Type III secretion systems are widely distributed, essential virulence determinants of Gram-negative bacteria. They are encoded by approximately 25 genes which share homology with those encoding flagellar basal bodies. Type III secretons serve to translocate, upon contact with host cells, proteins from the bacterial cytoplasm into the host cell cytoplasm. I have identified the type III secreton of Shigella flexneri and shown that during intimate contact with eukaryotic cells it inserts two bacterial pore forming proteins into the host membrane. Protein translocation appears to be a one-step process directly from the bacterial cytoplasm through the secreton imbedded in both bacterial membranes into the bacterially inserted pore in the host membrane. The questions to be addressed now are: 1) What is the detailed structure of the secreton and how is it assembled? For this, the secreton will be isolated and examined by high resolution electron microscopy. In parallel, its constituting gene products will be identified. Then, each subunit will be mapped in the detailed structure. Experiments are also planned to determine the order in which the gene products are required for assembly of the secreton. 2) How tight is the connection between secreton and pore? To assess this biochemical and structural studies using intact cells or the isolated secreton and pore complexes are planned. 3) What are the signals that target the substrates to the secreton and translocator pore? To answer this in vivo secretion and translocation assays for reporter protein substrates will be established. 4) How, in molecular detail, does type III secretion occur? To address this, type III machinery-mediated protein export across the bacterial inner membrane will be reconstituted and dissected in vitro. 5) What powers type III secretion and how is the energy utilized for substrate export? A model and specific experiments are proposed to address the role of the probable energizing component of type III secretion apparatuses in secreton assembly and substrate protein export. Thus, this proposal seeks to provide fundamental insight into the workings of type III secretons. This knowledge could be used to design specific inhibitors of these systems which would be important therapeutic agents at a time when antibiotic resistance is increasing.