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Usher Mechanics: Workings of an Escherichia coli Molecular Machine
Date of Award
Doctor of Philosophy (PhD)
The chaperone-usher pathway (CUP) constitutes one of the prevailing mechanisms of adhesive pilus assembly of Gram-negative bacteria. In each CUP assembly system dedicated periplasmic chaperones and outer-membrane usher proteins coordinate to assemble thousands of subunits into highly stable fibers and display them on the extracellular surface. CUP ushers contain five functional domains: a transmembrane β-barrel, a β-sandwich Plug resident within the transmembrane domain in the apo usher, an N-terminal periplasmic domain (NTD), and two C- terminal periplasmic domains, CTD1 and CTD2. Biolayer interferometry studies demonstrated that: i) usher domains of the P pilus usher, PapC, had differential affinities for the chaperone- subunit complexes, with the NTD having selective binding with highest affinity for chaperone- adhesin, while the Plug and CTD2 domains had lower affinities and little selectivity; ii) the CTD2 domain catalytically dissociates the NTD-chaperone-adhesin complex, thus allowing transfer of the chaperone-adhesin to the CTDs and binding of the next chaperone-subunit to the NTD; iii) the Plug domain can form a high affinity, stable interaction with the NTD domain, leading to a model where binding of the chaperone-adhesin to the NTD triggers the Plug domain to relocate to the periplasm where it binds stably to the NTD and helps to recruit additional chaperone-subunit complexes for pilus assembly; and iv) the chaperone-terminator complex shows selectivity for the Plug domain providing new insights into pilus termination and anchoring. PapC mutagenesis and antibiotic sensitivity studies revealed that the Plug domain had separable functions in pore gating and pilus assembly and identified a salt bridge important for stability of the Plug domain in the transmembrane pore and a periplasmic loop necessary for Plug opening. Studies of pilicides delineated in vitro effects on purified chaperone-subunit stabilities and polymerization that may be relevant to their mechanism of action in vivo. This dissertation work reveals the cooperative roles of domains of the usher in pilus assembly and pore-gating giving insights into the biogenesis of critical virulence factors.
Chair and Committee
Jeffrey Henderson, Daniel Goldberg, David Haslam, David Hunstad, Christina Stallings
Volkan, Ender, "Usher Mechanics: Workings of an Escherichia coli Molecular Machine" (2013). Arts & Sciences Electronic Theses and Dissertations. 124.