Expression of theHelicobacter pylorivirulence factor vacuolating cytotoxin A (vacA) is influenced by a potential stem-loop structure in the 5′ untranslated region of the transcript

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Abstract

Summary

The vacuolating cytotoxin, VacA, is an important virulence factor secreted by the gastric pathogenHelicobacter pylori. CertainvacAgenotypes are strongly associated with disease risk, but the association is not absolute. The factors determiningvacAgene expression are not fully understood, and the mechanisms of its regulation are elusive. We have identified a potential mRNA stem-loop forming structure in the 5′ untranslated region (UTR) of thevacAtranscript. Using site-directed mutagenesis, we found that disruption of the stem-loop structure reduced steady-state mRNA levels between two- and sixfold (P= 0.0005) and decreased mRNA half-life compared with wild type (P= 0.03). This led to a marked reduction in VacA protein levels and overall toxin activity. Additionally, during stressful environmental conditions of acid pH or high environmental salt concentrations, when general transcription ofvacAwas decreased or increased respectively, the stabilising effects of the stem-loop were even more pronounced. Our results suggest that the stem-loop structure in thevacA5′ UTR is an important determinant ofvacAexpression through stabilisation of thevacAmRNA transcript and that the stabilising effect is of particular importance during conditions of environmental stress.

The factors determining expression of the Helicobacter pylori virulence gene vacuolating cytotoxin A (vacA) are not fully understood. We identified a well-conserved potential stem-loop structure in the 5′ untranslated region (UTR) of the vacA transcript. More rapid mRNA decay was observed in a disrupted stem-loop mutant, showing its important role in stabilising the vacA transcript. This stabilising effect was particularly evident during acid and salt shock, which are typical stress conditions in the human stomach.

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