Glycosphingolipids from the Soil Microbiome, Understanding Structure and Biosynthesis

由人类肠道微生物组的一名成员产生鞘糖脂􀀂-􀀃􀀄􀀅􀀄􀀆􀀇􀀈􀀉􀀊􀀅􀀆􀀋􀀌􀀄􀀍􀀎􀀏􀀋􀀁􀀐􀀂-Gal)是一个有趣的结果，因为这些脂类被认为是免疫刺激抗原，它们的产生表明它们在宿主微生物组信号中发挥作用。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􀀂连接的半乳糖被新的糖或糖生物等位取代时，5-7这些结果表明，产生这些糖鞘脂类的细菌，如鞘氨醇单胞目的土壤栖息成员，8-10可能是新的生物活性代谢物的来源。在本项目中，我们设计了一种土壤富集素筛选方法，利用聚合酶链式反应扩增第一个参与鞘磷脂合成的基因-丝氨酸棕榈酰转移酶(SPT)基因11，12来鉴定鞘磷脂产生者。在我们实验室的QTOF LC-MS系统上对SPT+生物体进行后续的脂组筛选将鉴定出新的神经鞘糖脂。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鉴定我们的糖鞘糖脂中的糖头基。使用基于GNPS的分子网络，我们还将能够快速复制已知的神经鞘糖脂分子，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的糖头基团上。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。灵感来自于 “糖基因组学”方法将次生天然产物中的糖化学映射到生物合成基因簇，13我们还将询问我们的基因组，将其与LC-MS/MS分析鉴定的神经鞘糖脂进行比较，以确定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，因为鞘脂是主要的代谢物，它们的生物合成并不像二级天然产品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生产复杂的鞘糖脂中的作用。我们还将能够利用已知的细菌SPT基因的混杂来喂养非天然的脂分子，1使用LC-MS/MS监测来检测通过加入这些原料而产生的新型糖鞘糖脂，展示哪些菌株能够被操纵来生产对糖鞘脂的脂尾具有理想变化的化合物。从放大培养中分离得到的鞘糖脂将通过核磁共振分析进一步表征，以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从巨噬细胞中激发细胞因子的生物测定，作为显示我们的鞘糖脂文库的临床相关性的第一步。

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.