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Updated: Jul 29, 2025
07:02
An Assay for Quantifying Protein-RNA Binding in Bacteria
Published on: June 12, 2019
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Grad-seq identifies KhpB as a global RNA-binding protein in Clostridioides difficile that regulates toxin production
Vanessa Lamm-Schmidt1, Manuela Fuchs1, Johannes Sulzer1
1Faculty of Medicine, Institute of Molecular Infection Biology (IMIB), University of Würzburg, Josef-Schneider-Straße 2/D15, 97080 Würzburg, Germany.
Microlife
|May 24, 2023
Summary
Researchers identified KhpB as a widespread RNA-binding protein (RBP) in the Gram-positive pathogen Clostridioides difficile. Deleting khpB increased toxin A production, revealing KhpB
Area of Science:
- Bacteriology
- Molecular Biology
- Genomics
Background:
- RNA-binding proteins (RBPs) are crucial for bacterial gene regulation.
- RBP knowledge is limited in Gram-positive bacteria compared to Gram-negative species.
- The RNA biology of Clostridioides difficile, a significant human pathogen, is largely unexplored.
Purpose of the Study:
- To comprehensively map RNA-protein complexes in Clostridioides difficile.
- To discover novel RBPs and their targets in this important pathogen.
- To investigate the role of RBPs in C. difficile virulence.
Main Methods:
- Gradient sedimentation (Grad-seq) analysis of native RNA-protein complexes.
- Pulldown assays and global RIP-seq (RNA immunoprecipitation sequencing).
- Analysis of a khpB deletion strain to assess functional impact.
Main Results:
- Grad-seq provided distributions for ~88% of transcripts and ~50% of proteins.
- Identified KhpB as a pervasive, uncharacterized RBP in C. difficile.
- KhpB binds numerous mRNA and small RNA targets, including those involved in virulence pathways and toxin A production.
Conclusions:
- KhpB plays a significant role in regulating gene expression in C. difficile.
- Increased toxin A production and transcript levels in khpB deletion strains highlight its role in virulence.
- KhpB homologues represent a new class of globally acting RBPs in Gram-positive bacteria.
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