Identifying Galactosyrin - the inhibitor of a novel hydrolytic immune signaling pathway

植物对病原体的识别对它们的生存和我们的粮食安全至关重要。大多数植物通过鞭毛碎片识别细菌病原体。我们最近发现，释放这些免疫原性鞭毛蛋白片段的水解途径始于分泌的β-半乳糖苷酶BGAL1，它作用于覆盖鞭毛蛋白聚合物的末端多糖(《科学》，2019年4月)。细菌可以通过产生对BGAL1不敏感的多糖或通过产生BGAL1抑制剂来逃避识别。我们的发现是利用丁香假单胞菌感染本氏烟草，但这些发现具有超越这个模型系统的意义，因为鞭毛蛋白在植物界得到普遍认可，而BGAL1在植物中是保守的。此外，病原菌鞭毛中普遍存在多糖多态性，这与植物-病原菌的军备竞赛是一致的。BGAL1抑制剂(半乳糖苷)的阐明一直是人们高度关注的问题，因为它干扰了这一新的、保守的免疫途径。这一建议旨在阐明半乳糖蛋白的结构及其生物合成，并发现更多在感染过程中被抑制的水解酶。我们有令人兴奋的初步数据。首先，我们已经确定了调控基因和一个操纵子，该操纵子包含两个生物合成基因，负责半乳糖苷的生产。将生物合成基因转移到大肠杆菌中可以促进半乳糖苷的大量生产。第二，我们已经建立了一个健全的半乳糖苷富集方案，这是一种由细菌在最低限度的介质中生长时产生的稳定的碱性亲水分子。该提案的第一个也是主要的目标是通过两种途径鉴定半乳糖苷的结构：首先，使用制备高效液相色谱法进行经典分离，并与James McCullagh(化学，牛津大学)合作使用化学方法进行结构鉴定。同时，我们将与张培军(eBIC，Harwell)合作进行冷冻电子显微镜研究，以阐明半乳糖苷在LacZ编码的β-半乳糖苷酶的活性部位的结构，该酶被用作低温EM研究的标准。第二个目标是通过代谢组学、饲养实验、底物合成和异源表达来鉴定生物合成基因和相应的突变体，以确定它们的底物和产物。第三个目标是利用基于活性的蛋白质组学发现更多像BGAL1这样的水解酶，这些水解酶在感染期间或被细菌代谢物抑制，目前显示了&gt；150水解酶的活性。这项建议与BBSRC的优先项目‘农业和食品安全’相关，并通过增加我们对细菌植物病原体的基本抗性的理解来解决战略重点‘可持续地提高农业生产’。这个项目是可行的，因为我们可以获得大量耐热的半乳糖苷，并且已经建立了浓缩程序。我们还鉴定了生物合成簇及其调控因子。该项目也很重要，不仅是作为BBSRC任务支持基础研究的资产，而且还因为该项目将导致新的作物保护战略，例如通过设计抗半乳糖苷的BGAL1。此外，一种新型的BGAL抑制剂及其生物合成将具有重要的医学影响，因为BGAL抑制剂被用于治疗代谢疾病、癌症以及病毒和细菌疾病。

The recognition of pathogens by plants is pivotal to their survival and to our food security. Most plants recognize bacterial pathogens through fragments of flagella. We recently discovered that the hydrolytic pathway releasing these immunogenic flagellin fragments starts with the secreted beta-galactosidase BGAL1, which acts on the terminal glycan that covers the flagellin polymer (Science, April 2019). Bacteria can evade recognition by producing BGAL1-insensitive glycans, or by producing a BGAL1 inhibitor. We made our discoveries using Pseudomonas syringae infecting Nicotiana benthamiana, but these findings have implications beyond this model system because flagellin is universally recognized in the plant kingdom and BGAL1 is conserved in plants. Furthermore, glycan polymorphism is common to flagella of pathogenic bacteria, consistent with the plant-pathogen arms race. The elucidation of the BGAL1 inhibitor ('galactosyrin') has been of high interest because it interferes with this novel, conserved immune pathway. This proposal aims to elucidate the structure of galactosyrin, its biosynthesis and discover more hydrolases that are suppressed during infection. We have exciting preliminary data. First, we have identified the regulatory genes and an operon containing two biosynthesis genes responsible for galactosyrin production. Transfer of the biosynthesis genes into E. coli prompts galactosyrin production in large quantities. Second, we have established a robust enrichment protocol for galactosyrin, which is a stable, basic, hydrophilic molecule produced by bacteria when grown in minimal media.The first and main objective of this proposal is to identify the galactosyrin structure through two routes: First, by classical fractionation using preparative HPLC and structure elucidation using chemical methods in collaboration with James McCullagh (Chemistry, Oxford). In parallel we will perform cryo electron microscopy studies in collaboration with Peijun Zhang (eBIC, Harwell) to elucidate the galactosyrin structure when trapped in the active site of the beta-galactosidase encoded by LacZ, which is used as a standard in cryo-EM studies. The second objective is to characterize the biosynthesis genes and the corresponding mutants to identify their substrates and products using metabolomics, feeding experiments, substrate synthesis, and heterologous expression. The third objective is to discover more hydrolases like BGAL1 that are suppressed during infection or by bacterial metabolites, using activity-based proteomics, which currently displays >150 hydrolase activities.This proposal is relevant to the BBSRC priority program 'Agriculture and food security' and addresses the strategic priority 'Sustainably enhancing agricultural production' by increasing our understanding of basal resistance to bacterial plant pathogens. This project is feasible because we have access to large amounts of heat-resistant galactosyrin and enrichment procedures have been established. We also have identified the biosynthesis cluster and its regulators. This project is also important, not only as an asset to the BBSRC mission to support fundamental research, but also because this project will lead to novel crop protection strategies, e.g. by engineering galactosyrin-resistant BGAL1. In addition, a novel BGAL inhibitor and its biosynthesis will have an important medical impact as BGAL inhibitors are used to treat metabolic disorders, cancer, and viral and bacterial diseases.