Project Summary Pathogenic bacteria use specialized “nanomachines” to identify and interact with host cells. These machines are attractive drug targets because they are surface-exposed, widespread, and vital for pathogenicity. While one of these machines, the Type III Secretion System, has been well studied, other systems remain relatively poorly understood. Here, we propose to use electron cryotomography (ECT) to dissect the structures and functions of multiple pathogenic nanomachines. ECT is a powerful technique to image intact structures with macromolecular (2-5 nm) resolution inside cells. Previously, we applied ECT to the Type VI Secretion System (T6SS), where our images immediately revealed its "spring-loaded" contractile mechanism. Going a step further, we realized that we could combine ECT and high-resolution subtomogram averaging with available knowledge from other techniques to produce a complete architectural model of the Myxococcus xanthus Type IVa Pilus (T4aP). This produced a flood of new mechanistic insights and inspired us to apply the same approach to pathogenic secretion systems. In this project, we propose to use ECT to reveal the structure and function of pathogenic Type IV Pilus (T4P), Type VI Secretion (T6SS), and Type IV Secretion System (T4SS) machineries. For each system, we will image the entire, intact structure in situ. In most cases, this will be the first high-resolution imaging of these structures. We will then combine subtomogram averaging with difference analysis of mutants in which individual components are knocked out or tagged in order to produce architectural models of the structures. In cases where crystal structures of components (or homologs) are available, we will dock them into our maps to produce pseudo-atomic models of each machine. By comparing these structures with those of non-pathogenic relatives (solved previously or in the current study), we aim to identify adaptations underlying virulence functions. We will also apply state-of-the-art cryogenic-focused ion beam milling and correlated cryogenic fluorescence light microscopy and ECT to image secretion structures in action – in bacterial cells infecting eukaryotic hosts. This will provide the first such images of the critical human pathogens Helicobacter pylori and Legionella pneumophila, which we expect to provide invaluable insights into the operation of their pathogenic machinery in vivo. Together, we expect this project to produce a detailed mechanistic picture of the T4P, T6SS, and T4SS nanomachines that mediate pathogenesis, an important first step in identifying therapeutic targets in the future.