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的鞘糖脂 ( ) 是一种由人类肠道微生物组成员产生的鞘糖脂这是一个有趣的结果，因为已知这些脂质是免疫刺激抗原，并且它们由肠道微生物组产生，表明它们在宿主微生物组信号传导中发挥作用。1 ƀ-Gal 是免疫系统 CD1d 受体的典型激动剂，2-4 但合成工作表明 当连接的半乳糖被新的糖或糖生物等排体取代时，免疫信号中鞘糖脂的活性会发生巨大变化。 5-7 这些结果表明，产生这些鞘糖脂的细菌，例如鞘氨醇单胞菌目的细菌，8-10 可能是新的生物活性代谢物的来源。在这个项目中，我们设计了一种土壤富集筛选，使用丝氨酸棕榈酰转移酶 (SPT) 基因（第一个参与鞘脂合成的基因）的 PCR 扩增，11,12 来识别鞘脂生产者。在我们实验室的 QTOF LC-MS 系统上对 SPT+ 生物体进行后续脂质组学筛选将鉴定新型鞘糖脂。通过利用 MS/MS 碎片光谱分析，我们将能够从糖单体或糖碎片离子的中性损失中识别鞘糖脂中的糖头基。使用基于 GNPS 的分子网络，我们还能够快速复制已知的鞘糖脂分子，加快识别已知化学物质的过程，使我们能够将精力集中在新型糖头基上。对于我们分离的新型生物体，我们将使用牛津纳米孔技术的纳米孔平台进行全基因组测序（WGS），以创建可以搜索 SPT 基因的基因组数据集。灵感来自于 将次级天然产物中的糖化学映射到生物合成基因簇的“糖基因组”方法，13我们还将询问我们的基因组与通过 LC-MS/MS 分析鉴定的鞘糖脂进行比较，以识别 SPT 基因之后生物合成途径中的候选基因。尽管这带来了一些独特的挑战，因为鞘脂是主要代谢物，并且它们的生物合成并不像次级天然产物中常见的那样组织在紧密的生物合成基因簇中，但使用基因敲除或异源表达可以帮助确认这些基因在复杂鞘糖脂生产中的作用。我们还能够利用已知的细菌 SPT 基因混杂性来喂养非天然脂质分子，1 使用 LC-MS/MS 监测来检测通过掺入这些原料产生的新型鞘糖脂，从而证明哪些菌株可以被操纵来生产对鞘糖脂脂质尾部产生所需变化的化合物。从培养物放大中分离出的鞘糖脂将通过 NMR 分析进一步表征，以通过 MS/MS 碎片分析确认我们的结构分配。在项目结束时，我们的鞘糖脂将进行生物测定，以从巨噬细胞中诱导细胞因子，这是展示我们的鞘糖脂库的临床相关性的第一步。