Molecular Basis of Localized Adherence in E. coli

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DESCRIPTION (provided by applicant): The bundle-forming pilus (BFP) of enteropathogenic Escherichia coli (EPEC) is an important virulence factor and a member of the large type IV fimbria family. BFP are assembled by a complex 11-component machine and may serve an adhesive function. This proposal seeks an understanding of the architecture and function of the BFP biogenesis machine and of the function of BFP and bundlin in pathogenesis. Toward these ends, this proposal has four specific aims involving: 1. To define the structural and functional interactions among BfpC, BfpD, and BfpE. Prior experiments have established interactions among these three (3) critical components of the inner membrane subassembly of the BFP biogenesis machine. The proposed experiments will define binding sites for BfpD and BfpE in BfpC, and for BfpE and BfpC in BfpD and will determine the effect of BfpC and BfpE binding on the conformation of BfpD. 2. To explore the hypothesis that BfpU ferries bundlin across the cytoplasmic membrane. An exciting hypothesis that has emerged from studies to date states that BfpU caps the hydrophobic amino terminus of bundlin to allow the latter to be extracted from the cytoplasmic membrane and ferried to the growing pilus. Aspects of this hypothesis will be tested by examining the effect of BfpU on bundlin partitioning into membrane vesicles and by investigating interactions between periplasmic domains of the BFP biogenesis machine and both BfpU and a BfpU-bundlin complex. 3. To define the topology and binding interactions of BfpB. The topology of the outer membrane secretin protein BfpB will be defined by systematic insertion of epitopes and protease cleavage sites into the protein to determine which domains are surfaced-exposed. The binding sites for BfpG and BfpB in BfpB will be identified. 4. To deduce the structure of BFP and its function as an adhesion through studies of bundlin. Structural and mutagenesis data on the a1-bundlin monomer will be used to model the pilus itself. Further studies will investigate the binding of phospholipids to bundlin and determine the receptor binding sites on the protein. The structure of the distantly related B6 variant of bundlin will be solved to shed light on the role of sequence variation in pilus structure and immunogenicity.