# Rho dependent regulation of Clostridioides difficile virulence

> **NIH NIH F32** · UNIV OF NORTH CAROLINA CHAPEL HILL · 2024 · $74,284

## Abstract

Project Summary
Clostridioides difficile is a gram-positive, spore-forming, obligate anaerobic bacterium and opportunistic
pathogen. C. difficile infection (CDI) is associated with hospital antibiotic use and can cause severe diarrhea,
pseudomembranous colitis, and death. The primary causative agents of CDI symptoms are the secreted toxins
TcdA and TcdB, which are regulated by the flgB operon through sigma factor sigma-D. The bacterial
transcription termination factor Rho regulates the flgB operon by repressing transcription only when an
invertible genetic “switch” mapping to the flgB transcript 5’ UTR is in the “off” orientation. Genetic deletion of
Rho functionality restores flgB expression when the switch is oriented off. Loss of Rho functionality in C.
difficile results in growth, sporulation, and virulence defects independent of loss of regulation at the flgB
operon, indicating additional Rho-dependent regulation of C. difficile virulence factors. Efforts to identify a Rho
recognition sequence in C. difficile, including at the flagellar switch, based on a multi-organism consensus
have been unsuccessful, possibly due to the unusually GC-poor C. difficile genome (~29%). Rho-dependent
regulation is also likely to be highly influenced by interactions with termination/antitermination factors NusA and
NusG. Nus factors are essential for C. difficile growth but their influence on Rho-dependent termination and
effect on the virulence factors expression is unknown. I hypothesize that Rho is a key factor in C. difficile
pathogenesis through direct regulation of critical virulence genes in addition to the flagellar operon, and that
this regulation is dependent on, or attenuated by, Nus factors on a gene-by-gene basis. The objective of this
proposal is to identify and annotate Rho, NusA, and NusG dependent terminators in C. difficile to identify key
Rho and Nus factor regulated growth, sporulation, and virulence genes for further study, and determine a
predictive rut site sequence in C. difficile. I will compare RNA-seq and Term-seq data between the Rho-null
and control C. difficile strains to identify Rho dependent terminators and verify these sites in vitro. I will use
CRISPRi to generate NusA and NusG dependent transcript-depleted strains for Term-seq analysis. Completion
of this study will improve our understanding of C. difficile virulence regulation, provide further annotation of the
C. difficile transcriptome, and may reveal new potential targets for developing therapeutics to combat CDI. Dr.
Tamayo is a leader in the field of C. difficile biology and her expertise in genetic approaches complements my
graduate training in biochemical techniques. The University of North Carolina at Chapel Hill and the
Department of Microbiology and Immunology both contribute to a productive, collegial research environment
with numerous opportunities for career development.

## Key facts

- **NIH application ID:** 10825902
- **Project number:** 1F32AI181516-01
- **Recipient organization:** UNIV OF NORTH CAROLINA CHAPEL HILL
- **Principal Investigator:** Dylan Plaskon
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $74,284
- **Award type:** 1
- **Project period:** 2024-09-01 → 2027-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10825902

## Citation

> US National Institutes of Health, RePORTER application 10825902, Rho dependent regulation of Clostridioides difficile virulence (1F32AI181516-01). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10825902. Licensed CC0.

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