The gram-negative zoonotic bacteria, F tularensis is the causative agent of tularemia, a severe and sometimes fatal illness that can be caused by inoculation or inhalation of as few as 10 organisms. The extreme infectivity, ease of dissemination and capacity for disease and death, make the potential use of F tularensis as a biological weapon a serious concern. Despite this, there has been little basic science research on this pathogen. It is known that the macrophage is central for both the pathology and resolution of F tularensis infections. Accordingly, a molecular understanding of F tularensis interactions with macrophages is crucial if we hope to modify the course of disease with therapeutics. Chemical genetics provides a way to rapidly advance both fundamental and applied research on this genetically intractable microbe and we propose to use this approach, coupled with high resolution quantitative fluorescence microscopy to address the following two questions: First, how does F tularensis infect host cells? There is conflicting evidence regarding the entry mechanism used by this organism. It remains to be determined if F tularensis invades cells by phagocytosis, endocytosis or an active invasion mechanism. We will use fluorescence microscopy and pharmacological and molecular inhibitors to investigate the entry mechanisms, and we will use automated fluorescence imaging to screen a small molecule library for compounds that block macrophage infection. Knowledge of viral entry mechanisms is currently providing the basis for development of new therapeutics for HIV, so analogous information may be similarly helpful for protecting against F tularensis. Second, what are the properties of the intracellular niche occupied by F tularensis? Vacuoles inhabited by Mycobacteria, for example, are slightly acidic and this fact is being exploited for targeted delivery of antibiotics. There is scant data about the nature of the F tularensis vacuole. We will measure the pH of F tularensis-containing vacuoles using ratiometric fluorescence imaging, determine if vacuoles can fuse with early endosomes and/or lysosomes, and characterize the protein constituents of the vacuole. We will then use a fluorescence imaging-based screen to identify cell-permeable molecules that alter the intracellular fate of F tularensis. Small molecule modulators of F tularensis-macrophage interactions will be used in future experiments to identify virulence factors, and may provide leads for post-attack prophylaxes.