# Defining functional domains of a P. aeruginosa efflux pump using periplasmic nanobodies

> **NIH NIH R21** · HARVARD MEDICAL SCHOOL · 2021 · $254,250

## Abstract

PROJECT SUMMARY
Some of the most important human pathogens includes those that cause difficult to treat infections due to
their resistance to commonly used antibiotics. For Gram negative organisms, the main resistance
mechanism includes an impermeable cell envelope in conjunction with activities of expulsion (efflux)
pumps. This group includes Pseudomonas aeruginosa, a pathogen of immunocompromised individuals and
those with cystic fibrosis, with numerous efflux pumps encoded in its genome. This proposal aims at
creating new enabling tools for drug discovery, by exploring the feasibility of using periplasmically
expressed single domain antibodies (nanobodies) capable of blocking biological processes that take place
in this compartment. The work proposed here will target the periplasmic components of the MexXY-OprM
efflux pump, with the objective of identifying nanobodies that interfere with antibiotic efflux. In the first Aim,
recombinant MexX and the fused periplasmic domains of MexY will be purified and these will be used to
select binders from a synthetic library of nanobodies displayed on yeast surface. These will be transferred
to bacterial expression vectors and introduced into P. aeruginosa where the secretion of antibodies into the
periplasm should interfere with the assembly or function of MexXY-OprM, rendering the bacteria sensitive
to antibiotics that are substrates of this efflux pump. A number of genetic and biochemical approaches will
be used to map the nanobody epitopes on the Mex proteins. Nanobodies showing weak affinities will be
subjected to several round of affinity maturation. The project should generate not only information about
functional domains of an efflux pump but also tools suitable for screening and validation of small molecule
inhibitors of antibiotic efflux.

## Key facts

- **NIH application ID:** 10179317
- **Project number:** 5R21AI153471-02
- **Recipient organization:** HARVARD MEDICAL SCHOOL
- **Principal Investigator:** STEPHEN LORY
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $254,250
- **Award type:** 5
- **Project period:** 2020-06-03 → 2024-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10179317

## Citation

> US National Institutes of Health, RePORTER application 10179317, Defining functional domains of a P. aeruginosa efflux pump using periplasmic nanobodies (5R21AI153471-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10179317. Licensed CC0.

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