Project Summary/Abstract Francisella tularensis is a bacterium that causes tularemia, a disease which, when in its pneumonic form, can be fatal even with appropriate treatment. Due to its low infectious dose, ease of spread by aerosol, and high virulence, F. tularensis is classified as a Tier 1 Select Agent by the U.S. federal government. This R01 project builds on our earlier identification (by contact PI Horwitz's group) of the Francisella Type VI Secretion System (T6SS) and our subsequent determination (by Horwitz's and MPI Zhou's group) of the first atomic models of its sheath and its uniquely endowed central spike complex through cryo electron microscopy (cryoEM). T6SSs are large, complex, multi-protein nanomachines that Gram-negative bacteria use to sense environmental cues and deliver toxins into other bacteria or into eukaryotic hosts; in Francisella, they mediate phagosome escape and intracytoplasmic replication. They are important virulence determinants, present in 25% of Gram-negative bacteria and in an even higher percentage of those that are human pathogens. However, without knowing T6SS composition and structure, we cannot fully understand its mechanisms of pathogenesis nor effectively design countermeasures against a myriad of bacterial diseases. The T6SS of Francisella is both significant and attractive to study because of the high infectivity and lethality of Francisella species and its relative simplicity compared with other T6SSs. However, significant knowledge gaps remain, including the following: (1) an atomic model of the structure of the pre-contraction outer sheath; (2) the composition and an atomic model of the baseplate and membrane complex; and (3) the composition of the Francisella central spike and secreted effector protein complex and an atomic model of its interaction with the sheath, baseplate, and membrane complex in the pre-contraction state and during the contraction process. To fill these gaps, we propose to carry out three major structure-function studies on T6SS using Francisella novicida [and its closely related F. tularensis live vaccine strain (LVS)] as a model. First, we shall obtain the atomic model of the sheath and tube complex in purified T6SS in its pre-contraction state with cryoEM, and elucidate the energetics and mechanism of T6SS contraction by structural comparison with the contracted sheath and structure-guided mutagenesis. Second, using proximity labeling, crosslinking, affinity pull-down, immunoblotting, proteomics, and bacterial 2-hybrid analyses, we shall determine the composition and protein interactions of the baseplate and membrane core complex. This information will be used in conjunction with cryo electron tomography of T6SS-containing mini-cells to determine the composition and structure of the T6SS baseplate and membrane complex in their pre- and post-contraction states. Third, we shall determine the composition and structure of the Francisella T6SS central spike and secreted effect...