Glycosphingolipids from the Soil Microbiome, Understanding Structure and Biosynthesis

来自土壤微生物组的鞘糖脂，了解结构和生物合成

• 批准号: 10836832
• 负责人: Paul Davis Boudreau
• 金额: $ 27.49万
• 依托单位: UNIVERSITY OF MISSISSIPPI
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10836832
• 关键词: Agonist; Anabolism; Antigens; Bacteria; Biological Assay; Candidate Disease Gene; Centers of Research Excellence; Chemistry; Complex; Galactose; Gene Cluster; Genes; Genome; Glycosphingolipids; Human; Immune signaling; Immune system; Immunologic Stimulation; Ions; Laboratories; Libraries; Link; Lipids; Macrophage; Maps; Molecular; Monitor; Nanotechnology; Natural Products; Neutral Glycosphingolipids; Organism; Pathway interactions; Process; Production; Role; Signal Transduction; Soil; Source; Speed; Sphingolipids; Structure; System; Tail; Work; candidate identification; clinically relevant; cytokine; design; genome sequencing; genomic data; gut microbiome; host microbiome; knockout gene; lipidomics; member; microbiome; monomer; nanopore; novel; receptor; scale up; screening; serine palmitoyltransferase; sugar; whole genome

The production of the glycosphingolipid 􀀂-􀀃􀀄􀀅􀀄􀀆􀀇􀀈􀀉􀀊􀀅􀀆􀀋􀀌􀀄􀀍􀀎􀀏􀀋􀀁􀀐􀀂-Gal) by a member of the human gut microbiome was an intriguing result because these lipids are known to be immune stimulating antigens, and their production by the gut microbiome suggests a role in host-microbiome signaling.1 􀀂-Gal is the canonical agonist for the immune system’s CD1d receptor,2–4 but synthetic work has shown that when the 􀀂-linked galactose is replaced with novel sugars, or sugar bioisosteres, the activity of the glycosphingolipid in immune signaling can change dramatically.5–7 These results suggest that bacteria which produce these glycosphingolipids, such as soil dwelling members of the order Sphingomonadales,8–10 might be a source of novel bioactive metabolites. In this project we have designed a soil enrichment screen using PCR amplification of serine palmitoyltransferase (SPT) gene, the first gene involved in sphingolipid synthesis,11,12 to identify sphingolipid producers. Follow-on lipidomic screening of SPT+ organisms on our laboratory’s QTOF LC-MS system will identify novel glycosphingolipids. By utilizing MS/MS fragment spectra analysis we will be able to identify sugar headgroups in our glycosphingolipids from neutral losses of the sugar monomers or the sugar fragment ions. Using GNPS-based molecular networking we will also be able to rapidly dereplicate known glycosphingolipid molecules, speeding up the process of identifying known chemistry to allow us to focus our efforts on novel sugar headgroups. With the novel organisms we isolate we will conduct Whole Genome Sequencing (WGS) with the Oxford Nanopore Technology’s nanopore platform to create a genomic data set that can be searched for the SPT gene. Inspired by the “glycogenomic” approach of mapping sugar chemistry in secondary natural products to biosynthetic gene clusters,13 we will also interrogate our genomes compared against the glycosphingolipids identified by LC-MS/MS analysis to identify candidate genes in the biosynthetic pathway after the SPT gene. Though this poses some unique challenges as sphingolipids are primary metabolites and their biosynthesis is not organized in tight biosynthetic gene clusters as is common in secondary natural products, the use of gene knockouts or heterologous expression can help confirm the role of these genes in the production of complex glycosphingolipids. We will also be able to utilize the known promiscuity of bacterial SPT genes to feed in unnatural lipid molecules,1 using LC-MS/MS monitoring to detect the novel glycosphingolipids produced by the incorporation of these feedstocks, demonstrating what strains might be able to be manipulated into producing compounds with desirable changes to the lipid tail of the glycosphingolipids. Glycosphingolipids isolated from scale up of the cultures will be further characterized by NMR analysis to confirm our structure assignment by MS/MS fragmentation analysis. At the end of the project, our glycosphingolipids will be submitted to a bioassay for cytokine elicitation from macrophages as a first step towards showing the clinical relevance of our glycosphingolipid library.

人类肠道微生物组成员产生鞘糖脂􀀂-􀀃􀀄􀀅􀀄􀀆􀀇􀀈􀀉􀀊􀀅􀀆􀀋􀀌􀀄􀀍􀀎􀀏􀀋􀀁􀀐􀀂-Gal是一个有趣的结果，因为已知这些脂质是免疫刺激抗原，肠道微生物组产生的鞘糖脂表明在宿主-微生物组信号传导中起作用。1􀀂-Gal是免疫系统CD1d受体的典型激动剂，2-4但合成工作表明，当􀀂-linked半乳糖被新的糖或糖生物异构体取代时，鞘糖脂在免疫信号传导中的活性会发生显著变化。这些结果表明，产生鞘糖脂的细菌，如生活在土壤中的鞘单胞目细菌，可能是一种新的生物活性代谢物的来源。本项目利用丝氨酸棕榈酰转移酶（SPT）基因的PCR扩增设计了土壤富集筛选，该基因是鞘脂合成的第一个基因11,12，用于鉴定鞘脂的产生者。在我们实验室的QTOF LC-MS系统上对SPT+生物体进行后续的脂质组学筛选将鉴定出新的鞘糖脂。通过MS/MS片段光谱分析，我们将能够从糖单体或糖片段离子的中性损失中识别糖鞘脂中的糖头基团。利用基于gnps的分子网络，我们还将能够快速地去复制已知的鞘糖脂分子，加快识别已知化学物质的过程，使我们能够集中精力研究新的糖头基。通过分离的新生物，我们将利用牛津纳米孔技术的纳米孔平台进行全基因组测序（WGS），以创建一个可以搜索SPT基因的基因组数据集。受到