Molecular mechanisms of peptidoglycan endopeptidase regulation

PROJECT SUMMARY Most bacteria maintain a cell wall, an essential, mesh-like structure mainly comprising the polysaccharide peptidoglycan (PG). Some of our most powerful antibiotics, the b-lactams (penicillins, carbapenems and cephalosporins) target enzymes required for cell wall synthesis and derive their efficacy from their ability to not only inhibit cell wall biogenesis, but also to actively cause its destruction. Cell wall destruction after exposure to b-lactams is mediated by “autolysins”, a group of enzymes (amidases, lytic transglycosylases and endopeptidase) with the capacity to cut a variety of chemical bonds within the PG mesh. Under normal growth conditions, autolysins engage in important cell wall remodeling functions, such as PG mesh expansion during cell elongation; how these functions are regulated to ensure proper PG maintenance is poorly understood. We have shown that in the diarrheal pathogen Vibrio cholerae, the endopeptidases (EPs) ShyA and ShyC are required for cell elongation during normal growth, but are also key factors mediating cell wall breakdown after exposure to beta lactam antibiotics. How ShyA and ShyC are regulated to ensure proper cell wall maintenance in the absence of antibiotics is unknown. Here, we propose experiments to build a thorough understanding of mechanisms of endopeptidase regulation in V. cholerae on multiple levels. Since M23 EPs are well-conserved throughout Bacteria, our experiments will likely yield insights with broad relevance to other pathogens. Leveraging extensive preliminary screens, we will i) establish how EPs are co-ordinated with PG synthesis, ii) interrogate the functional interaction between EPs and other autolysins, and iii) determine the role of proteolytic processing in EP activity regulation. Taken together, these experiments will provide us with an extensive framework of how bacteria maintain the balance between cell wall synthesis and remodeling, which could ultimately lead to the discovery of new potential targets for antibiotics that modulate autolysin activity.