# Innovative Strategies to Combat Antibiotic-resistant Infections

> **NIH NIH U19** · WASHINGTON UNIVERSITY · 2024 · $133,690

## Abstract

ABSTRACT:
Antimicrobial resistance (AMR) represents a global public health crisis, with an estimated 10 million deaths per
year expected by 2050 without significant public health intervention and the development of new classes of
antibiotics. Towards combating this crisis, the parent grant funding of this supplement proposal aims to better
understand bacterial virulence factors in order to develop new strategies to prevent, diagnose, and treat
antibiotic-resistant infections. In project 2 of the parent U19 grant, we have further elucidated the mechanism by
which FimD, the outer membrane (OM) usher of the type I chaperone usher pathway (CUP) pilus system,
critical for both mouse and human cystitis, accommodates the tip adhesin during pilus biogenesis, and developed
monoclonal antibodies that inhibit this process by binding to and inhibiting usher function. While we have made
tremendous progress towards understanding usher function during pilus biogenesis, the process by which
ushers are folded and inserted into the OM has remained obscure. Each usher is -800 amino acids and consists
of five domains; i) a periplasmic N-terminal domain (NTD); ii) two periplasmic C-terminal domains (CTD1 and
CTD2); iii) a 24-stranded β-barrel translocation domain (TD); and iv) a plug domain (PD) that sits in the TD when
the usher is inactive and is moved into the periplasm to interact with the NTD when the usher is active. We
hypothesize that like other Gram-negative OM β-barrel proteins tested to date, all CUP ushers are folded and
inserted into the OM by the β-barrel Assembly Machinery (BAM). However, the presence of soluble periplasmic
terminal domains (NTD, CTD1, CTD2) and a luminal plug domain between strands 136 and 137 of the translocation
domain raises yet untested questions on how the β-barrel Assembly Machinery (BAM) accommodates these
domains and whether they play a role during usher folding by BAM. In this proposal, we will establish that ushers
are folded through the canonical BAM-mediated pathway by conducting photocrosslinking experiments between
BamA and FimD and investigate the role of the PD on FimD folding by comparing the crosslinking efficiency
between wild type and a plugless FimD (which is genetically deleted for the PD). We will also determine which
components of the BAM complex are required to accommodate the different soluble domains of the FimD usher.
These studies will not only enhance our understanding of the mechanism by which ushers are folded and inserted
into the OM by BAM, but also how BAM handles large multidomain OM proteins; revealing folding intermediates
that can be targeted for future therapeutic development.

## Key facts

- **NIH application ID:** 11094587
- **Project number:** 3U19AI157797-04S2
- **Recipient organization:** WASHINGTON UNIVERSITY
- **Principal Investigator:** SCOTT J. HULTGREN
- **Activity code:** U19 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $133,690
- **Award type:** 3
- **Project period:** 2021-03-01 → 2026-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/11094587

## Citation

> US National Institutes of Health, RePORTER application 11094587, Innovative Strategies to Combat Antibiotic-resistant Infections (3U19AI157797-04S2). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/11094587. Licensed CC0.

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