Abstract
Pseudomonas aeruginosa uses a type III secretion system to introduce the adenylyl and guanylyl cyclase exotoxin Y (ExoY) into the cytoplasm of endothelial cells. ExoY induces Tau hyperphosphorylation and insolubility, microtubule breakdown, barrier disruption and edema, although the mechanism(s) responsible for microtubule breakdown remain poorly understood. Here we investigated both microtubule behavior and centrosome activity to test the hypothesis that ExoY disrupts microtubule dynamics. Fluorescence microscopy determined that infected pulmonary microvascular endothelial cells contained fewer microtubules than control cells, and further studies demonstrated that the microtubule-associated protein Tau was hyperphosphorylated following infection and dissociated from microtubules. Disassembly/reassembly studies determined that microtubule assembly was disrupted in infected cells, with no detectable effects on either microtubule disassembly or microtubule nucleation by centrosomes. This effect of ExoY on microtubules was abolished when the cAMP-dependent kinase phosphorylation site (Ser-214) on Tau was mutated to a non-phosphorylatable form. These studies identify Tau in microvascular endothelial cells as the target of ExoY in control of microtubule architecture following pulmonary infection by Pseudomonas aeruginosa and demonstrate that phosphorylation of tau following infection decreases microtubule assembly.
Highlights
Cell shape is controlled by interactions between microfilaments, intermediate filaments and microtubules
To determine whether microtubule cytoskeletal alterations could be contributing to the formation of endothelial gaps, cells infected with P. aeruginosa exotoxin Y (ExoY)+ or P. aeruginosa ExoYK81M were analyzed using anti-tubulin immunofluorescence microscopy
Fewer microtubules were observed in cells infected with P. aeruginosa ExoY+, and microtubules were rarely observed extending completely to the cell cortex
Summary
Cell shape is controlled by interactions between microfilaments, intermediate filaments and microtubules. CAMP is capable of diffusion within the cytosol, multiple mechanisms are utilized for maintaining elevated levels of cAMP near the membrane relative to levels deeper in the cell, including anchoring adenylyl cyclases to the plasma membrane [7,9], steric inhibition of diffusion by intracellular membranes [10,11,12,13], and localization of phosphodiesterases to the cortical area of cells [14,15] Proper maintenance of this regional cAMP pool is critical for maintaining strength of the endothelial barrier. The essential nature of cAMP for barrier integrity can be seen by the action of inflammatory agonists, such as thrombin and bradykinin; these inflammatory agents activate signal transduction events that result in decreased levels of membrane-associated cAMP, resulting in weakened cell adhesion, the formation of gaps between neighboring endothelial cells, and tissue edema [2,5,16]
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