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A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment
Published on: May 25, 2017
Clostridium difficile toxin expression is inhibited by the novel regulator TcdC
Susana Matamouros1, Patrick England, Bruno Dupuy
1Unité de Génétique Moléculaire Bactérienne, Institut Pasteur, Paris, France.
Abstract:
Clostridium difficile, an emerging nosocomial pathogen of increasing clinical significance, produces two large protein toxins that are responsible for the cellular damage associated with the disease. The precise mechanisms by which toxin synthesis is regulated in response to environmental change have yet to be discovered. The toxin genes (tcdA and tcdB) are located in a pathogenicity locus (PaLoc), along with tcdR and tcdC. TcdR is an alternative RNA polymerase sigma factor that directly activates toxin gene expression, while the inverse relationship between expression of tcdR, tcdA and tcdB genes on the one hand and tcdC on the other has led to the suggestion that TcdC somehow interferes with toxin gene expression. This idea is further supported by the finding that many recent C. difficile epidemic strains in which toxin production is increased carry a common tcdC deletion mutation. In this report we demonstrate that TcdC negatively regulates toxin synthesis both in vivo and in vitro. TcdC destabilizes the TcdR-containing holoenzyme before open complex formation, apparently by interaction with TcdR or TcdR-containing RNA polymerase holoenzyme or both. In addition, we show that the hypertoxigenicity phenotype of C. difficile epidemic strains is not due to their common 18 bp in-frame deletion in tcdC.
Insights
Clostridium difficile toxin regulator TcdC negatively controls toxin production by destabilizing the TcdR-RNA polymerase complex. This finding clarifies toxin synthesis mechanisms and explains why epidemic strains with tcdC deletions are not hypertoxigenic.
Area of Science:
- Microbiology
- Molecular Biology
- Pathogenesis
Background:
- Clostridium difficile is a significant nosocomial pathogen causing disease via toxin production.
- Toxin synthesis regulation by environmental factors remains unclear.
- The pathogenicity locus (PaLoc) contains toxin genes (tcdA, tcdB) and regulatory genes (tcdR, tcdC).
Purpose of the Study:
- To elucidate the regulatory role of TcdC in Clostridium difficile toxin synthesis.
- To investigate the mechanism by which TcdC influences toxin gene expression.
- To determine if tcdC deletions in epidemic strains correlate with hypertoxigenicity.
Main Methods:
- In vivo and in vitro experiments were conducted to assess TcdC function.
- Analysis of TcdR-RNA polymerase holoenzyme interactions.
- Genomic analysis of C. difficile epidemic strains for tcdC mutations.
Main Results:
- TcdC was demonstrated to negatively regulate toxin synthesis.
- TcdC destabilizes the TcdR-containing holoenzyme prior to open complex formation.
- The common 18 bp in-frame deletion in tcdC of epidemic strains does not cause hypertoxigenicity.
Conclusions:
- TcdC acts as a negative regulator of Clostridium difficile toxin production.
- TcdC interferes with RNA polymerase holoenzyme function, inhibiting toxin gene transcription.
- The hypervirulence of certain C. difficile epidemic strains is not attributable to the observed tcdC deletion mutation.
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