The extreme C terminus of the Pseudomonas aeruginosa effector ExoY is crucial for binding to its eukaryotic activator, F-actin
The extreme C terminus of the Pseudomonas aeruginosa effector ExoY is crucial for binding to its eukaryotic activator, F-actin
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DOI：
10.1074/jbc.ra118.003784
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发表时间：
2018-12-21
影响因子：
4.8
通讯作者：
Mechold, Undine
中科院分区：
文献类型：
--
作者：
Belyy, Alexander;Santecchia, Ignacio;Mechold, Undine
细菌核苷酸环化酶毒素是一种强毒力因子，在进入真核细胞后受到内源性辅因子的刺激，催化产生大量的35-环核苷单磷酸。来自铜绿假单胞菌的效应子ExoY的活性由丝状形式的肌动蛋白（F-肌动蛋白）刺激。利用酵母表型分析，定点诱变，功能性生化测定和共聚焦显微镜，我们证明了ExoY的C末端的最后9个氨基酸与F-肌动蛋白的相互作用是至关重要的，因此，ExoY的体外酶活性和酵母模型中的毒性。我们观察到已融合至载体蛋白的铜绿假单胞菌ExoY的分离的C末端序列与F-肌动蛋白结合，并且对应于极端ExoY C末端的合成肽在体外抑制ExoY酶活性并与全长酶竞争F-肌动蛋白结合。有趣的是，我们注意到PA 14家族的各种铜绿假单胞菌分离株，包括高毒力菌株，含有ExoY变体，其突变改变了该效应子的C末端。我们发现这些天然存在的ExoY变体显示出显著降低的酶活性和毒性。我们的研究结果揭示了ExoY-F-肌动蛋白相互作用的分子基础，揭示了ExoY的极端C末端对于结合靶细胞中的F-肌动蛋白至关重要，并且一些铜绿假单胞菌分离株携带C末端突变的低活性ExoY变体。
Bacterial nucleotidyl cyclase toxins are potent virulence factors that upon entry into eukaryotic cells are stimulated by endogenous cofactors to catalyze the production of large amounts of 35-cyclic nucleoside monophosphates. The activity of the effector ExoY from Pseudomonas aeruginosa is stimulated by the filamentous form of actin (F-actin). Utilizing yeast phenotype analysis, site-directed mutagenesis, functional biochemical assays, and confocal microscopy, we demonstrate that the last nine amino acids of the C terminus of ExoY are crucial for the interaction with F-actin and, consequently, for ExoY's enzymatic activity in vitro and toxicity in a yeast model. We observed that isolated C-terminal sequences of P. aeruginosa ExoY that had been fused to a carrier protein bind to F-actin and that synthetic peptides corresponding to the extreme ExoY C terminus inhibit ExoY enzymatic activity in vitro and compete with the full-length enzyme for F-actin binding. Interestingly, we noted that various P. aeruginosa isolates of the PA14 family, including highly virulent strains, harbor ExoY variants with a mutation altering the C terminus of this effector. We found that these naturally occurring ExoY variants display drastically reduced enzymatic activity and toxicity. Our findings shed light on the molecular basis of the ExoY-F-actin interaction, revealing that the extreme C terminus of ExoY is critical for binding to F-actin in target cells and that some P. aeruginosa isolates carry C-terminally mutated, low-activity ExoY variants.