Development of a ppGpp capture compound to assist mapping the stringent response signalling network in Staphylococcus aureus

The alarmones guanosine tetra-phosphate (ppGpp) and guanosine penta-phosphate (pppGpp), collectively termed (p)ppGpp, are universally conserved second messenger nucleotides in prokaryotes. (p)ppGpp control the stringent response, a survival response that occurs in bacteria exposed to external stresses such as nutrient starvation. This response promotes cell survival through a form of quiescence and has been implicated in antimicrobial tolerance and persistent/chronic infections. Despite the discovery of these alarmones in 1969, numerous binding targets remain unidentified, and how these nucleotides facilitate a dormant state is incompletely understood. Here, I describe the synthesis of a (p)ppGpp target-protein capture compound via an enzymatic and chemical synthesis. This compound was validated against known ppGpp binding proteins before its use in pulldown assays with lysates of Staphylococcus aureus. Subsequent proteomics revealed many putative hits enriched by the compound. These hits were preliminarily screened for binding to [α-32P]-ppGpp to highlight proteins of interest. A number of binding candidates were purified and their binding to ppGpp was established using 31P-NMR. The binding affinity of one protein involved in carbon metabolism, HxlB, was investigated by microscale thermophoresis (MST) and an assay was designed to examine the impact of ppGpp on HxlB enzymatic activity. Similar capture compound approaches have previously been implemented in Escherichia coli. However, as the protein pathways utilised by E. coli often differ to those of S. aureus, applying this method to different species allows for the identification of new protein targets that aid in the understanding of this stress response. In addition to the capture compound, I also describe the synthesis and in vitro testing of the fluorescent chemosensor PyDPA, capable of quantifying (p)ppGpp levels in the presence of structurally similar nucleotides. This chemosensor allows us to circumnavigate issues associated with current (p)ppGpp quantification methods, with the eventual aim of probing (p)ppGpp synthesis in response to various stressors.