The interplay of sRNAs Hfq and RNase E in the control of gene expression; a novel mechanism linked to pathogenic bacterial virulence

With antibiotic resistance on the rise, research into understanding the workings of bacterial organisms is crucially important, as are new approaches to combating the infections they cause. When bacterial cells bring about infection, one of the first steps is that they must gain entry to the host cell. Recently scientists have found that an interlinked sequence of events occurs at the molecular level which aids the process of bacterial invasion into a host cell. They found that within the bacteria, messenger molecules (mRNA) played an important role in the invasion, but that these molecules were either degraded or stabilized by destruction (RNase E) or protection (Hfq) molecules respectively. It is also known that bacteria use signal molecules (sRNAs) to trigger either the destruction or the protection of the messenger molecules (mRNA). During the life of a bacterial cell, it is now understood that a complex sequence of interactions continually occurs between these molecules. Recent advances have taken the first steps to understanding this complex sequence of interactions, but quite how the events are communicated and regulated within the bacterial cell is still unknown. How does the destruction pathway work and how does the protector molecule prevent it? Are different signal molecules (sRNAs) treated differently? Can the protection pathway be interrupted in order to prevent the bacteria invading the host cell, and thereby preventing infection? Current data are lacking to answer these most fundamental questions. The aim of this research proposal is therefore to understand the interactions between the signal, messenger, protector and destructor molecules (sRNA, mRNA, Hfq and RNase E) found within a model bacterial cell. Only with this knowledge will it be possible to accurately inhibit the appropriate interactions to develop novel antibacterial approaches.

The aim of this project is to increase our understanding of a newly discovered mechanism of genetic regulation with potential applications in the field of antibacterial research. The essential ribonuclease RNase E has a critical role in initiating mRNA decay, and therefore serves an important role in the post-transcriptional control of gene expression. Recently, non-coding small RNAs (sRNAs) have been identified that can program RNase E to target specific mRNA transcripts for destruction. This targeting is mediated through interaction with the RNA chaperone Hfq. However, certain sRNAs have been shown to have entirely the opposite effect, in that they and their mRNA targets are stabilized by Hfq against cleavage by RNase E. This protective mode has been shown to be critical for the transcription of major virulence factors in various pathogenic bacteria, with Hfq deletion mutants displaying attenuation of invasive virulence. The interplay of sRNAs, their mRNA targets and Hfq results in a finely balanced mechanism of communication with RNase E to bring about either the destruction or the stabilization and subsequent translation of specific transcripts. The fundamental questions in this area are how this communication occurs and whether this mechanism, with a direct impact on pathogenic bacterial virulence, can be exploited in the search for novel antibacterial approaches and/or targets. The proposed research uses a toolbox of biochemical, biophysical and structural characterization techniques, initially to investigate and understand isolated interactions (e.g. sRNA-RNase E and sRNA-Hfq) and subsequently to analyse the key steps in the pathway as a whole (e.g. efficiency of sRNA-mRNA duplex formation in the presence and absence of Hfq and RNase E).