Gram-positive bacteria harbor a second messenger signal molecule called cyclic-di-adenosine monophosphate (c-di-AMP). C-di-AMP levels in bacterial cells regulate genomic integrity, cell wall homeostasis, biofilm formation, virulence, induction of host immune responses, and development of resistance to ?-lactam antibiotics. The intracellular concentration of this centrally important second messenger c-di-AMP is very critical to cellular physiology. However, the mechanisms of the precise regulation of c-di- AMP levels and c-di-AMP-mediated changes in gene expression are not completely understood. This is primarily due to the lack of structural information of proteins involved in these processes. A combination of three-dimensional structure determination with biochemical and genetic analysis is the best approach to accurately determine the details of how c-di-AMP levels are regulated and how does c-di-AMP exerts its affect on the activity of receptor proteins in mediating gene expression regulation. Therefore, we will use X-ray crystallography techniques coupled with novel in vitro and in vivo activity assays to determine a comprehensive view of how c-di-AMP metabolism and functions are accomplished in bacteria. These studies will pave our way for screening and development of compounds that can enhance or reduce the activity of key proteins thereby enabling the fine-tuning of c-di-AMP levels in the cell. Some of the many possible potential applications of such selected compounds include their use (a) as adjuvants to ?-lactam thereby restoring the efficacy of these antibiotics, (b) to facilitate the development of robust biofilms of commensal microflora, and (c) to develop commensal bacteria into efficient mucosal delivery systems of c-di-AMP vaccine adjuvant into host. The overall goal of our laboratory is to elucidate mechanistic details of c-di-AMP regulation and eventually exploit this knowledge to develop non-traditional methods that will specifically target the pathogenic bacteria while sparing the commensal bacteria and strengthening the host's immune response.
VijayParashar The proposal seeks to employ structural biology coupled with biochemical and genetic analysis to understand the mechanistic details of c-di-AMP regulation and function in Gram-positive bacteria. Structure- guided screening of compounds will be done to identify novel therapeutics that can perturb intracellular c-di- AMP concentrations and its activity. Such compounds will be potential drug candidates to combat bacterial infections by modulating signal transduction mechanisms.
