Functional analysis of small regulatory RNAs in Helicobacter pylori

The intense study of Helicobacter pylori, one of the most prevalent human pathogens, has contributed much to understanding of bacterial virulence mechanisms. Nevertheless, until last year, no regulatory RNAs had been reported in H. pylori, which was even regarded as a bacterium without riboregulation [1]. However, a recent differential RNA-seq approach based on high-throughput sequencing of cDNA led to the discovery of ~60 sRNAs candidates in H. pylori strain 26695 [2], including the Epsilon-subdivision counterpart of the regulatory 6S RNA as well as potential regulators of cis- and trans-encoded target mRNAs.

Here we present the functional characterization of H. pylori sRNA HPnc5490 using biochemical and genetic approaches. Conservation analysis shows that this sRNAs is highly conserved in diverse Helicobacter strains. Furthermore, bioinformatics-based target predictions indicate that HPnc5490 could directly bind to a G-repeat far upstream in the 5’UTR of tlpB mRNA, which encodes for one of the chemotaxis receptors. Studying changes in the transcriptome as well as in the proteome upon deletion of HPnc5490 revealed down-regulation of tlpB on the mRNA as well as protein level and confirmed tlpB as a first trans-encoded target for HPnc5490. In addition, chromosomal complementation of HPnc5490 in the rdxA locus restores repression of the TlpB protein. Initial in vitro structure probing and toeprinting experiments suggest that down-regulation of tlpB via HPnc5490 is rather based on structural rearrangements, transcript destabilization or transcription attenuation than on the translational inhibition by masking the ribosome binding site. Therefore, we are creating transcriptional and translational reporter gene fusions to analyze the mechanism of tlpB regulation by HPnc5490. Moreover, the construction of several sRNA mutants will help to validate the interaction site between tlpB and HPnc5490 in vivo.