Level of vacA transcription was determined by densitometric analysis of RNA dot blots. Level of VacA protein production was determined by densitometric analysis of immunoblots of 24 hour broth culture supernatants. For each isolate, nucleotide sequences were determined for the region between vacA and its 5' gene, cysS (the cysS-vacA intergenic region) and For 122 nucleotides of the 5' end of vacA. Finally, the vacuolating activity of each strain was determined by cell culture assay. Results: vacA transcription varied 6 fold among the clinical isolates. VacA RNA levels correlated closely with protein levels suggesting that variation in vacA production resulted mainly from variation in transcription. Level of vacA transcription was not related to vacA genotype (genotypes of wild type isolates were: 2 s1/m1; 4 s1/m2; 1 s2/m2; 1 s2/m1) nor was it associated with disease state or specific gastric histological features. Analysis of the cysS-vacA intergenic region revealed 70% identity across all 10 strains (including control strains 60190 and Tx30a). The -10 and -35 promoter regions were well conserved. Identity within the vacA 5' untranslated region was 77%. Despite high variation within the cysSvacA intergenic region, none of the genetic differences were associated with vacA transcription level. A potential stem-loop structure at the +4 site was conserved between all strains. Conclusion: Level of vacA...
ACCESSION #
9747639

 

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