Determinants of vacuolating cytotoxin production and its impact on Helicobacter pylori pathogenesis

Masters, Charlotte Grace (2010) Determinants of vacuolating cytotoxin production and its impact on Helicobacter pylori pathogenesis. PhD thesis, University of Nottingham.

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Abstract

Introduction: The vacuolating cytotoxin is an important H. pylori virulence factor and is naturally polymorphic, one of the most diverse regions being the signal region (type s1 or s2). Signal type determines vacuolating activity: type s1 strains are vacuolating and are associated with peptic ulcers and gastric adenocarcinoma; and type s2 strains are non-vacuolating. Heterogeneity in VacA levels between strains also exists, and different strains produce different amounts: this is due to differences in vacA transcription and differences in protein secretion between strains. A vacA promoter and a transcriptional start point (TSP) have been identified. Sequence analysis revealed that -35 and -10 motifs are well conserved. Mutagenesis of this region resulted in a decrease in vacA transcription in vitro. However, investigation of vacA expression between 8 different H. pylori strains using real-time PCR revealed that differences in vacA transcription observed between different strains were unrelated to the putative -35 and -10 motifs. VacA type s1 and s2 signal sequences differ in the cleavage recognition site at key positions. In type s1 the more favourable serine and proline residues are at positions -3 and -6 respectively, whereas type s2 strains have leucine present at -3 and glycine at -6. In addition to VacA type, the determinants of VacA production are of importance in H. pylori pathogenesis: It has been shown that larger amounts of toxin produce greater damage in animal models. However there is limited information on the level of vacA expression in vivo and disease.

Aims: To analyse the cysS – vacA intergenic regions of a large number of strains to identify motifs and natural polymorphic differences that contribute to different vacA mRNA levels between strains in vivo and in vitro using sequencing and real-time PCR. Then to investigate these regions of interest using site directed mutagenesis. Next, to determine whether naturally occurring differences in the signal peptide cleavage recognition sites between type s1 and s2 strains affect the secreted VacA levels between the two strains using isogenic mutants and ELISA and Western blotting. Finally, to determine whether there is an association between the level of vacA mRNA in vivo and pre-cancerous changes, infection density and disease outcome in infected patients.

Results and Conclusions: Analysis of the cysS – vacA intergenic region revealed that differences between strains in the -35 and -10 regions were not significantly associated with levels of vacA transcription, but that other regions were associated with differences in vacA production between strains. These regions differed in vitro and in vivo, which may imply in vivo regulation of vacA transcription. Strains with a fully palindromic inverted repeat upstream of the -35 motif expressed more vacA than other strains in vitro (P=0.01). Analysis of vacA transcription from the same strains in vivo revealed that strains with an A within a second stem loop located in the 5’ UTR of the vacA transcript expressed more vacA than those with a G at this position (P=0.006). Site directed mutagenesis confirmed that replacing the G with an A in an isogenic mutant strain resulted in an increase in vacA transcript levels. As found for other bacterial secreted proteins, these results show that positions -6 and -3 are important in the VacA signal sequence and may affect signal sequence processing efficiency. The level of vacA mRNA in vivo was significantly positively associated with the severity of chronic inflammation, neutrophilic activity, glandular atrophy and infection density in the infected patient. Differences in toxin production between H. pylori strains could help explain why only some infections result in disease.

Item Type:Thesis (PhD)
Supervisors:Atherton, J.
Letley, D.P.
Doherty, N.C.
Faculties/Schools:UK Campuses > Faculty of Medicine and Health Sciences > School of Clinical Sciences
ID Code:1139
Deposited By:Charlotte Grace Masters
Deposited On:15 Nov 2010 14:08
Last Modified:15 Nov 2010 14:08

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