Biochemical and structural characterization of the cell wall synthesis complex required for bacterial division

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PROJECT SUMMARY/ABSTRACT The processes that drive bacterial cell division should be prime targets for the design of novel antibiotics, but our understanding of how one bacterium becomes two is riddled with significant gaps that prevent its exploitation. Nearly all bacteria possess the same five-protein complex (FtsQLB-WI) to coordinate the synthesis of a peptidoglycan (PG) septum between dividing cells. Our laboratory has extensive experience with the purification and modification of lipid-bound PG precursors for the reconstitution of PG synthesis in vitro, and, we have intimate knowledge of this specific PG synthesis complex as we were the first to characterize FtsW as a PG glycosyltransferase. We are therefore uniquely poised to resolve the mechanism by which pathogens such as Streptococcus pneumoniae regulate the synthesis of PG during division. In Aim 1, I will generate a structure of the synthase enzyme subcomplex, FtsW and FtsI, bound to PG precursor substrate. FtsW is a novel member of the SEDS-family, and this structure will be the first to define how FtsW binds PG precursors and suggest a mechanism of action. We have already reconstituted the PG synthase activity of S. pneumoniae FtsWI in vitro and found that addition of FtsQLB is inhibitory. We hypothesize that both protein-protein and substrate interactions are required for de-repression of the FtsQLB-WI complex. In Aim 2, I will explore protein factors that regulate FtsWI synthase activity, including a systematic identification of domains within the non-enzymatic subcomplex FtsQLB that are required for FtsWI regulation, as well as in vivo co-immunoprecipitation assays against FtsQLB-WI to identify additional components required for FtsQLB-WI de-repression. In Aim 3, I will isolate components of the peptidoglycan cell wall predicted to bind regulatory domains within FtsQLB-WI to determine their influence on the activity of FtsQLB-WI in vitro. Together, these aims will generate a comprehensive model of septal PG synthesis in S. pneumoniae that can inform the design of novel antibiotics. The work proposed here will not only grant me the expertise in biochemistry I require to become an independent researcher, but additionally place me in proximity to other important means of professional development. At Harvard Medical School and in the Boston area, I will have ample access to teaching and mentoring experiences in preparation for a career in higher education. I will also foster collaborative professional relationships within my HMS community as well as the broader microbiology and undergraduate educator community. As a postdoctoral fellow under Dr. Suzanne Walker at HMS, I will develop the skills I require to be a well-rounded microbiology researcher and educator at an undergraduate institution.