Suppression of innate immunity by an ADP-ribosyltransferase type III effector

描述(申请人提供)：真核生物的先天免疫系统是病原体为了致病需要绕过的一个重要屏障。这个系统的几个组成部分在真核生物中是保守的。最近，通过III型蛋白分泌系统(TTSS)注射到宿主细胞中的细菌病原体效应物被证明能够抑制真核生物的先天免疫。植物病原菌丁香假单胞菌依赖TTSS在植物上致病。S P.P.PV.番茄DC3000效应基因HopU1与ADP核糖基转移酶(ADP-RTS)基因相似。这些基因编码动物的细菌病原体中一些最广为人知的毒素(例如霍乱毒素)。这个提议中的初步数据表明，HopU1是一个活性的ADP-RT，它能使几种植物蛋白发生ADP-核糖基化。质谱仪确定叶绿体和富含甘氨酸的RNA结合蛋白作为HopU1的体外底物。这些都是ADP-RTS的新型底物。此外，HopU1具有抑制植物天然免疫系统的多种反应的能力，其方式依赖于其ADP-RT活性位点。一个缺失HopU1底物的拟南芥突变体AtGRP7对丁香疫霉菌表现出更强的敏感性，这表明它是天然免疫的一个组成部分。AtGRP7是一种富含甘氨酸的RNA结合蛋白，表明该病原体针对参与RNA代谢的蛋白来抑制天然免疫。拟议实验的中心假设是，AtGRP7和可能是HopU1 ADP-RT III型效应器的其他靶点是先天免疫的组成部分。一些实验试图用生化和分子生物学的方法来阐明AtGRP7在先天免疫中所起的作用。由于可利用的资源、真核生物之间的天然免疫系统的相似性以及成本效益的研究，丁香疫霉-拟南芥病理系统是研究天然免疫系统的一个很好的模型。这些实验将有助于从根本上理解细菌致病和先天免疫的分子机制。 具体目标如下：(1)确定ADP-核糖化对AtGRP7功能的分子后果，阐明该蛋白在天然免疫中的作用；(2)鉴定HopU1的其他底物，并验证它们参与天然免疫；(3)分析HopU1对宿主-微生物相互作用的影响。 项目简介：确定丁香假单胞菌HopU1 ADP-核糖基转移酶的真核靶点将有助于我们理解细菌的发病机制，并可能揭示先天免疫系统的重要组成部分。HopU1的一个靶点属于一大类被称为富含甘氨酸的RNA结合蛋白的蛋白质，这些蛋白质目前还不太清楚，这项研究可能会增加我们对这些蛋白质的理解。由于植物和哺乳动物的先天免疫系统有相当大的相似性，我们预计我们的发现将与美国国立卫生研究院的任务相关，并对研究细菌致病和先天免疫的分子机制的研究人员产生广泛的兴趣。

DESCRIPTION (provided by applicant): The eukaryotic innate immune system represents an important barrier that pathogens need to circumvent in order to cause disease. Several components of this system are conserved in eukaryotes. Recently, bacterial pathogen effectors that are injected into host cells by type III protein secretion systems (TTSSs) have been shown to be capable of suppressing innate immunity in eukaryotes. The bacterial plant pathogen Pseudomonas syringae is dependent on a TTSS to cause disease on plants. The P. s. pv. tomato DC3000 effector gene hopU1 resembles ADP ribosyltransferases (ADP-RTs) genes. These genes encode some of the best understood toxins in bacterial pathogens of animals (e. g., cholera toxin). Preliminary data within this proposal show that HopU1 is an active ADP-RT and that it ADP-riboslylates several plant proteins. Mass spectrometry determined that chloroplast and glycine-rich RNA-binding proteins acted as in vitro substrates for HopU1. These are novel substrates for ADP-RTs. Moreover, HopU1 has the ability to suppress several responses of the plant innate immune system in a manner that is dependent on its ADP-RT active site. An Arabidopsis mutant lacking one HopU1 substrate, AtGRP7, displayed enhanced susceptibility to P. syringae suggesting that it is a component of innate immunity. AtGRP7 is a glycine-rich RNA-binding protein, which suggests this pathogen targets proteins involved in RNA metabolism to suppress innate immunity. The central hypothesis of the proposed experiments is that AtGRP7 and perhaps other targets of the HopU1 ADP-RT type III effector are components of innate immunity. Several of the experiments seek to elucidate the role AtGRP7 plays in innate immunity using biochemical and molecular biological approaches. The P. syringae-Arabidopsis pathosystem is an excellent model to study the innate immune system because of the resources available, the similarities between innate immune systems between eukaryotes, and the cost efficient research. These experiments will contribute to a fundamental understanding of the molecular mechanism of bacterial pathogenesis and innate immunity. The Specific Aims are the following: (1) Determine the molecular consequence of ADP- ribosylation on the function of AtGRP7 and elucidate the role this protein plays in innate immunity; (2) Identify additional substrates of HopU1 and verify their involvement in innate immunity; (3) Analyze the affect that HopU1 has on host-microbe interactions. Project Narrative: Identifying the eukaryotic targets for the P. syringae HopU1 ADP-ribosyltransferase will contribute to our understanding of bacterial pathogenesis and will likely reveal important components of the innate immune system. One HopU1 target belongs to a large group of proteins called glycine-rich RNA binding proteins, which are not well understood, and this research will likely increase our understanding of these proteins. Because there are considerable similarities between the innate immune systems in plants and mammals we expect that our findings will be relevant to the mission of the NIH and be broadly interesting to researchers studying molecular mechanisms of bacterial pathogenesis and innate immunity.