A spatially resolved single-cell transcriptomic technique for microbial pathogenesis

用于微生物发病机制的空间分辨单细胞转录组技术

基本信息

批准号：
10612336
负责人：
Jeffrey Moffitt
金额：
$ 22.13万
依托单位：
BOSTON CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-04-21 至 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10612336
关键词：
Acclimatization Address Antibiotic Resistance Architecture Atlases Bacillus subtilis Bacteria Bacterial Genes Bacterial Model Behavior Benchmarking Biology Cecum Cell Wall Cell physiology Cells Cessation of life Chemistry Childhood Citrobacter rodentium Clinical Colon Communities Complex Cues Detection Development Disparate Distal Environment Epithelial Cells Escherichia coli Escherichia coli Infections Gel Gene Expression Gene Expression Profiling Gene Expression Regulation Genes Goals Hydrogels Image In Situ Individual Infection Infection Control Intervention Invaded Lesion Life Link Location Mammalian Cell Maps Measurement Measures Messenger RNA Metabolic Pathway Methods Microbe Microbial Biofilms Microbiology Microscopy Modality Modeling Molecular Mus Names Optics Pathogenesis Pathogenicity Pathway interactions Phase Physiological Population Process Property RNA Regulation Repression Resolution Role Sampling Site Slice Source Stress Symptoms System Techniques Technology Time Tissues Virulence Factors Work bacterial community cell type clinically relevant density diarrheal disease empowerment enteric infection enteric pathogen enteropathogenic Escherichia coli gut microbiota host-microbe interactions human model imaging approach insight member microbial microbial community microbiome microbiota model organism novel novel therapeutics pathogen response single molecule transcriptome transcriptome sequencing transcriptomics ultra high resolution whole genome

项目摘要

Image-based approaches to single-cell transcriptomics are an emerging suite of technologies that allow large fractions of the transcriptome to be directly imaged and quantified within single cells. One such method— MERFISH—has emerged as a leader given its unique combination of high spatial resolution, high detection efficiency, single-molecule sensitivity, transcriptome-wide multiplexing, large throughput, and proven ability to discover, identify, functionally annotate, and map a diverse range of cell types within intact mammalian tissues. Such methods offer tremendous promise for the study of bacterial systems. They could discover and profile rare but clinically relevant populations of antibiotic resistant cells, define and characterize new mechanisms of virulence factor regulation from correlations in gene expression, and link the internal organization of the bacterial transcriptome to our growing understanding of its regulatory capacity. Moreover, such methods promise the ability to map bacterial gene expression in native contexts, revealing spatial gradients in bacterial behavior in microbial communities, cellular specialization in biofilms, host-pathogen interactions at infection sites, and the behavior of unculturable bacteria in their natural communities, to name only a few exciting applications. Unfortunately, there are no spatially resolved single-cell transcriptomic methods for bacteria. Thus, to address this need, we will create bacterial-MERFISH. We will use expansion microscopy—a super-resolution approach that physically expands samples to enhance optical resolution—to overcome RNA densities and will explore, optimize, and validate a suite of expansion chemistries and gel anchoring methods that promise bacterial volumetric expansions of 100- to 10,000-fold. We will develop and benchmark bacterial-MERFISH in two model bacteria, E. coli and B. subtilis, at two scales, ~200 genes and transcriptome-wide (~2000 genes). We will then demonstrate the discovery potential of bacterial-MERFISH with two focused studies of the mouse intestinal pathogen, C. rodentium—a model of human enteropathogenic E. coli infections. First, we will leverage single-molecule sensitivity and single-cell resolution to explore virulence factor (VF) regulation in C. rodentium cultures with the goal of characterizing multiple pathogenesis aspects, including a recently described sub-population of pathogenic ‘active’ EPEC in VF repression conditions. Second, we will explore gene expression in C. rodentium and the surrounding microbiome during intestinal infection in the mouse. We will infect mice harboring a defined microbiota—the Altered Schaedler Flora (ASF)—and profile whole-transcriptome gene expression in C. rodentium and key stress and metabolic pathway expression in all 8 members of the ASF in slices of the mouse cecum and colon at defined time points during infection. The single-cell, spatial-gene- expression atlases we will create promise new insights into local remodeling of pathogen, microbiome, and, eventually, host. With its combination of spatial resolution, sensitivity, and transcriptome-wide multiplexing, we anticipate that bacterial-MERFISH will find immediate use in the study of a wide range of bacterial systems.

基于图像的单细胞转录组学方法是一套新兴的技术，允许大量一种这样的方法可以在单细胞内直接成像和量化转录组的片段。 MERFISH——凭借其高空间分辨率、高检测能力的独特组合而成为领导者效率、单分子灵敏度、转录组范围内的多重分析、大通量以及经过验证的能力发现、识别、功能注释和绘制完整哺乳动物组织内多种细胞类型的图谱。这些方法为细菌系统的研究提供了巨大的前景，它们可以发现和分析。罕见但临床相关的抗生素耐药细胞群体，定义并表征了抗生素耐药性的新机制基因表达相关性的毒力因子调节，并将细菌的内部组织联系起来转录组有助于我们不断了解其调控能力。能够绘制本地环境中细菌基因表达的能力，揭示细菌行为的空间梯度微生物群落、生物膜中的细胞特化、感染部位的宿主-病原体相互作用以及不可培养的细菌在其自然群落中的行为，仅举几个令人兴奋的应用。不幸的是，没有针对细菌的空间分辨单细胞转录组学方法。为了满足这一需求，我们将创建细菌-MERFISH，我们将使用扩展显微镜——一种超分辨率技术。物理扩展样本以提高光学分辨率的方法——克服 RNA 密度，并将探索、优化和验证一套有望实现的扩展化学物质和凝胶锚定方法细菌体积膨胀 100 至 10,000 倍我们将开发细菌-MERFISH 并进行基准测试。两种模型细菌，大肠杆菌和枯草芽孢杆菌，在两个尺度上，约 200 个基因和转录组范围（约 2000 个基因）。然后，我们将通过两项重点研究来证明细菌-MERFISH 的发现潜力小鼠肠道病原体，啮齿类梭菌——人类肠道致病性大肠杆菌感染的模型。利用单分子敏感性和单细胞分辨率探索念珠菌毒力因子 (VF) 调控。啮齿类动物培养物的目标是表征多个发病机制方面，包括最近描述的 VF 抑制条件下致病性“活跃”EPEC 的亚群其次，我们将探索基因。我们将在小鼠肠道感染期间在啮齿类动物和周围微生物组中表达。受感染的小鼠含有特定的微生物群——改变的谢德勒菌群（ASF）——并分析了全转录组啮齿类动物中的基因表达以及 ASF 所有 8 个成员中的关键应激和代谢途径表达在感染过程中指定时间点的小鼠盲肠和结肠切片中。我们将创建的表达图谱有望为病原体、微生物组的局部重塑提供新的见解，最终，凭借其空间分辨率、灵敏度和转录组范围内的多重性的结合，我们预计细菌-MERFISH 将立即用于多种细菌系统的研究。