Pilus biogenesis in uropathogenic Escherichia coli : An electrophysiology study on the PapC usher

Uropathogenic Escherichia coli are the main causal agents of urinary tract infections. Pili are bacterial surface adhesive organelles that are critical virulence factors in the successful colonization of the urinary tract by uropathogenic E. coli. Pili are composite in structure and are formed from the polymerization of different subunits. The chaperone/usher pathway is a bi-component secretion system that is dedicated to the biogenesis and export of pili to the cell surface. It is comprised up of a periplasmic chaperone protein and an outer membrane usher protein that functions as the site of pilus assembly and simultaneous translocation. The usher exists as a dimer, where each monomer consists of a gated β-barrel pore with several domains. To shed insight into the mechanistic details behind pilus biogenesis we instituted the first electrophysiology study on the PapC usher channel. In this dissertation we present data on the pore properties of the PapC usher. Using planar lipid bilayer technique we found that PapC forms an ion conducting channel that is mostly in a closed state. However, the usher channel is dynamic and transiently opens to various conductance levels. A structure function relationship study was applied to investigate the roles of the α-helix, plug, N- and C-terminal domains on the usher channel behavior. Domain deletion mutants characterized using planar lipid bilayer showed the modulating effects of the α-helix, plug, N- and C-terminal domains on the usher channel behavior. In contrast to the wild type, domain deletion mutants displayed open channel behavior that was marked by frequent closing transitions. The ∆plug mutant formed a channel with an extremely large conductance indicating that the plug domain gates the usher closed and its removal creates a large pore. Single site substitution of the D234 residue resulted in an open channel as well, indicating the D234 residue plays a role in plug displacement. Following characterization of the usher, we investigated the modulatory effect of chaperone – subunit complexes on the usher channel behavior. We attained inconclusive results and found no clear-cut discernible effect on the usher channel behavior by the chaperone subunit complexes in the various assays reported in this dissertation. However, these results set the stage for more work on the modulation of the usher by substrates.Biology and Biochemistry, Department o