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Published on: December 10, 2016
A Drosophila Model for Clostridium difficile Toxin CDT Reveals Interactions with Multiple Effector Pathways
Ruth Schwartz1, Annabel Guichard2, Nathalie C Franc3
1Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA 92093-0335, USA.
Abstract:
Clostridium difficile infections (CDIs) cause severe and occasionally life-threatening diarrhea. Hyper-virulent strains produce CDT, a toxin that ADP-ribosylates actin monomers and inhibits actin polymerization. We created transgenic Drosophila lines expressing the catalytic subunit CDTa to investigate its interaction with host signaling pathways in vivo. When expressed in the midgut, CDTa reduces body weight and fecal output and compromises survival, suggesting severe impairment of digestive functions. At the cellular level, CDTa induces F-actin network collapse, elimination of the intestinal brush border, and disruption of intercellular junctions. We confirm toxin-dependent re-distribution of Rab11 to enterocytes' apical surface and observe suppression of CDTa phenotypes by a Dominant-Negative form of Rab11 or RNAi of the dedicated Rab11GEF Crag (DENND4). We also report that Calmodulin (Cam) is required to mediate CDTa activity. In parallel, chemical inhibition of the Cam/Calcineurin pathway by Cyclosporin A or FK506 also reduces CDTa phenotypes, potentially opening new avenues for treating CDIs.
Insights
Clostridium difficile toxin A (CDTa) disrupts gut function by targeting actin polymerization. Inhibiting calmodulin or Rab11 pathways shows potential for treating C. difficile infections (CDIs).
Area of Science:
- Molecular Biology
- Cell Biology
- Microbiology
Background:
- Clostridium difficile infections (CDIs) are a significant cause of infectious diarrhea.
- Hyper-virulent C. difficile strains produce toxin CDT, which disrupts the host cytoskeleton.
- CDT ADP-ribosylates actin monomers, inhibiting actin polymerization.
Purpose of the Study:
- To investigate the in vivo interaction of the CDTa catalytic subunit with host signaling pathways.
- To explore potential therapeutic targets for CDIs.
Main Methods:
- Creation of transgenic Drosophila melanogaster expressing the CDTa subunit in the midgut.
- Analysis of cellular phenotypes, including F-actin networks, brush borders, and intercellular junctions.
- Investigating the roles of Rab11, Crag (DENND4), Calmodulin (Cam), and the Cam/Calcineurin pathway.
Main Results:
- CDTa expression in Drosophila midgut caused reduced body weight, fecal output, and survival.
- CDTa induced F-actin collapse, brush border elimination, and junctional disruption.
- Rab11 redistribution was observed, and its suppression ameliorated CDTa phenotypes.
- Calmodulin was essential for CDTa activity, and its inhibition reduced CDTa-induced damage.
Conclusions:
- CDTa disrupts intestinal epithelial integrity and digestive function by targeting actin dynamics.
- The Rab11 and Calmodulin pathways are critical mediators of CDTa toxicity.
- Inhibition of the Calmodulin/Calcineurin pathway presents a potential therapeutic strategy for CDIs.
