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

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.

The main aim of this proposal is to identify the molecular structure of galactosyrin, a heat-stable, basic, small molecule inhibitor that targets a conserved secreted beta-galactosidase (BGAL1). BGAL1 the initiator of a novel hydrolytic pathway that releases the flagellin elicitor, which is universally recognized by plants as a signature of bacterial invasion. Our exciting preliminary data includes the identity of the two biosynthesis genes and a robust protocol for galactosyrin enrichment.
The FIRST OBJECTIVE is to purify galactosyrin to homogeneity and resolve its structure using NMR, HRMS and GC-MS and in parallel resolve its structure when bound to the LacZ-encoded BGAL enzyme using cryo-EM. Candidate galactosyrin will be re-synthesised and used for enzymatic essays and for complementation of bacterial growth in infection assays. We will also engineer a galactosyrin-insensitive BGAL1 and test this in elicitor release assays.
The SECOND OBJECTIVE is to elucidate the galactosyrin biosynthesis pathway by metabolic characterization and feeding experiments on the mutants lacking the biosynthesis genes and on E. coli producing galactosyrin, and by characterizing heterologously expressed biosynthesis enzymes. We will use GCMS and LCMS to identify putative substrates and produce putative substrates by synthetic chemistry to confirm the biosynthesis using RapidFire MS.
The THIRD OBJECTIVE is to discover more extracellular hydrolases that are suppressed during infection using activity-based proteomics using a cocktail of biotinyated probes and by including convolution activity-profiling assays and metabolite suppression experiments. The role of targeted hydrolases in immunity will be addressed by silencing with the aim to discover hydrolases with similar importance as BGAL1.

The primary and immediate impact of this research will be enhanced knowledge and understanding of how plant pathogens manipulate the host to cause disease. This knowledge will be communicated to the scientific community; commercial partners and general public in several ways (see Pathways-to-Impact).

This project will have a deep impact on PLANT SCIENCE and MICROBIOLOGY because we will resolve a novel biosynthetic route and a identify new type of BGAL inhibitor. This will result in the annotation of orphan genes and increased understanding of host manipulation by plant pathogens. This project will stress the importance of extracellular interactions in bacterial diseases, which are currently mostly studied from the perspective of host manipulation by cytonuclear effectors. This project will raise interests to study other bacterial plant pathogens for suppression of host hydrolases and their underlying molecular mechanisms. This project will also demonstrate the great potential to chemical proteomic approaches to uncover novel biological phenomena.

This project will have a significant impact on INDUSTRY and COMMERCIAL PARTNERS in two ways. First, this project will deliver novel strategies for crop protection, e.g. based on the introduction of inhibitor-insensitive BGAL to reduced pathogen susceptibility, or by developing agrochemicals that block galactosyrin biosynthesis. Second, the increased production of galactosyrin in bacteria, facilitated by the description of the galactosyrin biosynthesis genes and regulators, will make galactosyrin available in large quantities, facilitating research into the use of galactosyrin(-derivatives) for the treatment of human diseases that require regulation of glycan metabolism. For instance, a similar iminosugar, 1-deoxynojirimycin (DNJ), which inhibits alpha-glucosidase, was discovered in Mulberry leaves and is now produced in Bacillus species. DNJ is used as drug against HIV and its derivatives are used to treat diabetes (Miglitol) and Gauchers disease (Miglistat).

This project will make a substantial impact on the general public by the increased awareness of intimate relationships that pathogens engage with their host to cause disease. This project will also nicely illustrate that increased knowledge leads to new strategies for environmentally safe crop protection, stressing the relevance of basic research for the society.

The impact of this project on UK research potential resides in publications of high impact and the training of highly skilled personnel. These activities will strengthen the position of the UK to sustain its 'Knowledge Based Economy'.