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.

由􀀂􀀃􀀄􀀅􀀄􀀆􀀇􀀈􀀉􀀊􀀅􀀆􀀋􀀌􀀄􀀍􀀎􀀏􀀋􀀁􀀐􀀂人类肠道微生物组的成员产生鞘糖脂（Gal）是一个有趣的结果，因为已知这些脂质是免疫刺激抗原，并且它们由肠道微生物组产生表明在宿主微生物组信号传导中的作用。1􀀂-Gal是免疫系统的CD 1d受体的典型激动剂，2-4但合成工作已经表明，当􀀂连接的半乳糖被新的糖或糖生物电子等排体取代时，鞘糖脂在免疫信号传导中的活性可以发生显著变化。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土壤寄居成员，8-10可能是新的生物活性代谢物的来源。在这个项目中，我们设计了一个土壤富集屏幕使用PCR扩增丝氨酸棕榈酰转移酶（SPT）基因，第一个基因参与鞘脂合成，11，12，以确定鞘脂生产商。在我们实验室的QTOF LC-MS系统上对SPT+微生物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，我们还将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，使我们能够专注于新的糖头基。对于我们分离的新生物，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 除了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”方法之外，13我们还将询问我们的基因组，将其与通过LC-MS/MS分析鉴定的鞘糖脂进行比较，以鉴定SPT基因之后的生物合成途径中的候选基因。虽然这带来了一些独特的挑战，因为鞘脂是初级代谢产物，并且它们的生物合成不像次级天然产物中常见的那样以紧密的生物合成基因簇组织，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。我们还将能够利用已知的细菌SPT基因的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，证明哪些菌株可能能够被操纵以产生对鞘糖脂的脂质尾部具有期望变化的化合物。将通过NMR分析进一步表征从放大培养物中分离的鞘糖脂，以通过MS/MS裂解分析确认我们的结构归属。在项目结束时，我们的鞘糖脂将被提交给巨噬细胞因子诱导的生物测定，作为显示我们的鞘糖脂库的临床相关性的第一步。