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

描述（由申请人提供）：真核先天免疫系统是病原体需要绕过以引起疾病的重要屏障。该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已经显示能够抑制真核生物中的先天免疫。细菌性植物病原体假单胞菌依赖于TTSS在植物上引起疾病。附：PV.番茄DC3000效应基因hopU1类似于ADP核糖基转移酶（ADP-RTs）基因。这些基因编码动物细菌病原体中一些最好理解的毒素（例如，例如，在一个实施例中，霍乱毒素）。初步数据表明，HopU1是一个活跃的ADP-RT，它ADP核糖水解几种植物蛋白。质谱测定叶绿体和富含甘氨酸的RNA结合蛋白作为HopU1的体外底物。这些是ADP-RT的新型底物。此外，HopU1具有以依赖于其ADP-RT活性位点的方式抑制植物先天免疫系统的几种反应的能力。缺乏一个HopU1底物AtGRP7的拟南芥突变体显示出对P. dogingae的敏感性增强，表明它是先天免疫的一个组成部分。AtGRP7是一种富含甘氨酸的RNA结合蛋白，这表明该病原体靶向参与RNA代谢的蛋白质以抑制先天免疫。所提出的实验的中心假设是AtGRP7和HopU1 ADP-RT III型效应器的可能其他靶点是先天免疫的组分。几个实验试图阐明AtGRP7在先天免疫中的作用，使用生物化学和分子生物学方法。P.lingae-Arabidopsis pathosystem是研究天然免疫系统的理想模型，因为它具有资源丰富、天然免疫系统与真核生物相似以及研究成本低等优点。这些实验将有助于从根本上了解细菌致病和先天免疫的分子机制。 具体目的如下：（1）确定ADP-核糖基化对AtGRP7功能的分子后果，并阐明该蛋白在先天免疫中的作用;（2）鉴定HopU 1的其他底物，并验证它们参与先天免疫;（3）分析HopU 1对宿主-微生物相互作用的影响。 项目叙述：鉴定P.lingae 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。其中一个HopU1靶点属于一大类称为富含甘氨酸的RNA结合蛋白的蛋白质，这些蛋白质还没有得到很好的理解，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之间有相当大的相似性，我们希望我们的发现将与NIH的使命相关，并对研究细菌发病机制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.