Spatially resolved multiomics profiling of microbes and their host tissue

项目摘要/摘要 微生物细胞群可能是高度异质性的，这对菌株在复合体中的生存至关重要。 抗生素治疗等条件。显然，细胞间的异质性不能用 传统的批量测序技术。基于单细胞的微生物细胞方法正在出现，以解决 然而，这个问题是空间背景信息，对于理解微生物与宿主的相互作用至关重要， 没有被收集。到目前为止，我们仍然缺乏高分辨率的空间组学工具来研究微生物及其 栖息在哺乳动物体内的宿主。目前可用的大多数基于NGS的空间转录组平台不是 与细菌图谱兼容的原因有三：1)细菌细胞壁的厚度高度不同 以及阻止逆转录酶和引物等试剂进入细胞的组合物， 尤其是革兰氏阳性菌，细胞壁较厚；2)细菌细胞mRNAs稀疏，短小 半衰期；3)细菌的RNA序列中缺少PolyA尾巴。在过去的5年里，我开发了DBiT-seq (组织中的确定条形码)，第一个高分辨率的空间蛋白转录组平台，它具有 被广泛应用于神经科学、发育和人类癌症的研究。我进一步报道了 SPACE-CITE-SEQ技术可以共定位~300个表面蛋白和整个转录组 各种组织类型。我建议在未来五年内开发一种新的空间测序技术 名为microDBiT，这将是第一个空间蛋白质转录组平台，可以绘制微生物和 空间环境中的宿主细胞。在最初阶段，我们将设计和使用培养革兰氏切片 以阳性菌金黄色葡萄球菌和阴性菌大肠埃希菌为模型优化微DBiT关键步骤 包括细胞壁消化、mRNA多聚腺苷化、逆转录和细胞内连接。我们会 接下来，开发微DBiT协议，使用从以下来源获得的肠道组织进行微生物和宿主细胞共定位 细菌在无菌C57BL/6小鼠体内定植。最后，我们将应用microDBiT来定位该基因 炎症性肠病病原菌和患者细胞的表达谱。因为代谢物 被认为是影响宿主细胞行为的重要途径，同时我们将包括 一个宿主受体的抗体小组，研究代谢物将如何影响宿主的基因表达 细胞。这项技术最终将实现高通量和高分辨率的空间表征 微生物的异质性及其与宿主细胞的相互作用。从长远来看，我们将把MicroDBiT打造成 一个可以在不同组学水平上应用于不同微生物和宿主系统的综合平台 (基因组学、转录组学、表观基因组学等)。

PROJECT SUMMARY/ABSTRACT Microbial cell populations can be highly heterogeneous, which is crucial for strain survival in complex conditions such as antibiotic treatment. Apparently, the cell-to-cell heterogeneity cannot be revealed using traditional bulk sequencing techniques. Single-cell based approaches for microbial cells are emerging to tackle this question, however, the spatial context information, crucial for understanding the microbe-host interactions, is not collected. As of current, we still lack high resolution spatial omics tools to study microbes and their residing mammalian host. Most currently available NGS based spatial transcriptome platforms are not compatible with bacteria profiling due to three reasons: 1) Bacteria cell walls are highly diverse in thickness and composition, which prevents the reagents such as reverse transcriptase and primers to enter the cell, especially for Gram Positive ones with thick cell walls; 2) mRNAs of bacteria cells are sparse and have short half-life; 3) bacteria mRNA lacks poly-A tail in RNA sequence. During the past 5 years, I developed DBiT-seq (Deterministic barcoding in tissue), the first high resolution spatial proteo-transcriptome platform, which have been widely applied to neuroscience, development, and cancer studies in human. I further reported the Spatial-CITE-seq technique which can co-mapping ~300 surface proteins and the whole transcriptome of various tissue types. I propose in the next five years the development of a new spatial sequencing technology called microDBiT, which will be the first spatial proteo-transcriptome platform that can map microbes and the host cells within the spatial context. At the initial stage, we will design and use the slides of cultured Gram positive bacteria S.aureus and negative bacteria E. coli as a model to optimize the key steps of microDBiT protocol, including cell wall digestion, mRNA polyadenylation, reverse transcription and in cell ligation. We will next develop the microDBiT protocol for microbe and host cell co-mapping using gut tissues obtained from bacteria colonized germ-free C57BL/6 mice. Lastly, we will apply the microDBiT to map out the gene expression profile of pathogens and the patient cells in inflammatory bowel disease (IBD). Since metabolites of microbes are considered important pathways that influence the host cell behavior, we will meanwhile include an antibody panel of host receptors and study how the metabolites will influence the gene expression of host cells. This technique will ultimately enable high-throughput and high-resolution characterization of spatial heterogeneity of microbes and their interaction with the host cells. In the long run, we will build microDBiT into a comprehensive platform that could be applied to diverse microbe and host systems at different omics levels (Genomics, Transcriptomics, Epigenomics, etc.).