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Updated: Mar 28, 2026
Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291
Published on: December 10, 2016
Increased toxin expression in a Clostridium difficile mfd mutant
Stephanie E Willing1,2, Emma J Richards3, Lluis Sempere4
1Department of Life Sciences, Centre for Molecular Bacteriology and Infection, Imperial College London, London, SW7 2AZ, UK. Stephanie.Willing@rhul.ac.uk.
Background:
The symptoms of Clostridium difficile infection are mediated primarily by two toxins, TcdA and TcdB, the expression of which is governed by a multitude of factors including nutrient availability, growth phase and cell stress. Several global regulators have been implicated in the regulation of toxin expression, such as CcpA and CodY.
Results:
During attempts to insertionally inactivate a putative secondary cell wall polysaccharide synthesis gene, we obtained several mutants containing off-target insertions. One mutant displayed an unusual branched colony morphology and was investigated further. Marker recovery revealed an insertion in mfd, a gene encoding a transcription-coupled repair factor. The mfd mutant exhibited pleiotropic effects, in particular increased expression of both toxin A and B (TcdA and TcdB) compared to the parental strain. Western blotting and cellular cytotoxicity assays revealed increased expression across all time points over a 24 h period, with inactivation of mfd resulting in at least a 10 fold increase in cell cytotoxicity. qRT-PCR demonstrated the upregulation of both toxins occurred on a transcriptional level. All effects of the mfd mutation were complemented by a plasmid-encoded copy of mfd, showing the effects are not due to polar effects of the intron insertion or to second site mutations.
Conclusions:
This study adds Mfd to the repertoire of factors involved in regulation of toxin expression in Clostridium difficile. Mfd is known to remove RNA polymerase molecules from transcriptional sites where it has stalled due to repressor action, preventing transcriptional read through. The consistently high levels of toxin in the C. difficile mfd mutant indicate this process is inefficient leading to transcriptional de-repression.
Insights
The Mfd protein is a novel regulator of toxin production in Clostridium difficile. Inactivating mfd significantly increases toxin A and B expression and cytotoxicity, impacting infection severity.
Area of Science:
- Microbiology
- Molecular Biology
- Bacterial Pathogenesis
Background:
- Clostridium difficile infection symptoms are primarily caused by TcdA and TcdB toxins.
- Toxin expression is influenced by nutrient availability, growth phase, cell stress, and global regulators like CcpA and CodY.
Purpose of the Study:
- To investigate the role of the mfd gene in regulating toxin expression in Clostridium difficile.
- To characterize the effects of mfd inactivation on toxin production and cytotoxicity.
Main Methods:
- Generating insertion mutants in Clostridium difficile.
- Analyzing colony morphology.
- Marker recovery to identify gene insertions.
- Western blotting and cytotoxicity assays.
- Quantitative reverse transcription PCR (qRT-PCR).
- Complementation studies using a plasmid-encoded mfd gene.
Main Results:
- An off-target insertion in the mfd gene resulted in a branched colony morphology.
- The mfd mutant showed significantly increased expression of both TcdA and TcdB toxins.
- Cytotoxicity assays revealed at least a 10-fold increase in cell-killing activity in the mfd mutant.
- qRT-PCR confirmed toxin upregulation at the transcriptional level.
- Complementation restored wild-type phenotype, confirming mfd's role.
Conclusions:
- Mfd is identified as a new factor involved in regulating toxin expression in Clostridium difficile.
- The Mfd protein's function in resolving stalled RNA polymerase is crucial for preventing toxin de-repression.
- Inefficiency in this process within the mfd mutant leads to consistently high toxin levels and increased pathogenicity.
