# Chaperone-Assisted Pili Assembly in Pathogenic E. Coli

> **NIH NIH R01** · WASHINGTON UNIVERSITY · 2021 · $393,750

## Abstract

PROJECT SUMMARY/ABSTRACT:
Rising antibiotic resistance in bacterial pathogens highlights the urgent need to understand the molecular
mechanisms by which bacteria cause infections, in order to develop effective precision-based antibiotic-sparing
therapies. Gram-negative bacterial pathogens encode over 100 extracellular fibers termed chaperone/usher
pathway (CUP) pili able to recognize and colonize different host tissues and habitats, a prerequisite to infection.
Each CUP pilus is encoded as part of a gene cluster containing a designated periplasmic chaperone and outer
membrane (OM) usher protein that facilitates assembly of tens of hundreds of structural subunits into each final
pilus structure. In addition, most CUP pili are tipped by two-domain adhesins comprising: i) an N-terminal domain
that recognizes a receptor with stereochemical specificity; and ii) a C-terminal pilin domain. Chaperone-
subunit/adhesin complexes are formed through a donor strand complementation (DSC) interaction in which the
chaperone donates steric information to promote the folding of the pilin domains and are then delivered to the
OM usher which catalyzes subunit-subunit interactions via donor strand exchange (DSE). DSE occurs when an
amino-terminal extension (Nte) present on each subunit completes the Ig fold of its neighbor. Pilus biogenesis
catalyzed by the OM usher is a remarkably complex process involving the multiple domains of the usher
functioning as a nanomachine to assemble pili tipped with an adhesin. With the support from this grant,
considerable progress has been made towards elucidating the mechanism of pilus biogenesis, however, this
proposal seeks to fill key knowledge gaps: understanding the molecular workings of the usher. To do this, we
will elucidate three-dimensional structures of usher intermediates representing critical points in the assembly
cascade: i) pilus initiation; and ii) subunit incorporation/(DSE) using X-ray crystallography and single-particle
cryo-electron microscopy (Aim 1). We will elucidate the mechanisms by which both two-domain adhesins and
specialized single-domain pilins activate ushers in three distinct pilus systems (Aim 2). Understanding the
molecular biology of CUP pili has already led to a FimH-based vaccine that was developed to prevent recurrent
uropathogenic E. coli (UPEC) urinary tract infections (UTI). This was developed based on understanding that
type 1 pili tipped with FimH mediate bladder colonization. The vaccine has completed a Phase 1A/1B study and
received FDA allowance for compassionate use based on promising results. In addition, rationally designed
inhibitors of FimH function, termed mannosides, have been shown to be highly efficacious in treating and
preventing UTI in mouse models, while simultaneously being able to selectively deplete UPEC from the mouse
gastrointestinal tract reservoir. Here, small molecules that block usher function will be developed (Aim 3), which
would potentially block assembly of mul...

## Key facts

- **NIH application ID:** 10238966
- **Project number:** 5R01AI029549-31
- **Recipient organization:** WASHINGTON UNIVERSITY
- **Principal Investigator:** SCOTT J. HULTGREN
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $393,750
- **Award type:** 5
- **Project period:** 1991-03-01 → 2024-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10238966

## Citation

> US National Institutes of Health, RePORTER application 10238966, Chaperone-Assisted Pili Assembly in Pathogenic E. Coli (5R01AI029549-31). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10238966. Licensed CC0.

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