Enteropathogenic Escherichia coli (EPEC), a leading cause of infant diarrhea worldwide, can attach intimately to host cells, efface microvilli and disrupt the cytoskeleton in a process known as attaching and effacing. A complete characterization of the attaching and effacing effect would greatly advance our understanding of pathogen-host interactions and is the long term goal of this project. The eae gene cluster is required for attaching and effacing. Within this cluster are genes encoding at least two proteins that are secreted by EPEC, EspA and EspB. When expressed in host cells, EspB causes dramatic changes in cellular morphology. In the studies described in this proposal, the proteins encoded by the eae gene cluster will be used as tools to dissect the molecular and cellular events that occur during attaching and effacing. Each of the remaining genes of the cluster will be mutated to assign a role for each locus in attaching and effacing. Experiments to elucidate functional roles as chaperones, components of the secretion apparatus, and signaling proteins are described. Detailed studies of the precise role of EspB in pathogenesis will be performed to test the primary hypothesis that EspB acts inside the host cell to directly alter signaling proteins involved in cytoskeletal dynamics. The functions of different domains of the EspB protein will be explored in carefully planned structure-function studies. Finally, the role of the EspA protein in pathogenesis will be studied, exploring the hypothesis that EspA is required for EspB translocation to the cell cytoplasm. A detailed understanding of the molecular events that result in the attaching and effacing effect is likely to emerge from these studies. Knowledge of the precise events involved in attaching and effacing will lead to a better understanding of EPEC infection, of enterohemorrhagic E. coli infection, of interactions between bacteria and host cells, and of regulation of the host cell cytoskeleton. This information may result in new strategies for preventing and ameliorating infections.
