Abstract

Gram-negative bacteria produce chaperone-usher pathway (CUP) pili, which are extracellular adhesive protein fibers critical for host-pathogen interactions. An outer-membrane usher protein, together with a periplasmic chaperone, assembles thousands of pilin subunits into a pilus fiber. Each pilus is tipped by an adhesin that mediates host and tissue tropisms by binding to receptors with stereochemical specificity. Type 1 pili expressed by uropathogenic Escherichia coli (UPEC) are essential virulence factors in urinary tract infections (UTIs) and facilitate attachment to mannosylated glycoproteins on bladder epithelial cells. Although type 1 pili have been extensively studied, critical gaps remain in our understanding of the molecular mechanisms governing their assembly and termination at the outer membrane (OM) usher FimD, as well as in the development of antibiotic-sparing therapeutics targeting usher function. Chapter 1 provides molecular details of adhesin translocation across the outer membrane and elucidates a unique conformational state adopted by the adhesin during stepwise secretion through the usher pore. A cryo-electron microscopy (cryoEM) structure of a quaternary tip complex consisting of the usher FimD, chaperone FimC, adhesin FimH, and the tip adapter FimF reveal the usher caught in the act of secreting its cognate adhesin. Comparison with previous structures depicting the adhesin either first entering or having completely exited the usher pore reveals remarkable structural plasticity of the two-domain adhesin during translocation, and identify the “stretched-relaxed” conformation of FimH. A piliation assay demonstrated that the stretched-relaxed conformation of FimH is required for transport through the usher. These findings reveal the molecular details of adhesin translocation across the outer membrane and provide the basis for the design of rational therapeutics. Chapter 2 focuses on the development of antibiotic-sparing therapeutics targeting type 1 pili, in response to the global rise of multidrug-resistant bacteria. Fabs that bind the outer membrane usher FimD were generated with the goal of either inhibiting conformational changes required for usher function or blocking interactions between the usher and pilus subunits. Candidate Fabs were structurally characterized by cryoEM and functionally assessed for their ability to inhibit usher activity. One Fab targeting the N-terminal domain (NTD) of FimD was found to inhibit usher-mediated donor-strand exchange (DSE), a critical step in pilus assembly. This work demonstrates that Fab-mediated inhibition can effectively disrupt usher function and identifies a functionally important epitope on the outer membrane usher, providing a foundation for the development of small-molecule inhibitors. Chapter 3 investigates the molecular basis of FimI-mediated termination and anchoring of type 1 pili at the outer membrane usher FimD. A minimal termination complex was engineered that preserved native interactions between FimI and the preceding rod subunit FimA during incorporation into the FimD usher and was visualized by cryoEM. In the structure, FimH adopted a distorted orientation when directly linked to FimA, but this was corrected by reintroducing a tip adapter, suggesting that tip adapter subunits play a structural role in orienting FimH. FimI was found to bind the FimD C-terminal domain 2 (CTD2) in a manner similar to other subunits, suggesting it does not engage in unique interactions with the usher's periplasmic domains during termination and anchoring. Interestingly, when preceded by FimI, FimA occupied a significantly lower position within the usher pore than the analogous subunit from a similar structure. Deletion of the elongated residues in FimI’s N-terminal extension (Nte), a conserved feature among terminating subunits, did not affect the location or orientation of FimI or FimA within the usher. These findings provide a structural framework for understanding how FimI mediates termination and anchoring of the pilus, and establish a platform for further investigation into termination and anchoring of pili at the FimD usher. Overall, the studies presented here advance our understanding of type 1 pilus assembly by revealing key conformational intermediates during adhesin secretion, identifying functionally critical epitopes on the FimD usher that could serve as targets for antibiotic-sparing therapeutics, and contributing to a comprehensive model of type 1 pilus termination. By integrating highresolution cryoEM analysis with functional assays, this work not only addresses long-standing gaps in the mechanistic understanding of CUP pilus assembly but also highlights promising new avenues for therapeutic intervention. The insights gained provide a strong foundation for the rational design of usher-targeted inhibitors and open the door to broader investigations of conserved mechanisms across diverse CUP pili systems.

Committee Chair

Scott Hultgren

Committee Members

Peng Yuan; David Hunstad; David Kast; Tom Brett; Zhongsheng You

Degree

Doctor of Philosophy (PhD)

Author's Department

Biology & Biomedical Sciences (Molecular Cell Biology)

Author's School

Graduate School of Arts and Sciences

Document Type

Dissertation

Date of Award

8-15-2025

Language

English (en)

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