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

研究真核细胞中细菌泛素连接酶的跨界平台

• 批准号: RGPIN-2020-04359
• 负责人: Bhavsar, Amit
• 金额: $ 2.19万
• 依托单位: University of Alberta
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=760779
• 关键词: Cross; kingdom; platforms; study; bacterial

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.

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特性，将泛素（一种由 76 个氨基酸组成的小真核蛋白）与底物蛋白共价连接。连接泛素部分的缺乏和结构可能会改变底物功能，或导致其降解，并且分子工具和探针的数量对理解 BUL 功能提出了挑战。我们正在利用从三个不同且互补的真核平台中获得的新见解来建立真核生物中 BUL 功能的综合模型。 平台 1. 哺乳动物细胞中沙门氏菌 BUL 功能的机制研究我们正在将细菌泛素连接酶引入哺乳动物细胞中。可靠的基于质粒的表达系统或细菌传递，我们正在检查模式识别受体（PRR）、NOD2和NLRP3是否是该方法的底物。 S.enterica BUL，SspH2。我们将分离 SspH2 和相互作用的宿主蛋白的复合物，后者将通过质谱或抗体检测来鉴定。我们还将使用质谱来鉴定被 SspH2 泛素化的特定 PRR 残基。随后将突变这些残基，以确定泛素转移是否在 SspH2 破坏先天免疫中发挥作用。 平台 2. 使用酵母开发工具来研究 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，并且将发现转化为哺乳动物系统的潜力很高。我们已经证明，肠沙门氏菌 BUL（SspH1）对酵母具有深远的毒性。我们正在使用该平台中可用的强大筛选工具来开发抑制 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的潜在缺陷。平台 3. 检查肠沙门氏菌 BUL 与植物免疫相互作用 植物是肠沙门氏菌的新兴宿主，越来越多的报道表明植物具有可被肠沙门氏菌污染的免疫反应。肠沙门氏菌 3 型分泌系统效应器 我开发了将肠沙门氏菌 BUL 引入烟草的系统，并发现 SspH2 增强了植物中的免疫样反应。我们还将通过基因表达和功能测定来检查免疫反应，以确定 SspH2 如何破坏植物免疫，这些平台共同构成了一系列有用的模型系统和资源，可用于获得丰富的信息。了解 BUL 如何在宿主细胞中发挥作用，这一知识将为宿主与病原体之间的相互作用提供新的线索，从而可用于有针对性的破坏。