Clostridioides difficile Zinc-binding PBPs as anti-sporulation targets

Clostridioides difficile is the leading cause of nosocomial infection. The use of b-lactam antibiotics especiallycephalosporins is a major risk factor for infection but high dietary zinc concentrations have also been shown toincrease infection rates. Current treatments of C. difficile infection (CDI) are plagued by high recurrencefrequency due to C. difficile spores which are the main vehicle of transmission and highly resilient against harshenvironmental conditions. We hypothesize that penicillin-binding proteins (PBPs) the target of b-lactamantibiotics may play important roles in C. difficile cephalosporin resistance while serving as valuable targets fornovel antibiotic development especially targeting C. difficile sporulation in recurrent infection. Despite PBPsbiological and clinical importance our understanding of PBPs has mostly relied on studies of a few modelorganisms. Most recently we have demonstrated for the first time that the structures of several C. difficile PBPscontain a zinc ion in their active site a feature never observed in PBPs from model organisms but likely prevalentin 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 rolesof PBPs and other peptidoglycan transpeptidases in the pathogens 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. Takentogether the proposed studies will deepen our understanding of PBPs across bacterial species and provide newknowledge about the unique properties of C. difficile PBPs particularly those related to the risk factors of CDIand future drug discovery to address current treatment challenges. The results will also facilitate the developmentof new combination therapies for recurrent CDI by using existing CDI drugs such as vancomycin and anti-sporulation inhibitors.