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Sequential inactivation of Rho GTPases and Lim kinase by Pseudomonas aeruginosa toxins ExoS and ExoT leads to endothelial monolayer breakdown

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Abstract

Pseudomonas aeruginosa is a major human opportunistic pathogen and one of the most important causal agents of bacteremia. For non-blood-borne infection, bacterial dissemination requires the crossing of the vascular endothelium, the main barrier between blood and the surrounding tissues. Here, we investigated the effects of P. aeruginosa type 3 secretion effectors, namely ExoS, ExoT, and ExoY, on regulators of actin cytoskeleton dynamics in primary endothelial cells. ExoS and ExoT similarly affected the Lim kinase-cofilin pathway, thereby promoting actin filament severing. Cofilin activation was also observed in a mouse model of P. aeruginosa-induced acute pneumonia. Rho, Rac, and Cdc42 GTPases were sequentially inactivated, leading to inhibition of membrane ruffling, filopodia, and stress fiber collapse, and focal adhesion disruption. At the end of the process, ExoS and ExoT produced a dramatic retraction in all primary endothelial cell types tested and thus a rupture of the endothelial monolayer. ExoY alone had no effect in this context. Cell retraction could be counteracted by overexpression of actin cytoskeleton regulators. In addition, our data suggest that moesin is neither a direct exotoxin target nor an important player in this process. We conclude that any action leading to inhibition of actin filament breakdown will improve the barrier function of the endothelium during P. aeruginosa infection.

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Acknowledgments

This work was supported by grants from the Commissariat à l’Energie Atomique, the Institut National de la Santé et de la Recherche Médicale, the Centre National de la Recherche Scientifique and Joseph Fourier University. Part of this work was also supported by the Alliance pour les sciences de la VIE et de la SANté program in infectiology. We thank Anne-Sophie Ribba for the moesin antibody, Laurence Lafanechère for Pyr1, Cécile Gauthier-Rouvière, Kenzaku Mizuno, Roland Wedlich-Soldner, Hélène Delanoë-Ayari and Gregg Gunderson for expression plasmids, and Arne Rietsch for Pseudomonas aeruginosa mutant strains.

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Authors and Affiliations

  1. INSERM, U1036, Biology of Cancer and Infection, Grenoble, France

    P. Huber, S. Bouillot, S. Elsen & I. Attrée

  2. CNRS, ERL 5261, Bacterial Pathogenesis and Cellular Responses, Grenoble, France

    P. Huber, S. Bouillot, S. Elsen & I. Attrée

  3. Université Joseph Fourier-Grenoble I, Grenoble, France

    P. Huber, S. Bouillot, S. Elsen & I. Attrée

  4. CEA, DSV/iRTSV, Grenoble, France

    P. Huber, S. Bouillot, S. Elsen & I. Attrée

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Correspondence to P. Huber.

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Huber, P., Bouillot, S., Elsen, S. et al. Sequential inactivation of Rho GTPases and Lim kinase by Pseudomonas aeruginosa toxins ExoS and ExoT leads to endothelial monolayer breakdown. Cell. Mol. Life Sci. 71, 1927–1941 (2014). https://doi.org/10.1007/s00018-013-1451-9

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