Pseudomonas aeruginosa Exotoxin Y Is a Promiscuous Cyclase That Increases Endothelial Tau Phosphorylation and Permeability
Pseudomonas aeruginosa Exotoxin Y Is a Promiscuous Cyclase That Increases Endothelial Tau Phosphorylation and Permeability
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DOI：
10.1074/jbc.m111.301440
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发表时间：
2012-07-20
影响因子：
4.8
通讯作者：
Stevens, Troy
中科院分区：
文献类型：
--
作者：
Ochoa, Cristhiaan D.;Alexeyev, Mikhail;Stevens, Troy
外毒素Y （ExoY）是一种III型分泌系统效应物，在90%的铜绿假单胞菌分离株中发现。虽然已知ExoY可引起内皮间隙和血管渗漏，但其发生的机制尚不清楚。利用细菌传递和密码子优化条件表达的ExoY，我们报道了这种毒素是一种双可溶性腺苷酸和鸟苷酸环化酶，导致细胞内cAMP和cGMP积累。ExoY的酶活性导致内皮Tau丝氨酸214的磷酸化，不溶性Tau的积累，内皮细胞间间隙的形成，以及大分子通透性的增加。为了确定是cAMP还是cGMP信号导致了Tau磷酸化和屏障破坏，研究人员对肺微血管内皮细胞进行了工程改造，使其有条件地表达野生型合成cGMP的鸟苷基环化酶或突变的合成cAMP的鸟苷基环化酶。硝普钠刺激cgmp生成环化酶导致瞬时Tau丝氨酸214磷酸化和间隙形成，而刺激camp生成环化酶则诱导Tau丝氨酸214磷酸化、间隙形成和大分子通透性的显著增加。这些结果表明cAMP信号是Tau磷酸化的主要刺激物。因此，ExoY是一种混杂环化酶和水肿因子，它利用cAMP，并在一定程度上利用cGMP诱导内皮Tau的过度磷酸化和不溶性。由于过度磷酸化和不溶性Tau是阿尔茨海默病等神经退行性Tau病的标志，急性假单胞菌感染引起内皮的病理生理后遗症，以前只在慢性神经退行性疾病中发现。
Exotoxin Y (ExoY) is a type III secretion system effector found in similar to 90% of the Pseudomonas aeruginosa isolates. Although it is known that ExoY causes inter-endothelial gaps and vascular leak, the mechanisms by which this occurs are poorly understood. Using both a bacteria-delivered and a codon-optimized conditionally expressed ExoY, we report that this toxin is a dual soluble adenylyl and guanylyl cyclase that results in intracellular cAMP and cGMP accumulation. The enzymatic activity of ExoY caused phosphorylation of endothelial Tau serine 214, accumulation of insoluble Tau, inter-endothelial cell gap formation, and increased macromolecular permeability. To discern whether the cAMP or cGMP signal was responsible for Tau phosphorylation and barrier disruption, pulmonary microvascular endothelial cells were engineered for the conditional expression of either wild-type guanylyl cyclase, which synthesizes cGMP, or a mutated guanylyl cyclase, which synthesizes cAMP. Sodium nitroprusside stimulation of the cGMP-generating cyclase resulted in transient Tau serine 214 phosphorylation and gap formation, whereas stimulation of the cAMP-generating cyclase induced a robust increase in Tau serine 214 phosphorylation, gap formation, and macromolecular permeability. These results indicate that the cAMP signal is the dominant stimulus for Tau phosphorylation. Hence, ExoY is a promiscuous cyclase and edema factor that uses cAMP and, to some extent, cGMP to induce the hyperphosphorylation and insolubility of endothelial Tau. Because hyperphosphorylated and insoluble Tau are hallmarks in neurodegenerative tauopathies such as Alzheimer disease, acute Pseudomonas infections cause a pathophysiological sequela in endothelium previously recognized only in chronic neurodegenerative diseases.