DEVELOPMENT OF TOOLS FOR MONITORING TCS ACTIVATION

Abstract

A two-component system (TCS), consisting of a histidine kinase (HK) and a response regulator (RR), is vital for cellular signal transduction in prokaryotes. In bacteria, TCS regulates virulence, antibiotic resistance, and biofilm formation. Therefore, it has been considered as an attractive novel antibiotic therapeutic target. Nonetheless, the technical difficulty in measuring the TCS activation, due to the chemical instability of the phosphohistidine (pHis) and phosphoaspartate (pAsp) intermediates, hindered the search for TCS inhibitors. Therefore, high-throughput methods for monitoring the TCS activation would facilitate discovering TCS inhibitors as novel therapeutic candidates. In this regard, this thesis describes the development of biochemical methods to monitor TCS activation. Chapter I discusses the development and application of an HK activity assay method, named PHACYL (PHosphatase-Autophosphorylation Coupled assaY without Labeling). We validated the assay using E. coli EnvZ as a model system. Further, we generalized this assay for other HKs, E. coli PhoQ, and E. faecium VanS. With these positive results, we applied PHACYL to high-throughput screening (HTS) of VanS inhibitors. Since VanS is a master regulator of vancomycin resistance, its inhibitors would be novel antibiotic candidates. From a library of 2123 clinical compounds, we identified 11 hit compounds, and one lead compound showed promising in vivo antimicrobial activity against vancomycin-resistant E. faecium (VRE). Chapter II discusses our progress towards the development of the RR activity measurement methods. Since the RR activation by phosphorylation leads to its dimerization, we aimed at developing tools to monitor the RR dimerization. As a reporter for dimerization, we employed the homo-molecular fluorescence compensation (Homo-FC), which can reconstruct its GFP structure only when two identical proteins are very close to each other, turning on the fluorescence signal. Therefore, the fusion of Homo-FC to RR would constitute a convenient dimerization monitoring tool. We designed and prepared various fusion proteins using E. coli OmpR as a model RR and demonstrated that they could be phosphorylated. Unfortunately, the fusion proteins did not dimerize, presumably due to the suboptimal linkers. Overall, our studies contributed to the development of chemical biology tools for studying TCS activation. These studies will provide new directions for TCS research, especially in the discovery of TCS inhibitors as novel antibiotics.