Project No.2234

Primary Supervisor

Dr Seung Lee – University of Southampton

Co-Supervisor(s)

Dr Christopher Mulligan – University of Kent

 

Summary

Two-component signalling systems (TCS) are main signalling pathways in bacteria, which control major physiological processes, particularly antimicrobial resistance.

TCSs consist of a sensor histidine kinase (HK) and a response regulator (RR). The former senses environmental stimuli, and self-phosphorylates its own histidine residue. Then it transfers the phosphate to an aspartate residue of RR. Upon phosphorylation, RR binds to the promoter region of downstream genes, triggering transcription. Unlike kinase networks in eukaryotes, for which various specific antibodies for important phosphorylated kinases or proteins exist, the lack of proper tools hampers

the progress in studying prokaryotic TCSs. The development of antibodies take long time and high cost, posing a significant risk in research. As an alternative, aptamers are small nucleic acids or peptides made to have high affinity and specificity to epitopes, which rival those of antibodies. Their development time and cost are only a fraction of those for antibodies. Our project is to develop aptamers specific for phosphohistidine of HptS and phosphoaspartate of HptR, constituting the HptRS TCS in Staphylococcus aureus. We will use a well-established SELEX process, a method to select aptamers with affinity to desired targets from a large oligosaccharide library. Since phosphohistidine and phosphoaspartate are unstable, we will synthesise chemically stable analogues of these amino acid residues, and incorporate them in target peptide fragments derived from the sensor HK, HptS containing phosphohistidine and that of RR, HptR containing phosphoaspartate, respectively. HptRS TCS is involved in hexose-6-phosphate uptake of S. aureus, and increasing evidence indicates it also affects the production of various virulence factors. We also identified small molecule agonists and antagonists of HptRS. Thus, in this project, aptamers specific for HptR and HptS will be developed, and tested against these molecules along with genetic knockdown, which will underpin the proof-of-principle of our approach as a general strategy.