# Type III effector-cofactor dynamics within the cellular environment

> **NIH NIH R01** · MEDICAL COLLEGE OF WISCONSIN · 2020 · $440,793

## Abstract

Project Summary
Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that is problematic for individuals who
are immunocompromised. Importantly, P. aeruginosa is responsible for pathology associated with chronic
colonization of individuals with cystic fibrosis. The organism is well adapted to health care settings by being
able to survive on minimal nutrients, form microbial communities on surfaces and express intrinsic resistance
to antibiotics and disinfectants. P. aeruginosa has a large repertoire of destructive enzymes and toxins that aid
bacterial replication by neutralizing innate immune cells in human hosts. The mechanisms of action include
interference with or inhibition of signal transduction, host protein synthesis, cytoskeletal function or membrane
dynamics. Our studies focus on the cytotoxic enzymes or effectors injected by the type III secretion system of
P. aeruginosa. These enzymes, ExoS and ExoU, demonstrate broad substrate specificity and can recognize
both prokaryotic and eukaryotic targets. The property of broad substrate specificity requires that additional
control elements must be in place to keep the effector inactive before delivery to the appropriate host
environment. The type III effector, ExoU, a potent phospholipase, is used as a model system to study the
dynamic changes that mediate activation of enzyme activity in host cells.
We identified ubiquitin (Ub), which is synthesized only by eukaryotes, as the activator required for ExoU-
mediated phospholipase activity in host cells. The long-term objectives of this research are to mechanistically
describe Ub and substrate induced conformational changes that occur to activate ExoU. We have used
molecular modeling, continuous wave and double electron electron resonance spectroscopy to build an initial
model of ExoU activation. The model makes specific predictions and provides testable hypotheses that will be
challenged in iterative biochemical and biophysical analyses and model refinement with novel mono- and diUb
probes. Ub-mediated activation is postulated to serve as a specific, therapeutic target, not limited to ExoU. This
property extends to ExoU-orthologs that are encoded in the genomes of a variety of Gram negative human
pathogens and opportunists. Overall, these studies aim to contribute a structure-function basis for the rational
design of inhibitors that may be applicable for treatment of infections caused by resistant, problematic
organisms.

## Key facts

- **NIH application ID:** 9989014
- **Project number:** 5R01AI104922-08
- **Recipient organization:** MEDICAL COLLEGE OF WISCONSIN
- **Principal Investigator:** Jimmy Feix
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $440,793
- **Award type:** 5
- **Project period:** 2013-05-23 → 2022-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9989014

## Citation

> US National Institutes of Health, RePORTER application 9989014, Type III effector-cofactor dynamics within the cellular environment (5R01AI104922-08). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9989014. Licensed CC0.

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