Gram-positive bacteria harbor a second messenger signal molecule calledcyclic-di-adenosine monophosphate (c-di-AMP). C-di-AMP levels in bacterial cellsregulate genomic integrity, cell wall homeostasis, biofilm formation, virulence, inductionof host immune responses, and development of resistance to ?-lactam antibiotics. Theintracellular concentration of this centrally important second messenger c-di-AMP is verycritical 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 completelyunderstood. This is primarily due to the lack of structural information of proteins involvedin these processes. A combination of three-dimensional structure determination withbiochemical and genetic analysis is the best approach to accurately determine thedetails of how c-di-AMP levels are regulated and how does c-di-AMP exerts its affect onthe activity of receptor proteins in mediating gene expression regulation. Therefore, wewill use X-ray crystallography techniques coupled with novel in vitro and in vivo activityassays to determine a comprehensive view of how c-di-AMP metabolism and functionsare accomplished in bacteria. These studies will pave our way for screening anddevelopment of compounds that can enhance or reduce the activity of key proteinsthereby enabling the fine-tuning of c-di-AMP levels in the cell. Some of the manypossible potential applications of such selected compounds include their use (a) asadjuvants to ?-lactam thereby restoring the efficacy of these antibiotics, (b) to facilitatethe development of robust biofilms of commensal microflora, and (c) to developcommensal bacteria into efficient mucosal delivery systems of c-di-AMP vaccineadjuvant into host. The overall goal of our laboratory is to elucidate mechanistic details of c-di-AMPregulation and eventually exploit this knowledge to develop non-traditional methods thatwill specifically target the pathogenic bacteria while sparing the commensal bacteria andstrengthening the host's immune response.
Vijay Parashar The proposal seeks to employ structural biology coupled with biochemical and genetic analysis tounderstand 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 bacterialinfections by modulating signal transduction mechanisms.