# Linking Gene Expression Profiles to Cell Fate in Clostridioides difficile Using Time-Lapse Microscopy

> **NIH NIH R21** · TUFTS UNIVERSITY BOSTON · 2021 · $218,601

## Abstract

Clostridioides difficile is a spore-forming bacterial pathogen that is the leading cause of healthcare-associated
infections in the United States. While C. difficile’s ability to produce potent cytotoxins has long been known to
allow it to cause inflammatory diarrheal disease, little is known about the properties of C. difficile that allow it to
grow and survive in the competitive gut environment. Like many gut bacteria, C. difficile generates phenotypically
distinct sub-populations within a seemingly clonal population. This observation has led to the hypothesis that C.
difficile uses phenotypic heterogeneity to promote its survival in the dynamic gut environment. Unfortunately,
testing this hypothesis has been complicated by the absence of methods for following the fate of specific sub-
populations over time. Time-lapse microscopy has traditionally been used to address this question, but existing
methods cannot be used to study the growth of C. difficile because it cannot grow in the presence of atmospheric
oxygen. We have overcome this technological challenge by developing a simple anaerobic time-lapse
microscopy method for visualizing C. difficile growth at the single-cell level. By combining this method with an
automated lineage tracking pipeline, we have measured the growth properties of individual C. difficile cells for
the first time. These analyses indicate that C. difficile cell size and elongation rates are tightly controlled during
growth in rich media, but they become markedly heterogeneous in the presence of physiological stress. To
understand how C. difficile adapts to different physiological stressors at the single-cell level, this proposal will
use novel anaerobic imaging reporters to link the gene expression profile of individual cells to their cellular fate.
We will also use this system to study C. difficile spore outgrowth into vegetative cells and test whether outgrowing
cells are more vulnerable to physiological stressors than vegetative cells, a question that has not yet been studied
in any system to our knowledge. Determining the answers to these questions in C. difficile will inform strategies
for inhibiting C. difficile infections, while the methods established in this proposal will have broad utility for
studying the physiology of other anaerobes.

## Key facts

- **NIH application ID:** 10223787
- **Project number:** 1R21AI153853-01A1
- **Recipient organization:** TUFTS UNIVERSITY BOSTON
- **Principal Investigator:** Aimee Shen
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $218,601
- **Award type:** 1
- **Project period:** 2021-01-15 → 2022-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10223787

## Citation

> US National Institutes of Health, RePORTER application 10223787, Linking Gene Expression Profiles to Cell Fate in Clostridioides difficile Using Time-Lapse Microscopy (1R21AI153853-01A1). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10223787. Licensed CC0.

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