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.

我的研究计划旨在了解细菌泛素连接酶（BUL）蛋白如何通过细菌产生和传递，改变真核细胞的生物学。 Buls可以将泛素（一种小的76-氨基酸真核生物蛋白）共价连接到底物蛋白。链接的泛素部分的数量和结构可以修改底物功能，或导致其降解。底物降解以及缺乏分子工具和问题，对理解宿主中的小组功能构成了挑战。我们正在使用从三个潜水员那里获得的新见解，并使用完整的真核生物来制作真核生物中的小组功能的全面模型。平台1。沙门氏菌的机械研究在哺乳动物细胞中的功能。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中。使用这种方法，我们正在研究模式识别受体（PRR），NOD2和NLRP3是否是S. enterica Bul，SSPH2的底物。我们将分离SSPH2的复合物和相互作用的宿主蛋白，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识别SSPH2泛素化的特定PRR保留。随后，我们将突变这些残留物，以确定泛素转移是否在SSPH2的先天免疫颠覆中起作用。平台2。使用酵母来开发工具来研究块功能。酵母长期以来一直是探索细菌效应的细胞活性的模型，因为它通常是可探索的，并且将发现转化为哺乳动物系统的潜力很高。我们已经表明，肠链球菌SSPH1在酵母中施加深刻的毒性。我们正在使用此平台中可用的强大筛选工具来开发抑制SSPH1功能的试剂。我们已经确定了两个抑制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程中的潜在缺陷。该平台也适合小分子筛选。平台3。检查S. enterica Bul-dlant免疫学相互作用。植物是肠链球菌的新兴宿主，越来越多地报告了肠链链酵母污染的农产品。植物具有可以通过S. enterica 3型分泌系统效应来颠覆的免疫血液。我已经开发了将S. enterica Buls引入烟草的系统，发现SSPH2增强了植物中的类似免疫样反应。在此平台中，我们将使用质谱法来鉴定SSPH2的植物蛋白底物。我们还将通过基因表达和功能测定检查免疫反应，以确定SSPH2如何颠覆植物的免疫性。这些平台共同是模型系统和资源的有用数组，可以完全了解Buls在宿主单元中的功能。这些知识将为宿主 - 病原体相互作用提供新的启示，这些相互作用可以探索针对有针对性的破坏。