Enteroaggregative E. coli is an emerging enteric pathogen, whose virulence mechanisms are only partially elucidated. We have shown that EAEC adherence to the human intestinal mucosa is mediated by Aggregative Adherence Fimbriae (AAFs), but that the strong autoagglutination phenotype mediated by AAF fimbriae is modulated by a hydrophilic protein coat that is non-covalently attached to the surface of the bacterium. The coat protein is a 10.2 kDa species which we have named dispersin, the product of the aap gene on the EAEC virulence plasmid. We have recently solved the solution structure of dispersin and have shown it to be a tightly folded rod-like structure with two prominent beta-sheets. We have discovered that translocation of dispersin to the bacterial cell surface requires a putative ABC transporter complex, which includes a cognate homolog of the outer membrane channel TolC. We have proposed a molecular model for the AatA channel based on the TolC crystal structure. Given the novelty and importance of dispersin to EAEC pathogenesis, and the emerging importance of TolC family members and ABC transporters, we propose to characterize further both the structure-function aspects of the dispersin protein as well as functional characteristics of its secretion. These studies will provide contributions to understanding of each of these important areas. Work will be organized in three distinct aims. Aim 1: Structure-function analysis of dispersin. Here, we will address requirements for secretion and for function. The NMR structure will be used to build hypotheses and NMR spectroscopy will be used to evaluate the effects of mutagenesis. Aim 2: Structure-function analysis of AatA. In this aim, we will verify the TolC-based model of the AatA translocator of dispersin. We will also perform site-directed mutagenesis of specific regions of AatA suggested to be important and to confer diversity of function compared with TolC itself. Aim 3: Characterization of the inner membrane ABC complex. In aim, we will characterize the inner membrane complex required for dispersin secretion, the atypical ABC transporter represented by AatPBCD. Each protein will be localized and their roles addressed using mutagenesis and biochemical strategies.