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Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance
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Published on: January 3, 2012

Clostridium difficile toxin A binds colonocyte Src causing dephosphorylation of focal adhesion kinase and paxillin

Ho Kim1, Sang Hoon Rhee, Charalabos Pothoulakis

  • 1Department of Life Science, College of Natural Science, Daejin University, Pochen, Kyungkido, Republic of Korea.

Experimental Cell Research
|June 2, 2009
PubMed

Insights

Clostridium difficile toxin A disrupts colon cell focal adhesion by directly inhibiting Src kinase activity, leading to FAK and paxillin inactivation. This mechanism contributes to toxin A

Area of Science:

  • Cell biology
  • Molecular mechanisms of bacterial toxins
  • Gastrointestinal pathology

Background:

  • Clostridium difficile toxin A (TcdA) disrupts colonocyte tight junctions via Rho protein glucosylation.
  • The impact of TcdA on colonocyte focal adhesion and associated signaling pathways remains incompletely understood.

Purpose of the Study:

  • To investigate the molecular mechanisms by which TcdA affects focal adhesion formation in human colonocytes.
  • To elucidate the role of TcdA in the dephosphorylation of focal adhesion kinase (FAK) and paxillin.

Main Methods:

  • Exposure of NCM460 human colonocytes to TcdA.
  • Assessment of FAK and paxillin phosphorylation status.
  • Enzyme activity assays for protein tyrosine phosphatase.
  • UDP-2'3' dialdehyde to block glucosyltransferase activity.
  • GST-pull down assays and in vitro kinase assays to study Src activity.

Main Results:

  • TcdA exposure led to complete dephosphorylation of FAK and paxillin and reduced protein tyrosine phosphatase activity.
  • Blocking TcdA's glucosyltransferase activity did not prevent FAK and paxillin dephosphorylation.
  • TcdA directly binds to the catalytic domain of Src, inhibiting its kinase activity.
  • Src kinase inhibition by TcdA precedes FAK/paxillin inactivation.

Conclusions:

  • TcdA inhibits colonocyte focal adhesion through direct binding to and inactivation of Src kinase, independent of Rho glucosylation.
  • This Src-mediated pathway contributes to the disruption of the colonocyte cytoskeleton and barrier function observed in C. difficile infections.

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