A large number of bacterial pathogens cause disease by delivering proteins into host cells for the purposes of disarming immune defenses or establishing sites for microbial replication in tissues. Among the most important delivery systems is the multisubunit type III secretion system. Although the bacterial determinants involved in moving these misregulators into host cells are known, there is almost no information regarding how the eukaryotic cell contributes to bacterial protein movement across the host cell membrane, nor how the host facilitates recognition of targets with the cell. This application is intended to address this gap, in that it represents the first systematic attempt to identify all hot cell proteins that collaborate to ensure efficient translocation of bacterial proteins across the plasma membrane and movement to their intended targets. The proposed studies focus on protein delivery by Yersinia pseudotuberculosis, an enteropathogenic organism that causes systemic disease after spread from the intestine. They take advantage of a novel strategy developed in the course of these studies, in which a Fluorescence Resonance Energy Transfer (FRET) reporter is used to measure the amount of activity in single cells after delivery of the protein YopE during bacterial encounter. Cells having shRNAs that interfere with YopE activity were isolated, and a number of human proteins involved in assembly of a bacterial channel in the host cell plasma membranes were identified. The genes identified in the screen point to a model in which insertion of the translocation channel requires signaling from chemokine receptors, which are predicted to organize host cytoskeletal proteins to promote assembly of the channel. The Aims of the application are directed toward identifying the complete set of eukaryotic proteins necessary for function of the bacterial translocation system, with the goal that these proteins can be shown to be involved in one of three processes: 1) assembly of the channel; 2) folding of substrates exiting the channel; or 3) targeting of the translocated bacteria proteins to their intracellular targets. Of particular interest is determining whether chemokine receptors act as recognition sites for the channel, or whether they act as signaling molecules to allow other host proteins to support channel assembly. Finally, a hypothesis will be tested that assembly of the translocation channel in host cells requires multiple cell surface signals to amplify a cytoskeletal response. By identifying important collaborative relationships between bacterial proteins and their host cell partners, it is hoped that sites for therapeutic interventio against microbial infections can be identified. Furthermore, the data obtained are intended to single out collaborative human proteins that may allow the identification of individuals with variant sensitivities to infectious diseases.