Self-trimerization of ExsD limits inhibition of the Pseudomonas aeruginosa transcriptional activator ExsA in vitro.
Self-trimerization of ExsD limits inhibition of the Pseudomonas aeruginosa transcriptional activator ExsA in vitro.
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DOI：
10.1111/febs.12103
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发表时间：
2013-02
期刊：
影响因子：
--
通讯作者：
Schubot FD
中科院分区：
文献类型：
--
作者：
Bernhards RC;Marsden AE;Esher SK;Yahr TL;Schubot FD
机会性病原体铜绿假单胞菌是医院感染的主要原因之一。 III 型分泌系统 (T3SS) 通过向宿主细胞注入强效细胞毒素来抑制宿主的先天免疫反应，从而帮助急性铜绿假单胞菌感染。所有 T3SS 相关基因的表达严格依赖于转录因子 ExsA。因此，ExsA 和调节 ExsA 功能的生物过程具有重大的生物医学意义。目前的工作重点是 ExsA-ExsC-ExsD-ExsE 信号级联，该级联将宿主细胞接触与 T3SS 基因表达的上调联系起来。在 T3SS 诱导之前，抗激活蛋白 ExsD 与 ExsA 结合，并通过干扰 ExsA 二聚化和启动子相互作用来阻断 ExsA 依赖性转录。宿主细胞接触后，ExsD 被 T3SS 伴侣 ExsC 隔离，导致 ExsA 释放和 T3SS 上调。先前的研究表明 ExsD-ExsA 相互作用不是自由可逆的。由于未发现独立折叠的 ExsD 和 ExsA 相互作用，因此推测这两种蛋白质的折叠中间体形成复合物。在这里，我们首次证明仅 ExsD 就足以在体外抑制 ExsA 依赖性转录，并且不需要其他细胞因子。更重要的是，我们表明独立折叠的 ExsD 和 ExsA 能够相互作用，但仅在 37°C 而不是在 30°C。在 ExsD 晶体结构的指导下，我们设计了该蛋白质的单体变体，并证明 ExsD 三聚化可防止 ExsD 在 30°C 下抑制 ExsA 依赖性转录。我们认为这种独特的机制在 T3SS 调控中发挥着重要作用。
The opportunistic pathogen Pseudomonas aeruginosa ranks among leading causes of nosocomial infections. The type III secretion system (T3SS) aids acute P. aeruginosa infections by injecting potent cytotoxins into host cells to suppress the host's innate immune response. Expression of all T3SS-related genes is strictly dependent upon the transcription factor ExsA. Consequently, ExsA and the biological processes that regulate ExsA function are of great biomedical interest. The presented work focuses on the ExsA-ExsC-ExsD-ExsE signaling cascade that ties host cell contact to the up-regulation of T3SS gene expression. Prior to T3SS induction, the anti-activator protein ExsD binds to ExsA and blocks ExsA-dependent transcription by interfering with ExsA dimerization and promoter interactions. Upon host cell contact, ExsD is sequestered by the T3SS chaperone ExsC resulting in the release of ExsA and an up-regulation of the T3SS. Previous studies have shown that the ExsD-ExsA interactions are not freely reversible. Because independently folded ExsD and ExsA were not found to interact, it has been hypothesized that folding intermediates of the two proteins form the complex. Here we demonstrate for the first time that ExsD alone is sufficient to inhibit ExsA-dependent transcription in vitro and that no other cellular factors are required. More significantly, we show that independently folded ExsD and ExsA are capable of interacting, but only at 37°C and not at 30°C. Guided by the crystal structure of ExsD, we designed a monomeric variant of the protein and demonstrate that ExsD trimerization prevents ExsD from inhibiting ExsA-dependent transcription at 30°C. We propose that this unique mechanism plays an important role in T3SS regulation.
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