The GAP portion of Pseudomonas aeruginosa type III secreted toxin ExoS upregulates total and surface levels of wild type CFTR.
The GAP portion of Pseudomonas aeruginosa type III secreted toxin ExoS upregulates total and surface levels of wild type CFTR.
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III 型铜绿假单胞菌分泌的毒素 ExoS 的 GAP 部分上调野生型 CFTR 的总水平和表面水平。
DOI：
10.1159/000343357
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Guggino,WiliamB
中科院分区：
文献类型：
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作者：
Tukaye,DeepaliN;Kwon,Sang-Ho;Guggino,WiliamB
背景铜绿假单胞菌（PA）感染在致命的医院获得性肺炎中占很大比例。 PA III 型分泌毒素 (TTST) ExoS 是一种双功能蛋白，具有 N 端 GTP 酶激活蛋白 (GAP) 和 C 端 ADP 核糖基转移酶 (ADPRT) 活性，通过靶向小分子量 G 蛋白 (SMWGP)，显着增强 PA 毒力。在本研究中，我们研究了 ExoS 的 GAP 部分 (ExoS-GAP) 介导细胞毒性的机制之一。方法使用瞬时转染系统在稳定表达 CFTR 的 CFBE41o-Kir 2.2 和 MDCK 细胞系中研究 ExoS-GAP 对 CFTR 运输的影响。结果瞬时转染 ExoS-GAP 增加了稳定表达 CFTR 的上皮细胞中成熟野生型 CFTR 的总蛋白和表面蛋白水平。野生型（WT）CFTR。 ExoS-GAP 的作用对支气管上皮细胞中的 CFTR 具有特异性，因为它不影响另一种膜蛋白 Na+/K+ ATP 酶的总蛋白水平。 ExoS GAP 结构域 (R146K) 中的点突变已知会破坏其催化 GAP 活性，从而消除了 ExoS-GAP 对 WT CFTR 的影响。 Bafilomycin A1 的溶酶体抑制研究表明，ExoS-GAP 减少了成熟 WT CFTR 的溶酶体降解，同时成熟 WT CFTR 的总水平增加。然而，ExoS-GAP 并没有增加Δ F508CFTR 的总蛋白水平。结论 PA TTST ExoS 的 GAP 部分增加了体外哺乳动物细胞系统中野生型 CFTR 的总蛋白水平和表面水平。 ExoS-GAP 对 WT CFTR 总蛋白水平的影响为理解 PA 感染的毒性病理生理学提供了新的见解。
BackgroundPseudomonas aeruginosa (PA) infections account for a large percentage of fatal hospital acquired pneumonias. One of the PA Type III secreted toxin (TTST) ExoS, a bifunctional protein with N-terminal GTPase activating protein (GAP) and C-terminal ADP rybosyl transferase (ADPRT) activities, significantly contributes to PA virulence by targeting small molecular weight G-proteins (SMWGP). In this study, we have looked at one of the mechanisms by which the GAP portion of ExoS (ExoS-GAP) mediates cellular toxicity.MethodsThe effects of ExoS-GAP on CFTR trafficking were studied in CFBE41o-Kir 2.2 and MDCK cell lines stably expressing CFTR using a transient transfection system.ResultsTransient transfection of ExoS-GAP increased the total and surface protein levels of mature wild type CFTR in epithelial cells stably expressing wild type (WT) CFTR. The effect of ExoS-GAP was specific to CFTR in bronchial epithelial cells since it did not affect the total protein levels of Na+/K+ ATPase, another membrane protein. A point mutation in the ExoS GAP domain (R146K), known to disrupt its catalytic GAP activity, abolished the effect of ExoS-GAP on WT CFTR. Lysosomal inhibition studies with Bafilomycin A1 indicate that ExoS-GAP decreased lysosomal degradation of the mature WT CFTR with concomitant increase in the total levels of mature WT CFTR. However, ExoS-GAP did not increase the total protein levels of∆ F508CFTR.ConclusionThe GAP portion of the PA TTST ExoS increases the total and surface levels of wild type CFTR in vitro mammalian cell system. The effect of ExoS-GAP on WT CFTR total protein levels provides new insight into understanding the virulent pathophysiology of PA infections.