Expression of the Helicobacter pylori virulence factor vacuolating cytotoxin A (vacA) is influenced by a potential stem‐loop structure in the 5′ untranslated region of the transcript

Abstract

SummaryThe vacuolating cytotoxin, VacA, is an important virulence factor secreted by the gastric pathogen H elicobacter pylori. Certain vac A genotypes are strongly associated with disease risk, but the association is not absolute. The factors determining vac A gene 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 the vac A transcript. 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 of vac A was decreased or increased respectively, the stabilising effects of the stem‐loop were even more pronounced. Our results suggest that the stem‐loop structure in the vac A 5′ UTR is an important determinant of vac A expression through stabilisation of the vac A mRNA transcript and that the stabilising effect is of particular importance during conditions of environmental stress.

Helicobacter pylori vacAtranscription is genetically-determined and stratifies the level of human gastric inflammation and atrophy

“…In our previous work, site-directed mutagenesis was used to replace guanine with adenine at +28 in the vacA 5′ UTR of strains 60190 and SS1, allowing more favourable pairing with uracil at position +6 and potentially increasing the stability of the stem-loop. We found that isogenic mutants with A(+28) had significantly higher steady-state levels of vacA mRNA than their wild-type controls with G(+28) 24. This supports our current finding that clinical isolates with adenine at +28 are associated with increased vacA mRNA level in gastric biopsies from patients infected with H. pylori .…”

“…At the third pairing position from the base, the 5′ stem is consistently thymine (uracil in mRNA) but the 3′ pairing nucleotide (position +28) can be either guanine (G(+28)) or adenine (A(+28)), the latter potentially stabilising the stem-loop more efficiently. In our previous work, we showed that substituting adenine for guanine at position +28 significantly increased vacA mRNA steady-state levels in two different strain backgrounds 24. Here we report that strains possessing A(+28) expressed more vacA in gastric biopsies than those with G(+28) (p<0.01; figure 4A).…”

“…One explanation could be sequence constraints imposed by important secondary structure formation in this untranslated mRNA region. Indeed, we have recently identified a potential stem-loop structure from +4 to +30 in the vacA 5′ UTR which we showed was important for stabilising the vacA mRNA transcript 24. Interestingly, the only polymorphism significantly associated with gastric vacA mRNA level and inflammation, G/A(+28), was located within this stem-loop sequence.…”

“…24 We showed that this potential stem-loop is important for stabilising vacA mRNA: disrupting its formation led to reduced mRNA half-life and decreased steady-state vacA transcript levels 24. At the third pairing position from the base, the 5′ stem is consistently thymine (uracil in mRNA) but the 3′ pairing nucleotide (position +28) can be either guanine (G(+28)) or adenine (A(+28)), the latter potentially stabilising the stem-loop more efficiently.…”

RNA-Mediated cis Regulation in Acinetobacter baumannii Modulates Stress-Induced Phenotypic Variation

“…It has been shown that other bacterial genes employ 5= UTRs to stabilize mRNA transcripts (42)(43)(44). One well-studied example is the 5= UTR of ompA in E. coli, which contains a stem-loop that stabilizes the ompA transcript by preventing 5= enddependent degradation as well as increasing the life span of other RNA transcripts (42,45,46).…”