PROJECT SUMMARY Shigella causes bacillary dysentery with high worldwide morbidity and childhood mortality with complications that include cognitive and developmental impairment in children suffering multiple diarrheal episodes each year. Shigella’s main virulence factor is its type III secretion system (T3SS), which is used to deliver effector proteins into host cells to promote pathogen entry. T3SSs are shared by many Gram negative pathogens with the injectisome comprising a(n): 1) external needle and tip complex for delivering translocators and effectors; 2) basal body that spans the bacterial envelope; and 3) cytoplasmic sorting platform (SP) that energizes and controls secretion. We pioneered visualizing the SP by cryo-electron tomography (cryo-ET) and since then we and others have identified the components of the SP. We now propose studies to explore structural differences between the “on” and “off” secretion states for the in situ injectisome with parallel biochemical analysis of the SP sub-assemblies and the first visualization of the Shigella injectisome-host membrane interface in situ. We will use cryo-ET methods to view the SP pods at a 1-nm or better resolution and then use biochemical and molecular methods as we develop models of assembly and function. In our investigation of the SP, we will explore the movement of protein domains at the SP interface with the inner membrane ring. In parallel, we will extend our study to examine another important cytoplasmic component of the injectisome - the export gate nonamer formed by MxiA, which undergoes structural rearrangements in the absence of the SP. We will also exploit a system we’ve generated for trapping different secretion substrates within the in situ injectisome so that we can determine how the overall structure compares for three different states. These are the “on” injectisome (ipaD null strain) and two forms of “off” injectisomes (mxiH null strain and effector-blocked strains), which will provide functional insight into SP and export gate communication and association. We propose three complementary aims: 1) Define the makeup, intermediate states and structural requirements at the SP/inner-membrane ring (IR) interface that allow SP assembly and guide type III secretion. 2) Correlate export gate structural features with secretion status using complementary cryo-ET and molecular methods; and 3) Identify the structural changes associated with trapping substrates within the in situ injectisome and begin generating the first high-resolution picture of the injectisome-host membrane interface in situ using cryo-ET. Improved cryo-ET methods provide an unprecedented view of substructures within the Shigella injectisome in situ to reveal elements that cannot be studied using purified needle complexes and this is reflected in the preliminary data presented here. We can now visualize these sub-structures, target them for molecular analysis and purify them for in vitro biochemical and biophysi...