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

我的研究项目旨在了解细菌产生和传递的细菌泛素连接酶(BUL)蛋白如何改变真核细胞的生物学。牛可以将泛素(一种由76个氨基酸组成的真核小蛋白)共价连接到底物蛋白上。连接的泛素部分的数目和结构可以改变底物的功能，或导致其降解。底物降解，以及缺乏分子工具和探针，对了解BUL在宿主中的功能构成了挑战。我们正在使用从三个不同的、互补的真核细胞平台获得的新见解来建立真核细胞中BUL功能的综合模型。平台1.沙门氏菌在哺乳动物细胞中BUL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，我们正在检查模式识别受体(PRR)NOD2和NLRP3是否是肠杆菌BUL SspH2的底物。我们将分离SspH2和相互作用的宿主蛋白的复合体，后者将通过质谱学或抗体检测进行鉴定。我们还将使用质谱仪来鉴定被SspH2泛素化的特定PRR残基。我们随后将对这些残基进行突变，以确定泛素转移是否在SspH2对先天性免疫的颠覆中发挥作用。平台2.利用酵母开发工具研究BUL功能。长期以来，酵母一直被用作探索细菌效应器细胞活性的模型，因为它在遗传上容易处理，而且将发现转化为哺乳动物系统的潜力很高。我们已经证明，肠链球菌BUL，SspH1，在酵母中具有强烈的毒性。我们正在使用这个平台提供的强大的筛查工具来开发抑制SspH1功能的试剂。我们已经确定了两种抑制SspH1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展中的潜在缺陷。该平台也适用于小分子筛选。平台3.研究沙门氏菌与BUL-植物免疫的相互作用。植物是肠杆菌的一个新兴宿主，越来越多的报告显示受肠杆菌污染的农产品。植物具有可被肠链球菌3型分泌系统效应器颠覆的免疫反应。我已经开发了将肠链球菌导入烟草的系统，并发现SspH2增强了植物中的免疫类反应。在这个平台上，我们将使用质谱仪来鉴定SspH2的植物蛋白底物。我们还将通过基因表达和功能分析来检查免疫反应，以确定SspH2如何颠覆植物免疫。这些平台结合在一起是一个有用的模型系统和资源阵列，通过它们可以充分了解Buls在宿主细胞中的功能。这一知识将为可用于靶向干扰的宿主-病原体相互作用提供新的线索。

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 BUL-plant 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.