Probing the mechanism of bacterial metalloregulation and virulence regulation

A chemical biology approach is employed to understand biological processing such as bacterial metal ion sensing, antibiotic resistance and pathogenesis. As the first part of this dissertation, Chapter One to Chapter Three center on "Studying Bacterial Metalloregulation". Chapter One introduces the general strategy I developed on construction of metal ion fluorescent reporters based on the MerR family of metalloregulatory proteins. In Chapter Two, I performed functional study of a newly identified lead binding protein PbrR691 from MerR family and demonstrated that this is an exceptional selective lead recognition protein. Chapter Three reports spectroscopic investigation towards understanding the molecular mechanism for the high selectivity exhibited by PbrR691 against lead(II). The second part of this dissertation is Chapter Four and Chapter Five. The content of this part is "Probing the Virulence and Antibiotic Resistance Regulation in Pathogen". Chapter Four focuses on a global regulator MgrA in S. aureus and demonstrated that MgrA regulates a variety of genes including antibiotic resistance and virulence factors in this human pathogen. Structural and functional investigation of this key protein illustrated that an oxidation sensing mechanism is used by MgrA to tune a broad spectrum of genes to cope with the stress induced by the environment or the host. Due to the important role and the unique mechanism of MgrA in S. aureus, I seek to find a novel approach for the treatment of S. aureus infection by targeting MgrA. Chapter Five shows the strategy on developing small molecule modulators for tuning MgrA's function inside S. aureus. Chapter Six forms the third part of this dissertation with the emphasis on "Identifying Metal Participated Drug Resistance Regulation". The activation mechanism of MarR remained unclear despite extensive study on this important regulatory protein. Inspired by my previous results on identifying MgrA (MarR's close homologue) as a redox switch in S. aureus, I subsequently investigated MarR in E. coli. Instead of direct sensing of oxidative stress, my results proved that MarR is ready to react with copper ion. A copper participated activation mechanism is proposed for MarR to tune its downstream genes including antibiotic efflux pumps.