Department of Cell Biology and Neuroscience, University of South Alabama, Mobile, Alabama, United States of America ; Center for Lung Biology, University of South Alabama, Mobile, Alabama, United States of America.
PLoS One. 2013 Sep 4;8(9):e74343. doi: 10.1371/journal.pone.0074343. eCollection 2013.
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.
铜绿假单胞菌使用 III 型分泌系统将腺苷酸环化酶和鸟苷酸环化酶外毒素 Y（ExoY）导入内皮细胞质中。ExoY 诱导 Tau 过度磷酸化和不溶性、微管解体、屏障破坏和水肿，尽管微管解体的确切机制仍知之甚少。在这里，我们研究了微管行为和中心体活性，以测试 ExoY 破坏微管动力学的假设。荧光显微镜确定感染的肺微血管内皮细胞中的微管比对照细胞少，进一步的研究表明，感染后微管相关蛋白 Tau 过度磷酸化并从微管上解离。组装/拆卸研究确定感染细胞中的微管组装被破坏，而中心体对微管的拆卸或微管的成核没有检测到影响。当 Tau 上的 cAMP 依赖性激酶磷酸化位点（Ser-214）突变为不可磷酸化形式时，ExoY 对微管的这种作用被消除。这些研究确定了微血管内皮细胞中的 Tau 是铜绿假单胞菌肺部感染后控制微管结构的 ExoY 的靶标，并表明感染后 Tau 的磷酸化会降低微管组装。
