Bacteria rely on signal transduction pathways to respond to environmental cues. Their ability to specifically and sensitively detect the presence or absence of a single chemical entity amongst the background of thousands of other molecules is essential for their survival. Two-component systems (TCSs; generally 20-120 discrete systems per organism) are crucial for the translation of this complex molecular environment into bacterial action, ranging from growth to antibiotic resistance to virulence. Despite the central role of TCSs to an organism’s adaptive response, technical challenges have hampered their study, limiting our understanding of how the combined transmission of the signals from many TCSs are coordinated by the cell to orchestrate survival and pathogenesis. A small number of TCSs have been identified as important targets for the development of antibacterial agents. However, it is clear that the complex process of bacterial signaling holds many additional targets that will be critical for combating the rapidly approaching “post-antibiotic era.” The goal of the proposed research is to develop and apply the tools and techniques required to study the regulation of the histidine kinases, the key proteins required for TCS-mediated bacterial signaling. Histidine kinases directly sense external stimuli and transmit this chemical message inside of the cell, promoting the regulation of gene expression. Ultimately, the tools and knowledge amassed in this work will enable us to understand and predict how a signal is propagated into bacterial action and to hijack the TCSs for the treatment of infectious disease. These goals will be accomplished by pursuit of three Aims. Aim 1. Develop ATP-based molecules as activity-based probes for assessment of histidine kinase activation. Aim 2. Globally map histidine kinase activation from environmental stressors, antibiotic exposure, and host interactions to identify the key HKs required for rapid bacterial adaptation and survival. Aim 3. Utilize multi-histidine kinase inhibitors to determine the proteins that are major contributors to pathogenicity and virulence phenotypes. It is clear that there is a dire need for new approaches to the treatment of antibiotic-resistant infections – the antibiotic pipeline has focused on the “death phenotype,” which has led to the rapid rise of antibiotic resistance and a severe lack of molecules that function through novel mechanisms-of-action. Control of behavioral phenotypes, especially pathogenesis and virulence, is an exceptionally promising but unrealized approach for the control of infectious disease. Our highly interdisciplinary and innovative approach will enable us to assemble a critical understanding of TCS-mediated signal transduction, with particular emphasis on characterization of the HKs required in pathogenesis- and virulence-associated processes.