Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA.
Proc Natl Acad Sci U S A. 2011 Dec 27;108(52):21212-7. doi: 10.1073/pnas.1114023109. Epub 2011 Dec 7.
Effectors delivered into host cells by the Legionella pneumophila Dot/Icm type IV transporter are essential for the biogenesis of the specialized vacuole that permits its intracellular growth. The biochemical function of most of these effectors is unknown, making it difficult to assign their roles in the establishment of successful infection. We found that several yeast genes involved in membrane trafficking, including the small GTPase Ypt1, strongly suppress the cytotoxicity of Lpg0695(AnkX), a protein known to interfere severely with host vesicle trafficking when overexpressed. Mass spectrometry analysis of Rab1 purified from a yeast strain inducibly expressing AnkX revealed that this small GTPase is modified posttranslationally at Ser(76) by a phosphorylcholine moiety. Using cytidine diphosphate-choline as the donor for phosphorylcholine, AnkX catalyzes the transfer of phosphorylcholine to Rab1 in a filamentation-induced by cAMP(Fic) domain-dependent manner. Further, we found that the activity of AnkX is regulated by the Dot/Icm substrate Lpg0696(Lem3), which functions as a dephosphorylcholinase to reverse AnkX-mediated modification on Rab1. Phosphorylcholination interfered with Rab1 activity by making it less accessible to the bacterial GTPase activation protein LepB; this interference can be alleviated fully by Lem3. Our results reveal reversible phosphorylcholination as a mechanism for balanced modulation of host cellular processes by a bacterial pathogen.
军团菌属肺炎嗜肺军团菌的 Dot/Icm 型 IV 型转运器将效应器递送到宿主细胞中，对于允许其在细胞内生长的特殊空泡的生物发生至关重要。这些效应器的大多数生化功能尚不清楚，这使得难以确定它们在建立成功感染中的作用。我们发现，几个参与膜运输的酵母基因，包括小 GTPase Ypt1，强烈抑制 Lpg0695(AnkX)的细胞毒性，当过量表达时，该蛋白严重干扰宿主囊泡运输。用可诱导表达 AnkX 的酵母菌株中纯化的 Rab1 进行质谱分析表明，这种小 GTPase在 Ser(76)处被磷酰胆碱部分进行翻译后修饰。使用胞苷二磷酸胆碱作为磷酰胆碱的供体，AnkX 以 cAMP(Fic)结构域依赖性方式催化磷酰胆碱向 Rab1 的转移。此外，我们发现 AnkX 的活性受 Dot/Icm 底物 Lpg0696(Lem3)的调节，Lem3 作为脱磷酰酶，可逆转 AnkX 介导的 Rab1 修饰。磷酰化通过使 Rab1 更不易被细菌 GTP 酶激活蛋白 LepB 识别，从而干扰 Rab1 的活性；这种干扰可以通过 Lem3 完全缓解。我们的结果揭示了可逆磷酰化作为细菌病原体对宿主细胞过程进行平衡调节的一种机制。
