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Date of Award
Doctor of Philosophy (PhD)
Chaperone-usher pathway (CUP) pili are extracellular proteinaceous fibers ubiquitously found on Gram-negative bacteria. Type 1 and P pili are produced by uropathogenic strains of E. coli and are tipped with the FimH and PapG adhesins, respectively, to mediate host and tissue tropism to the bladder and kidney. During pilus assembly hundreds of individual pilus subunits or pilins are first exported across the inner membrane to the periplasm where each pilin adopts an incomplete immunoglobulin (Ig)-like structure lacking a seventh C-terminal _-strand. In a process termed donor-strand complementation (DSC), the chaperone, a boomerang shaped protein comprised of two complete Ig-like domains, provides in trans its G1 _-strand to complete the pilinճ Ig-like fold in a non-canonical fashion. Chaperone-pilin complexes are then guided to an outer membrane (OM) nanomachine called the usher, which catalyzes subunit-subunit interactions through a reaction called donor-strand exchange (DSE) wherein an N terminal extension on every subunit completes the canonical Ig-fold of its neighboring subunit in a zip-in zip-out mechanism that drives the dissociation of the chaperone. The usher contains five functional domains: a 24-stranded transmembrane _-barrel translocation domain (TD), a _-sandwich plug domain (PD) that resides in the pore of the TD in the apo-usher, an amino-terminal periplasmic domain (NTD) and two carboxy-terminal periplasmic domains (CTD1 and CTD2). DSE events are coordinated by the usher and require all five functional domains of the usher for productive interactions between pilus subunits during fiber polymerization. Taken together, the chaperone-usher pathway represents an ideal system in which to study the mechanisms by which binding and allosteric interactions drive sequential steps in a multi-domain assembly machine without ATP or other energy inputs at the bacterial outer membrane.
Usher activation is a complex multi-step mechanism, which begins with the interactions between the chaperone-adhesin and usher NTD and results in: i) chaperone-adhesin association with NTD in the periplasm; ii) transfer of chaperone-adhesin from the NTD to CTDs; and iii) PD displacement, thereby priming the usher to coordinate pilus assembly at the outer membrane. In the P pilus system, efficient activation of the PapC usher is thought to occur when the PapDG chaperone-adhesin complex binds to the usher NTD followed by binding of the PapDF chaperone-pilin complex. However, the mechanism by which activation leads to the transfer of the PapDG complex from the NTD to the CTD and the movement of the PD out of the TD channel to catalyze pilus assembly is unknown. This dissertation presents the first crystal structure of the full-length PapC usher in a pre-activation state, with PapDG bound by both the NTD and CTD2 domains and the PD still located within the TD pore. This structure provides insight into the mechanism by which the outer membrane usher transfers substrates from its N-terminus to C-terminus via an unexpected interaction between the two periplasmic domains. Addition of PapDF to the PapC-PapDG pre-activated complex resulted in DSE between PapG and PapF. Mutations in the NTD-CTD2 interface reduced pilus assembly in vivo and diminished the rate of PapG-PapF DSE in vitro. Thus, this study reveals important molecular details on the mode by which the chaperone-adhesin complex specifically initiates allosteric interactions to activate the usher.
Previously, bicyclic 2-pyridone compounds termed pilicides were determined to be inhibitors of pilus biogenesis by specifically blocking chaperone-subunit interactions with the N-terminal tail of the usher NTD. In light of the PapC-PapDG pre-activation structure, structure-activity relationship (SAR) analysis has been utilized to design even more potent inhibitors that bind to this critical chaperone-usher interface. Furthermore, this structure provides a template for the design of novel small molecules that sterically block the NTD-CTD2 interface to selectively hinder the usher from acting as an assembly platform for the development of antibiotic-sparing therapeutics. In summary, this dissertation elucidates the intricate workings of a bacterial nanomachine that catalyzes CUP pilus assembly and opens the door for the development of potent inhibitors that block assembly of a major bacterial virulence factor.
Chair and Committee
Scott J. Hultgren
Thomas J. Brett, Michael Caparon, David Hunstad, Christina Stallings,
Omattage, Natalie Saranga, "Ushering CUP Pilus Assembly: Dissecting the Mechanics of a Bacterial Nanomachine" (2018). Arts & Sciences Electronic Theses and Dissertations. 1643.
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