SUFU 2018: The Uropathogenic Escherichia Coli Pilus Usher Controls Pilus Assembly Through a 2-Step Verification Process During Activation

Austin, TX ( Uropathogenic strains of Escherichia coli use the chaperone-usher (CU) pathway to construct adhesive surface structures termed pili or fimbriae. Pili facilitate binding to bladder and kidney epithelial cells, thereby promoting urinary tract colonization and infection. Dr. Glenn Werneburg and colleagues suggested an essential and well-regulated step of pilus assembly is the activation of the usher, in which the usher undergoes a transformation from a plug-gated membrane pore to an engaged assembly machine. Werneburg and colleagues also suggested understanding the mechanism of how the usher is activated and controls the assembly of pili, they monitored pilus subunit interactions with different usher domains using genetic, biochemical, and biophysical approaches including Förster Resonance Energy Transfer (FRET).

Using an inter- and intramolecular FRET-based affinity approach, they identified the necessary conditions for usher activation. They also identified particular regions of the activating pilus subunit (adhesin) that are essential for discrete steps of the activation process. They also demonstrated that the C-terminal domain (pilin domain) of the activating subunit is necessary for subunit recruitment to the usher platform (Kd=194 nM), and that the N-terminal domain (lectin domain) of the activating subunit is necessary for subsequent usher plug expulsion (act1/2=4 nM) and priming for pilus assembly (Kd=4 nM). Further, using a mutagenesis approach, the author was able to demonstrate that the addition of the lectin domain is sufficient to confer usher-activating potential to a non-activating pilus subunit.

They concluded that the usher employs a “2-step verification” process in which it recognizes and verifies both the N and C domains of the initiating adhesin subunit, each during a discrete step in the activation process. This process may ensure that every assembled pilus has an adhesin at its tip, in a location to facilitate urinary tract adhesion and UTI. These discoveries open avenues for informed drug design to target these newly-elucidated mechanistic steps, thus potentially preventing or treating UTI. These FRET-based approaches are poised for the screening of molecules to disrupt this process.

Presented by: Glenn Werneburg, PhD

Authors: Glenn Werneburg, PhD, Hemil Chauhan, BS, Nadine Henderson, MS and David Thanassi ,PhD, Stony Brook University School of Medicine, Stony Brook, NY

Written by: Bilal Farhan, MD, Female Urology Fellow and Voiding Dysfunction, Department of Urology, University of California, Irvine at the Society of Urodynamics, Female Pelvic Medicine & Urogenital Reconstruction Winter Meeting (SUFU 2018), February 27-March 3, 2018, Austin, Texas