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Pasteurella multocida toxin increases endothelial permeability via Rho kinase and myosin light chain phosphatase
1Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Universität München, Germany. messler@klp.med.uni-muenchen.de
Abstract:
Pasteurella multocida toxin (PMT) has been shown to induce actin reorganization through activation of the GTPase Rho. Here we investigated the involvement of the Rho target proteins Rho kinase and myosin light chain (MLC) phosphatase in the PMT-induced increase in endothelial permeability and the underlying actin reorganization of endothelial cells. Stimulation of endothelial layers with PMT enhanced transendothelial permeability > 10-fold, and this was abolished by pretreatment with the specific Rho inactivator C3 transferase from Clostridium botulinum. The PMT-induced increase in endothelial permeability was associated with 1) inactivation of MLC phosphatase, 2) an increase in MLC phosphorylation, and 3) endothelial cell retraction and actin stress fiber formation. PMT-stimulated actin reorganization could be prevented by 1) pretreatment of cells with C3 transferase, 2) microinjection of the Rho binding domain and the pleckstrin homology domain of Rho kinase, and 3) microinjection of constitutively active MLC phosphatase. Together, these results suggest that PMT activates Rho/Rho kinase, which inactivates MLC phosphatase. The resulting increase in MLC phosphorylation causes endothelial cell retraction and a rise in endothelial permeability.
Insights
Pasteurella multocida toxin (PMT) increases endothelial permeability by activating Rho GTPase, leading to cell retraction. This involves Rho kinase inactivating MLC phosphatase, causing increased MLC phosphorylation and actin reorganization.
Area of Science:
- Cell biology
- Microbiology
- Biochemistry
Background:
- Pasteurella multocida toxin (PMT) disrupts host cell functions.
- PMT is known to activate the GTPase Rho, influencing the actin cytoskeleton.
Purpose of the Study:
- To investigate the role of Rho kinase and myosin light chain (MLC) phosphatase in PMT-induced endothelial permeability and actin reorganization.
- To elucidate the molecular mechanisms by which PMT affects endothelial cell structure and barrier function.
Main Methods:
- Endothelial cell cultures were stimulated with PMT.
- Transendothelial permeability was measured.
- Involvement of Rho, Rho kinase, and MLC phosphatase was assessed using C3 transferase, specific inhibitors, and microinjection techniques.
- Actin reorganization and MLC phosphorylation were analyzed.
Main Results:
- PMT treatment increased transendothelial permeability over 10-fold.
- This increase was blocked by C3 transferase, indicating Rho dependence.
- PMT induced MLC phosphatase inactivation, increased MLC phosphorylation, and promoted actin stress fiber formation and cell retraction.
- Preventing PMT-induced actin reorganization was achieved by inhibiting Rho, Rho kinase, or activating MLC phosphatase.
Conclusions:
- PMT activates the Rho/Rho kinase pathway, leading to MLC phosphatase inactivation.
- Increased MLC phosphorylation results in endothelial cell retraction and elevated endothelial permeability.
- This study clarifies a key mechanism of PMT-induced vascular leakage.
