Cross-kingdom platforms to study bacterial ubiquitin ligases in eukaryotic cells

我的研究计划旨在了解细菌泛素连接酶（BUL）蛋白质，由细菌产生和传递，改变真核细胞的生物学。BULs可以共价连接泛素，一个小的76个氨基酸的真核蛋白，底物蛋白。连接的泛素部分的数量和结构可以改变底物功能，或导致其降解。底物降解，以及缺乏分子工具和探针，对理解宿主中的BUL功能提出了挑战。我们正在使用从三个不同的，互补的真核生物平台获得的新见解，使BUL在真核生物中的功能的全面模型。 一号站台。肠道沙门氏菌BUL在哺乳动物细胞中功能的机制研究。我们正在使用可靠的基于质粒的表达系统或细菌递送将细菌泛素连接酶引入哺乳动物细胞。使用这种方法，我们正在研究模式识别受体（PRR），NOD 2和NLRP 3，是否是S。肠BUL、SspH 2.我们将分离SspH 2和相互作用的宿主蛋白质的复合物，后者将通过质谱或抗体检测来鉴定。我们还将使用质谱法来鉴定被SspH 2泛素化的特定PRR残基。我们随后将突变这些残基，以确定泛素转移是否在SspH 2的先天免疫颠覆中发挥作用。 2号站台。利用酵母开发工具研究BUL功能。酵母长期以来一直作为探索细菌效应子细胞活性的模型，因为它在遗传上易于处理，并且将发现转化到哺乳动物系统的潜力很高。我们证明了S.肠BUL，SspH 1，在酵母中发挥深刻的毒性。我们正在使用该平台中可用的强大筛选工具来开发抑制SspH 1功能的试剂。我们已经确定了两种抑制SspH 1毒性的底物变体，并将使用它们来探测细胞形态和细胞周期进程中的潜在缺陷。该平台也适用于小分子筛选。 三号站台。检查S。肠道BULplant免疫相互作用。植物是S.肠道沙门氏菌（S.被肠道污染的农产品植物的免疫反应可以被S.肠道3型分泌系统效应物。我已经开发了系统来介绍S。将肠杆菌BULs导入烟草中，发现SspH 2增强了植物中的免疫样应答。在这个平台中，我们将使用质谱来鉴定SspH 2的植物蛋白底物。我们还将通过基因表达和功能测定来研究免疫反应，以确定SspH 2如何破坏植物免疫。 这些平台一起是一个有用的模型系统和资源阵列，可以充分了解BULs在宿主细胞中的功能。这一知识将为宿主-病原体相互作用提供新的线索，这些相互作用可以用于靶向破坏。

My research program aims to understand how bacterial ubiquitin ligase (BUL) proteins, produced and delivered by bacteria, alter the biology of eukaryotic cells. BULs can covalently link ubiquitin, a small 76-amino acid eukaryotic protein, to a substrate protein. The number and structure of linked ubiquitin moieties can modify substrate function, or cause its degradation. Substrate degradation, and a lack of molecular tools and probes, pose a challenge to understanding BUL functions in the host. We are using new insights gained from three diverse, and complementary eukaryotic platforms to make comprehensive models of BUL functions in eukaryotes. Platform 1. Mechanistic study of Salmonella enterica BUL function in mammalian cells. We are introducing bacterial ubiquitin ligases into mammalian cells using reliable plasmid-based expression systems or bacterial delivery. Using this approach we are examining whether the pattern recognition receptors (PRR), NOD2 and NLRP3, are substrates of the S. enterica BUL, SspH2. We will isolate complexes of SspH2 and interacting host proteins, the latter of which will be identified by mass spectrometry or antibody detection. We will also use mass spectrometry to identify specific PRR residues that are ubiquitinated by SspH2. We will subsequently mutate these residues to determine if ubiquitin transfer plays a role in innate immune subversion by SspH2. Platform 2. Using yeast to develop tools to study BUL function. Yeast have long-served as a model to explore the cellular activity of bacterial effectors because it is genetically tractable and the potential to translate discoveries to mammalian systems is high. We have shown that the S. enterica BUL, SspH1, exerts profound toxicity in yeast. We are using robust screening tools available in this platform to develop reagents that inhibit SspH1 function. We have identified two substrate variants that suppress SspH1 toxicity and will use them to probe potential defects in cell morphology and cell cycle progression. This platform is also amenable to small molecule screening. Platform 3. Examining S. enterica BULplant immunity interactions. Plants are an emerging host for S. enterica with increasing reports of S. enterica-contaminated produce. Plants possess immune responses that can be subverted by S. enterica Type 3 secretion system effectors. I have developed systems to introduce S. enterica BULs into tobacco and found that SspH2 enhanced an immune-like response in planta. In this platform we will use mass spectrometry to identify plant protein substrates of SspH2. We will also examine immune responses via gene expression and functional assays to determine how SspH2 subverts plant immunity. Together these platforms are a useful array of model systems and resources, with which to gain a fulsome understanding of how BULs function in host cells. This knowledge will shed new light on host-pathogen interactions that could be exploited for targeted disruption.