Characterizing the Cyclic-di-AMP Pathway in Clostridioides difficile

About This Grant

Project summary/abstract Antimicrobial resistance (AMR) is a global public health issue. It is estimated that AMR would cause up to 10 million deaths by 2050, where south Asia and Latin America and the Caribbean being the highest overall mortality rate. AMR situation in Thailand is not better than others low- and middle-income countries (LMICs) owing to a high proportion of irrational drug use and the lack of policies in the past. Clostridioides difficile, a spore-forming, toxin-producing gram-positive, presents the threat on AMR as it is mostly multidrug resistance. C. difficile infection (CDI) can cause a wide range of symptoms ranging from severe diarrhea to life- threatening colitis. CDI is often associated with post-antibiotic exposure, therefore, it represents the major cause of nosocomial infections. Current treatment of CDI is limited to only fidaxomicin, vancomycin, and metronidazole. This urges the community to seek for novel therapeutic agents, better yet with new molecular targets. Therefore, the long-term goal of this proposed study is to explore c-di-AMP pathway as a novel drug target for C. difficile, while providing me the trainings and platform to become an independent researcher in the field of structural biology with the focus on drug mechanism study. The central hypothesis of this project is that c-di-AMP pathway is essential for bacterial osmotic homeostasis and response to environmental and antibiotic stress; and targeting this pathway is detrimental to the bacterium. The overall objectives of the study are (1) to characterize the functions and elucidate the structures of key component of the c-di-AMP receptors, TrkA and busAA, in C. difficile and (2) to identify small molecules that target these proteins as a proof-of- concept for draggability of the c-di-AMP pathway. To achieve the objectives and prove the hypothesis of this study, three specific aims will be pursued: (1) to elucidate the structure and function of c-di-AMP binding proteins involved in K+ transporter regulation by using cryo-EM technique and molecular biology/biochemistry characterizations; (2) to investigate the roles of c-di-AMP components in C. difficile physiology by generating and using knockout stains of c-di-AMP binding proteins and microbiology and biochemistry characterizations; and (3) to identify small molecule agonists for c-di-AMP binding proteins to synergize the effect of other antibiotic using virtual screening, protein characterization, and drug combination approaches. The proposed project is innovative because c-di-AMP is a recently discovered pathway and many aspects of this pathway are yet to be explored. The pathway has a potential for drug development, not just for C. difficile, but for other bacteria with limited treatment options and molecular targets. This project will also foster and incubate emerging cryo-EM research in Thailand.