| Summary: | <p>Adhesion to abiotic and biotic surfaces is essential for numerous bacterial developmental processes including biofilm formation. While the general role of adhesins in biofilm formation has been explored, the arrangement of these structures on the cell surface and how these structures interact with substrates at the molecular level remain unclear. In this project, the CupE chaperone usher fimbriae of Pseudomonas aeruginosa and the holdfast polysaccharide of Caulobacter crescentus were investigated as models of proteinaceous and polysaccharide-mediated adhesion respectively.</p>
<p>Using structural biology methods, including electron cryomicroscopy (cryo-EM) and electron cryotomography (cryo-ET), this thesis provides molecular details of a previously uncharacterized mechanism of polysaccharide adhesion. The holdfast is a polysaccharide adhesin which is anchored to the surface of C. crescentus by a defined molecular complex made up of the HfaABD proteins. The HfsDAB secretion proteins are required for assembly and localization of the HfaABD anchor, which spans the outer membrane of the stalk tip. The anchor protein HfaB is the major component of the anchor complex located on the periplasmic side of the outer membrane, while HfaA and HfaD are located on the cell surface. HfaB is the critical component of the complex, without which no HfaABD complex is observed in cells. Finally, anchor densities are present along the cell envelope of other alphaproteobacteria including Asticcacaulis and Hirschia spp., supporting a conserved mechanism in holdfast anchoring.</p>
<p>Additionally, this thesis provides insight into the regulation, structure and physiological role of archaic CupE fimbriae in P. aeruginosa. Deletion of the PA2133 phosphodiesterase gene upregulates CupE filaments under static conditions, indicating that filament assembly is promoted upon c-di-GMP elevation and surface sensing. CupE fimbriae are a major contributor to biofilm formation in DPA2133 mutants, with increased biofilm formation at the air-liquid interface, as well as cell clustering and organisation at the microscopic level. The diameter of CupE filaments is ~4 nm, consistent with fimbriae of the archaic s-clade. The cryo-EM structure of native CupE, at a resolution of 4.5 Å, reveals a single-start, helical morphology that does not resemble the tubular structure of classical chaperone usher assemblies. These findings provide the first structural insights into a fully assembled, archaic chaperone usher filament in bacteria and support CupE fimbriae function as an adhesin during biofilm formation.</p>
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