# Real-time metabolic analysis of virulence activation in P. aeruginosa using fluorescence lifetime imaging microscopy (FLIM)

> **NIH NIH R21** · UNIVERSITY OF CALIFORNIA-IRVINE · 2020 · $171,050

## Abstract

Project Summary
Pseudomonas aeruginosa is an opportunistic bacterial pathogen that infects a broad range of host cell types.
The ability of P. aeruginosa to kill host cells is tightly regulated by several mechanisms including cell-to-cell
communication and surface attachment. Metabolic pathways also appear to have a significant role in
controlling the activity of host killing factors. However, the regulatory connections between central metabolism
and virulence activation are not understood. What are the metabolic dynamics during P. aeruginosa activation?
How do central metabolic pathways contribute to virulence induction? The work proposed here attempts to
address these questions by simultaneously measuring metabolic activity and virulence states in P. aeruginosa.
Metabolic activity will be measured using fluorescence lifetime imaging microscopy (FLIM), which measures
the activity of the metabolite nicotinamide adenine dinucleotide (NAD(P)H), and through biochemical assays.
The FLIM technique does not damage cells and does not require any chemical treatments of the cells. FLIM
has been used extensively to measure metabolic activity in mammalian cells but has been used in bacteria in
only a few cases. In particular, the use of FLIM to track NAD(P)H activity in P. aeruginosa has not been
established.
In Aim 1, our experiments will characterize the extent to which FLIM measures the relative abundance of
NAD(P)H in P. aeruginosa. We will determine the extent to which molecules that are secreted by P. aeruginosa
contribute to the FLIM signal and the extent to which inhibiting metabolic activity produces changes in the FLIM
signal. In Aim 2, we will characterize how metabolic activity correlates with the activation of virulence. We will
induce virulence in the major P. aeruginosa virulence systems and measure metabolic activity using FLIM and
biochemical assays. In preliminary experiments, we observed that metabolic activity indeed correlates with the
virulence state. Moving forward, we will test the converse question: does altering metabolic activity affect
virulence? We will metabolically engineer P. aeruginosa to have altered NAD(P)H activity and measure
potential changes in virulence.
If successful, this work will establish a non-invasive procedure for real-time quantification of metabolic activity
in live P. aeruginosa cells and will lay the foundation for future metabolism-virulence studies in P. aeruginosa
and other pathogens. The long-term goals of this work are to develop the FLIM technique to measure
metabolic dynamics in both hosts and pathogens, as our experiments enable the simultaneous collection of
metabolic data from both organisms. We will extend the technique to monitor host and bacterial metabolism
during acute and chronic infections in tissue and whole organism models. The results of this work will broadly
impact our understanding of P. aeruginosa physiology, bacterial metabolism, and regulation of virulence by
metabolism, and will ...

## Key facts

- **NIH application ID:** 9850929
- **Project number:** 5R21AI139968-02
- **Recipient organization:** UNIVERSITY OF CALIFORNIA-IRVINE
- **Principal Investigator:** Albert Siryaporn
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $171,050
- **Award type:** 5
- **Project period:** 2019-01-16 → 2021-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9850929

## Citation

> US National Institutes of Health, RePORTER application 9850929, Real-time metabolic analysis of virulence activation in P. aeruginosa using fluorescence lifetime imaging microscopy (FLIM) (5R21AI139968-02). Retrieved via AI Analytics 2026-06-11 from https://api.ai-analytics.org/grant/nih/9850929. Licensed CC0.

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