Conformation and functional dynamics of Class B PBPs in enterococcal antimicrobial resistance

About This Grant

PROJECT SUMMARY The continued and inevitable emergence of antibiotic resistance demands a vigorous and sustained effort to identify fundamentally new targets and strategies for innovative antimicrobial therapeutics. Antibiotic-resistant enterococci are major causes of hospital-acquired infections. Enterococci are successful hospital-acquired pathogens in part because of their intrinsic resistance to commonly used antibiotics that target the bacterial cell envelope, such as cephalosporins. However, many questions remain regarding the genetic and biochemical basis for cephalosporin resistance in enterococci. Our published and preliminary data reveal that each of the three class B penicillin-binding proteins (bPBPs) encoded in the enterococcal genome are required for cephalosporin resistance, and moreover that they are each required for a different reason (i.e. they perform non-redundant functions). Many questions remain regarding the mechanisms by which the bPBPs are regulated, although recent structural studies suggest that dynamic conformational changes play a key role. A non-enzymatic domain found in bPBPs, commonly referred to as the pedestal domain, has emerged as a site of protein-protein interactions with regulatory factors. Crystallographic studies have revealed that two prominent lobes of the bPBP pedestal domain can adopt distinct “closed” and “open” conformations, and in at least one case interaction with a regulatory partner requires adopting the open conformation. However, studies assessing the conformational dynamics of the pedestal domain in a full-length bPBP in the context of a membrane have not been reported. The major knowledge gaps to be addressed in the proposed research are (i) to determine the conformational dynamics and in vivo function of the pedestal domains for each of the three enterococcal bPBPs; and (ii) to understand how the bPBP pedestal domains contribute to enterococcal cephalosporin resistance, thereby defining new mechanisms of resistance critical to all clinically relevant enterococci. By doing so, we will provide new insights into the fundamental biological processes that drive key antibiotic resistance in enterococci and define new strategies for innovative therapeutics designed to impair enterococcal cephalosporin resistance.