ExoY, an adenylate cyclase secreted by the Pseudomonas aeruginosa type III system
ExoY, an adenylate cyclase secreted by the Pseudomonas aeruginosa type III system
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DOI：
10.1073/pnas.95.23.13899
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发表时间：
1998-11-10
影响因子：
11.1
通讯作者：
Frank, DW
中科院分区：
文献类型：
--
作者：
Yahr, TL;Vallis, AJ;Frank, DW
外切酶S调节子是铜绿假单胞菌一组协同调控的毒力基因，其编码的蛋白质包括III型分泌和转运装置、基因表达调节因子和效应蛋白。效应蛋白包括两种具有ADP核糖基转移酶活性的酶（ExoS和ExoT）和一种急性细胞毒素（ExoU）。在本研究中，我们鉴定了ExoY作为调节子的第四效应蛋白，ExoY与百日咳杆菌（Bordetella pertussis，CyaA）和炭疽杆菌（Bacillus anthracis，EF）的胞外腺苷酸环化酶同源，三种腺苷酸环化酶之间的同源性限于两个短区域，其中一个具有ATP结合基序。重组ExoY（rExoY）催化cAMP的形成，其比活性与CyaA的基础活性相似。与CyaA和EF相反，rExoY活性不被刺激或激活qv钙调蛋白。一个500倍的活性刺激后，检测到添加从中国仓鼠卵巢（CHO）细胞的胞质提取物。这些结果表明，不同于钙调蛋白的真核因子增强rExoY催化。ExoY的推定活性位点内的残基的定点诱变消除了腺苷酸环化酶活性。用铜绿假单胞菌的产ExoY菌株感染CHO细胞导致cAMP的细胞内积累。CHO细胞内的cAMP积累依赖于完整的III型易位装置，表明ExoY直接易位到真核细胞质中。
The exoenzyme S regulon is a set of coordinately regulated virulence genes of Pseudomonas aeruginosa, Proteins encoded by the regulon include a type III secretion and translocation apparatus, regulators of gene expression, and effector proteins, The effector proteins include two enzymes with ADP-ribosyltransferase activity (ExoS and ExoT) and an acute cytotoxin (ExoU). In this study, we identified ExoY as a fourth effector protein of the regulon, ExoY is homologous to the extracellular adenylate cyclases of Bordetella pertussis (CyaA) and Bacillus anthracis (EF), The homology among the three adenylate cyclases is limited to two short regions, one of which possesses an ATP-binding motif, In assays for adenylate cyclase activity, recombinant ExoY (rExoY) catalyzed the formation of cAMP with a specific activity similar to the basal activity of CyaA. In contrast to CyaA and EF, rExoY activity was not stimulated or activated qv calmodulin. A 500-fold stimulation of activity was detected following the addition of a cytosolic extract from Chinese hamster ovary (CHO) cells. These results indicate that a eukaryotic factor, distinct from calmodulin, enhances rExoY catalysis. Site-directed mutagenesis of residues within the putative active site of ExoY abolished adenylate cyclase activity. Infection of CHO cells with ExoY-producing strains of P, aeruginosa resulted in the intracellular accumulation of cAMP. cAMP accumulation within CHO cells depended on an intact type III translocation apparatus, demonstrating that ExoY is directly translocated into the eukaryotic cytosol.