Protein Structure/Function

Type 1 and P pili are made up of a thick, rigid rod with a thinner, flexible tip fibrillum. At the distal end of the pilus is the adhesin, which is made up of a receptor binding domain and a pilin domain. In collaboration with Gabriel Waksman and Stefan Knight, we have determined the crystal structures of FimC/FimH, PapD/PapK, PapD/PapE, PapE/PapK, SfaE and the PapGII receptor-binding domain. Crystal structures of chaperone-subunit complexes revealed that the pilin domain has an incomplete Ig fold where the seventh strand of a canonical Ig fold is missing. The chaperone temporarily donates its beta strand to the pilin domain to complete the Ig fold of the pilin domain in a process termed donor strand complementation. These crystal structures also suggest a mechanism of pilus assembly, termed donor strand exchange, in which each subunit contributes its N-terminal sequence to complete the Ig fold of its immediate neighbor in the pilus. Information derived from these crystal structures has allowed us to develop several classes of inhibitors and vaccines.

Biogenesis of P and type 1 pili

Interactions between bacterial adhesin proteins and host cell receptors are critical in the pathogenesis of virtually every bacterial disease. In many gram-negative bacteria these adhesins are presented at the tip of adhesive pili, which radiate from the cell surface. P and type I pili are assembled via the chaperone-usher pathway. This pathway is used by many different pathogenic bacteria to assemble over 30 diverse adhesive surface organelles that mediate the attachment of pathogenic bacteria to host tissues. Using the P and type 1 pilus biogenesis systems as models, we have delineated many of the fine molecular details of the chaperone/usher pathway, including the structure and mechanism of action of the periplasmic chaperones required for pilus assembly.

Pili are comprised of repeating immunoglobulin (Ig)-like subunits that are missing their C-terminal beta strand. The fiber is assembled by a donor strand exchange reaction whereby every subunit donates its amino terminal extension to complete the Ig fold of its neighbor, thus forming a non-covalent Ig-like polymer. The absence of the C-terminal beta strand makes folding of the subunit dependent upon the periplasmic chaperone that is comprised of two Ig-like domains. The chaperone transiently donates its edge beta strand to complete the Ig fold of the subunit in a process we termed donor strand complementation. Chaperone-subunit complexes are then targeted to outer membrane assembly sites called ushers that form channels in the outer membrane. Here, the donor strand exchange reaction takes place, driving the translocation of the growing fiber.

Curli, a bacterial amyloid

Curli are a novel class of proteinaceous surface fimbriae produced by both Escherichia coli and Salmonella spp. These extracellular fibers mediate biofilm formation and have been shown to bind certain host proteins. Curli fibers are composed of a major subunit (CsgA) and a minor subunit (CsgB), and require several accessory proteins for biogenesis (CsgG,F,E). Once formed, curli fibers are extremely stable and highly resistant to depolymerization. The Hultgren lab has recently discovered that purified curli fibers exhibit characteristics of amyloid proteins (Science v. 295 p. 851). Thus the curli system in E. coli represents a unique and easily tractable model system to study general features of amyloid formation, and in turn may provide clues to the pathogenesis of amyloid diseases such as Alzheimer’s, Parkinson’s and Huntington’s disease.

Currently, work in the Hultgren lab is aimed at elucidating the mechanisms of biogenesis of curli fibers, both the protein-protein interactions important in polymerization of the amyloid subunits, and the roles of accessory proteins in building these structures at the cell surface. Additionally, future work will address the role these fibers may play in the pathogenesis of UPEC.

The Cpx periplasmic stress-response system

The CpxRA system is a two-component signal transduction pathway that consists of a sensor kinase (CpxA) residing in the inner membrane, and a response element (CpxR) in the cytoplasm. This pathway responds to stresses in the cell envelope of some Gram-negative bacteria. In Escherichia coli, the Cpx pathway also closely monitors the assembly of the P pilus, which is essential in the establishment of pyelonephritis by uropathogenic E. coli (UPEC). Interestingly, consensus Cpx binding motifs have likewise been found upstream of other virulence factors, such as hemolysin and cytotoxic necrotizing factor. Thus, the Cpx system may play an important role in the regulation of additional factors significant in pathogenesis. Studies are underway in this lab to examine the molecular details of the Cpx response to, and its effects on, P pilus biogenesis, as well as the roles it may play in the pathogenesis of UPEC.