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.

糖磷脂脂的产生是一个有趣的结果，因为已知这些脂质是免疫刺激抗原的，并且它们由肠道微生物组产生表明在宿主 - 微生物组信号中起作用。1◎-gal是gal- gal是一种免疫系统的cd1d issict insctic in Cd1d受体，但在2 – 4中表现出了同步，并表明被免疫信号中的糖脂果脂的活性代替，糖脂的活性可能会发生巨大变化。5–7这些结果表明，产生这些糖磷脂的细菌，例如sphingomonadales的污垢居民，8-10，8-10可能是新颖的生物活性生物活性的生物活性质体。在这个项目中，我们使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，这是第一个参与鞘脂合成的基因，11,12，以识别鞘脂生产商。在我们实验室的QTOF LC-MS系统上对SPT+生物的脂质筛选跟随脂质筛选将鉴定出新型的糖磷脂。通过利用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鉴定出糖脂脂中的糖头组。使用基于GNPS的分子网络，我们还将能够快速脱皮的已知糖脂分子，从而加快鉴定已知化学性质的过程，从而使我们能够将精力集中在新的糖头组上。通过新的生物体，我们将与牛津纳米孔技术的纳米孔平台进行整个基因组测序（WGS），以创建可以搜索SPT基因的基因组数据集。受到启发 在SPT基因后，SPT基因后的生物合成途径中鉴定的糖磷脂与糖磷脂相比，我们还将在生物合成基因簇中映射糖化化学的“糖基础”方法，13我们还将询问我们的基因组。尽管这使一些独特的挑战定位，因为鞘脂是主要的代谢产物，并且它们的生物合成并未在紧密的生物合成基因簇中组织起来，因为在次要天然产物中通常是常见的，但基因敲除或异源表达的使用可以帮助确认这些基因在复杂的糖脂生产中的作用。我们还将能够利用细菌SPT基因的已知滥交以非自然的脂质分子为食，1使用LC-MS/MS监测来检测通过将这些原料掺入这些原料所产生的新型糖脂脂，以表明哪些菌株可以将裂纹变成所需的毛皮变化。 NMR分析将进一步表征从培养物的规模中分离出来的糖磷脂，以通过MS/MS碎片分析确认我们的结构分配。在项目结束时，我们的糖磷脂将被提交给生物测定，以从巨噬细胞中诱导细胞因子诱导，这是迈向糖化脂质脂质库的临床相关性的第一步。