Abstract
Microbial resistance to antibiotics is a serious challenge for both the health and defence sectors. Unfortunately, there are insufficient new antibiotics in the pipeline to replace those that are no longer effective. Consequently, there is a need to initiate programmes to develop antibiotics that target novel pathways. The enzymes that are responsible for RNA degradation, ribonucleases such as the endoribonuclease RNase E, frequently play critical roles in pathogenic bacterial virulence and are potential unexplored antibacterial targets. The aim of this thesis is to explore RNase E as a potential antibacterial target.RNase E from Escherichia coli has been well characterised, far less is known about RNase E homologues from other bacteria. In the current study, RNase E catalytic domains from four pathogenic bacteria have been characterised using bioinformatical and biophysical techniques. Globally, the structural and catalytic properties of the RNase E homologues are similar, however, there are subtle species-specific differences.
In bacteria, there is increasing evidence of communication between the activity of ribonucleases and cellular metabolites. Within this study evidence is presented that RNase E is specifically inhibited by glucosamine-6-phosphate, a precursor of bacterial cell envelope elements. Glucosamine-6-phosphate has been shown, using in silico docking, to bind and occlude RNase E’s active site resulting in the inhibition of multiple RNase E homologues activity in vitro, suggesting a potentially evolutionarily conserved regulatory mechanism.
Novel small molecule inhibitors of RNase E, identified through a combination of
structure-based virtual high-throughput and in vitro inhibition screening, are predicted to target two sites of importance of RNase E. Each of the identified inhibitors inhibit RNase E from a selection of bacterial pathogens, demonstrating potential as broad-spectrum inhibitors.
A series of locked nucleic acid (LNA)-gapmers (DNA-LNA-DNA) have been designed to prevent E. coli RNase E synthesis through the combined action of translational blocking and RNase H-mediated RNA cleavage. The LNA gapmers successfully block translation and facilitate RNase H cleavage in in vitro assays. Although sequence specific against E. coli RNase E analogous gapmers targeting the rne gene from other pathogens could be designed.
In summary, this study has explored RNase E as an antibacterial target and lays the foundation work towards producing a successful antibacterial strategy against RNase E by either inhibiting its catalytic activity or protein synthesis. It is hoped that this approach begins the pathway towards the production of novel antibacterials targeting the area of RNA regulation as method of overcoming the challenge of antimicrobial resistance.
| Date of Award | 1 Mar 2019 |
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| Original language | English |
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| Supervisor | Anastasia Callaghan (Supervisor) |