# Enabling High-Throughput Analysis and Single-Cell Imaging of Bacterial Signals

> **NIH NIH R01** · UTAH STATE HIGHER EDUCATION SYSTEM--UNIVERSITY OF UTAH · 2020 · $350,750

## Abstract

PROJECT SUMMARY
 Enabling High-Throughput Analysis and Single-Cell Imaging of Bacterial Signals
Cyclic dinucleotides (CDNs) are an emerging class of signaling molecules at the intersection of bacterial and
host interactions. Within bacterial cells, CDNs act as chemical signals that control distinct cellular programs for
colonization (cyclic di-GMP), stress response (cyclic di-AMP), and surface contact (cyclic AMP-GMP).
Furthermore, these three bacterial CDNs and a newfound mammalian CDN called cGAMP are found to stimulate
an innate immune signaling pathway in mammalian cells through a protein receptor called STING (Stimulator of
Interferon Genes). Thus, understanding how CDN levels are regulated by environmental and host inputs would
advance our knowledge of bacterial-host interactions, on both the side of bacterial pathogens and the host
immune response. However, the major roadblock to obtaining these critical mechanistic insights has been the
difficulty in observing changes in the levels of these chemical signals across scales and systems. Thus, the
broad goals of this proposal are to develop luminescent and fluorescent biosensors that enable high-throughput
analysis and imaging of CDNs from many to single cells (Aim 1), from cultures to within hosts (Aim 2), and from
individual species to communities (Aim 3). We previously established that a new type of genetically-encoded
biosensors, RNA-based fluorescent (RBF) biosensors, have sufficient sensitivity and selectivity to track and
quantitate low abundance, intracellular metabolites including CDNs. Building on our earlier invention of turn-on
RBF biosensors for cyclic di-GMP and cyclic di-AMP, we will develop design strategies to make ratiometric RBF
biosensors for these CDNs that can report on the signaling status of bacterial pathogens within hosts (Aim 2). In
collaboration with Prof. Portnoy at UC Berkeley, we will study Listeria monocytogenes, the causative agent of
listeriosis, within mammalian cells. In collaboration with Prof. Stevenson at U Kentucky, we will study Borrelia
burgdorferi, the causative agent of Lyme disease, in the tick. To enable the study of CDN signaling in diverse
bacteria and in model microbial communities, we will employ a broad-host vector system for genomic integration
of RBF biosensor genes (Aim 3). Furthermore, to enable the study of the innate immune signal cGAMP, we will
perform high-throughput selections to make novel RBF biosensors (Aim 4). Finally, we will develop
bioluminescent resonance energy transfer (BRET) biosensors that can be applied to quantitate cyclic di-GMP in
crude lysates and have future potential for whole animal imaging (Aim 1). In collaboration with Prof. Waters at
Michigan State, we will use these novel BRET biosensors to analyze the response of Vibrio cholerae, the
causative agent of cholera, to human intestinal bile acids.

## Key facts

- **NIH application ID:** 10001046
- **Project number:** 5R01GM124589-05
- **Recipient organization:** UTAH STATE HIGHER EDUCATION SYSTEM--UNIVERSITY OF UTAH
- **Principal Investigator:** Ming Chen Hammond
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $350,750
- **Award type:** 5
- **Project period:** 2017-09-01 → 2022-09-23

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10001046

## Citation

> US National Institutes of Health, RePORTER application 10001046, Enabling High-Throughput Analysis and Single-Cell Imaging of Bacterial Signals (5R01GM124589-05). Retrieved via AI Analytics 2026-05-28 from https://api.ai-analytics.org/grant/nih/10001046. Licensed CC0.

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