PROJECT SUMMARY The rise of antibiotic resistance in hospital-acquired enterococcal pathogens constitutes a serious clinical threat. In particular, enterococci are now extremely likely to display resistance to vancomycin, which has historically functioned as the antibiotic of last resort for these pathogens, leaving the clinician with few viable therapeutic options. Vancomycin resistance in these organisms invariably involves the acquisition of resistance operons encoded on mobile DNA elements. These operons (which are ultimately derived from antibiotic-producing soil microbes) encode enzymes that remodel the bacterial cell wall, and thereby confer resistance to the antibiotic. Importantly, expression of these remodeling enzymes is controlled by a two- component system, VanS/VanR, that senses the presence of the antibiotic and responds to this signal by increasing transcription of the resistance genes. Little is known about how VanS, the sensor histidine kinase of this two-component system, detects vancomycin; indeed, fundamental details remain obscure, including whether VanS directly binds vancomycin, or whether it instead detects some downstream consequence of vancomycin action. This proposal focuses on the two main forms of vancomycin-resistant enterococci (VRE) found in patients, namely A- and B-type resistance. Using a combination of biochemical, biophysical, and microbiological approaches, the proposed experiments will address the molecular mechanisms underlying antibiotic recognition in these two types of VRE. The proposal builds upon preliminary observations revealing that the A and B resistance phenotypes appear to rely on fundamentally different mechanisms of antibiotic sensing. Hence, one Aim will focus on the VanS protein from B-type enterococci (VanSB), which is shown to bind directly to vancomycin. The details underlying this interaction will be elucidated, as will the effects of antibiotic action upon the enzymatic activities of VanSB. A second Aim focuses on VanSA, which does not respond directly to the antibiotic. Accordingly, the precise identity of the activating signal will be probed. A third Aim will provide structural information about the VanS and VanR proteins, in order to inform and direct the functional efforts. Overall, the proposed work will yield fundamental insights into the functioning of an antibiotic-sensing pathway; provide potential entry points for the rational redesign of therapeutics that will enable them to evade detection by pathogens; and inform the development of antibiotic adjuvants that can overcome bacterial surveillance systems and restore efficacy to well-tested drugs such as vancomycin.