Clostridioides difficile Zinc-binding PBPs as anti-sporulation targets

Abstract Clostridioides difficile is the leading cause of nosocomial infection. The use of b-lactam antibiotics, especially cephalosporins, is a major risk factor for infection, but high dietary zinc concentrations have also been shown to increase infection rates. Current treatments of C. difficile infection (CDI) are plagued by high recurrence frequency due to C. difficile spores, which are the main vehicle of transmission and highly resilient against harsh environmental conditions. We hypothesize that penicillin-binding proteins (PBPs), the target of b-lactam antibiotics, may play important roles in C. difficile cephalosporin resistance while serving as valuable targets for novel antibiotic development, especially targeting C. difficile sporulation in recurrent infection. Despite PBP’s biological and clinical importance, our understanding of PBPs has mostly relied on studies of a few model organisms. Most recently, we have demonstrated, for the first time, that the structures of several C. difficile PBPs contain a zinc ion in their active site, a feature never observed in PBPs from model organisms but likely prevalent in other bacteria. We have also developed a series of diazabicyclooctane (DBO) compounds as potent anti- sporulation reagents by inhibiting PBP3, a PBP critical for sporulation. Here we propose to 1) elucidate the roles of PBPs and other peptidoglycan transpeptidases in the pathogen’s complex life cycle and determine the b- lactam resistance profile through biochemical/structural analysis and gene knockout/knockdown experiments; 2) apply a structure-based design approach to develop new PBP inhibitors targeting C. difficile sporulation. Taken together, the proposed studies will deepen our understanding of PBPs across bacterial species and provide new knowledge about the unique properties of C. difficile PBPs, particularly those related to the risk factors of CDI and future drug discovery to address current treatment challenges. The results will also facilitate the development of new combination therapies for recurrent CDI by using existing CDI drugs such as vancomycin and anti- sporulation inhibitors.