# Identification of CodY-Regulated Factors that Control Sporulation in Clostridioides difficile

> **NIH NIH F31** · EMORY UNIVERSITY · 2024 · $48,974

## Abstract

PROJECT SUMMARY/ABSTRACT
Clostridioides difficile is an anaerobic spore-forming pathogen that causes around 500,000 infections, 13,000
deaths, and healthcare costs of around 1 billion dollars annually in the United States. C. difficile infections cause
severe diarrhea and inflammation of the colon. Infection is transmitted by ingestion of C. difficile spores, which
can survive oxygen, disinfectants, chemicals, and radiation. After ingestion, spores germinate into vegetative
cells within the intestine. These vegetative cells colonize the gastrointestinal tract, generating toxins and forming
more spores. Spore formation is triggered by nutritional deprivation of the pathogen in the gastrointestinal tract.
Nutrient availability is are sensed in C. difficile by specific transcriptional regulators, such as CodY. CodY is a
global transcription regulator that is present in low G + C Gram-positive bacteria and responds to intracellular
concentrations of branched-chain amino acids and GTP. Although it is known that C. difficile codY mutants
hyper-sporulate, the means by which CodY represses sporulation is not currently understood. Based on the
sporulation repression by CodY and its role as a transcriptional regulator, I hypothesize that CodY
regulates sporulation by controlling the expression of a sporulation factor(s). In preliminary studies, I
performed transcriptomic analyses of codY and wild-type strains, in combination with mapping of CodY-binding
sites and transcriptional knockdown. Through these experiments I identified direct CodY-regulated factors that
impact sporulation. The top three candidate genes are CDR20291_1334, CDR20291_1851-1853, and
CDR20291_2485-2486. To test the hypothesis, I propose to complete these Specific Aims: Aim 1, I will assess
the roles of CDR20291_1334, CDR20291_1851-1853, and CDR20291_2485-2486 in sporulation by generating
null mutants and subsequently analyzing their contribution to sporulation and transcriptional profiles. Also, I will
characterize the basic functions of these genes in C. difficile using growth curves, fluorescent microscopy, and
co-immunoprecipitation experiments. In Aim 2, I will compare CodY binding to these target genes on wild-type
alleles and variants containing mutated CodY binding sites by electrophoretic mobility shift assay, followed by
assessment of the impact of CodY-dependent regulation on sporulation frequency via complementation studies
with these genes containing wild-type and mutated CodY boxes. The completion of these aims will identify CodY-
regulated genes that control sporulation and uncover their role in sporulation regulation through CodY.

## Key facts

- **NIH application ID:** 10978198
- **Project number:** 5F31AI179158-02
- **Recipient organization:** EMORY UNIVERSITY
- **Principal Investigator:** Marcos Paulo Monteiro
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $48,974
- **Award type:** 5
- **Project period:** 2023-07-01 → 2025-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10978198

## Citation

> US National Institutes of Health, RePORTER application 10978198, Identification of CodY-Regulated Factors that Control Sporulation in Clostridioides difficile (5F31AI179158-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10978198. Licensed CC0.

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