Iehab N. Talukder

1st Year Graduate Student

Department: Cold Spring Harbor Laboratory

Graduate Program: Neuroscience

Advisor: Hiro Furukawa (rotation)


Abstract (rotation):

Title: The N terminus of the Outer Membrane Usher Plays a Critical Role in Pilus Biogenesis Beyond the Initial Targeting of Chaperone-Subunit Complexes

The pathogenesis of uropathogenic Escherichia coli arises from its ability to recognize and attach to host kidney and bladder epithelia by means of P and type 1 pili, causing pyelonephritis and cystitis, respectively. Both pili are made up of multiple subunits forming a helical rod and a distal tip fibrillum containing the adhesin subunit. In the periplasm, each of these subunits forms a complex with a chaperone, which is then targeted to an outer membrane (OM) usher protein for assembly and secretion of the pilus to the bacterial surface. The usher has a ß-barrel structure with a central channel for secretion. It contains exposed periplasmic and extracellular loops with distinct domains for initial binding of chaperone-subunit complexes and their subsequent assembly into a composite pilus. Previous work demonstrated that the usher N terminus is required for initial binding of chaperone-subunit complexes. I have constructed four usher mutants of the type 1 pilus system with mutations in the N-terminal region. Three of these mutants are completely defective for pilus assembly. Pilus extraction and hemagglutination assays showed that they are unable to assemble any pilus component on the bacterial surface. However, they are able to bind to the chaperone-adhesin complex, the first subunit to be targeted to the usher during pilus assembly, and undergo the proper initial conformational change, as proven by trypsin susceptibility experiments of the usher. These results demonstrate that the usher N terminus is not only the initial targeting site for chaperone-subunit complexes, as previously thought, but also plays a significant role in subsequent steps in pilus biogenesis.