A spatially resolved single-cell transcriptomic technique for microbial pathogenesis

基于图像的单细胞转录组学方法是一套新兴的技术， 在单个细胞内直接成像和定量的转录组部分。其中一种方法- MERFISH-由于其独特的高空间分辨率、高检测能力和高分辨率的 效率，单分子灵敏度，转录组范围内的多重，大通量，并证明有能力， 在完整的哺乳动物组织中发现、鉴定、功能注释和绘制各种细胞类型。 这些方法为细菌系统的研究提供了巨大的希望。他们可以发现并侧写 罕见但临床相关的抗生素耐药细胞群体，定义和表征新的机制， 毒力因子的调控来自于基因表达的相关性，并联系着细菌的内部组织 转录组，我们越来越了解它的调节能力。此外，这些方法保证了 在自然环境中绘制细菌基因表达的能力，揭示了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，没有空间分辨的单细胞转录组学方法的细菌。从而 为了满足这一需求，我们将创造细菌MERFISH。我们将使用膨胀显微镜-一种超分辨率 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、优化和验证一套膨胀化学和凝胶锚定方法， 细菌体积膨胀100到10,000倍。我们将开发和基准细菌MERFISH在 两种模式菌E. coli和B.枯草芽孢杆菌，在两个尺度上，~200个基因和全转录组（~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。 小鼠肠道致病菌C.啮齿类动物-人肠道致病性E.大肠杆菌感染。一是 利用单分子敏感性和单细胞分辨率来探索C. 啮齿类培养物，其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"活性" EPEC亚群。第二，我们将探索基因 表达于C.啮齿动物和周围的微生物组在肠道感染的小鼠。我们将 感染携带确定的微生物群--改变的Schaedler植物群（ASF）--的小鼠，并分析全转录组 C.啮齿类和关键应激和代谢途径的表达在所有8个成员的ASF 在感染过程中的规定时间点的小鼠盲肠和结肠切片中。单细胞空间基因 表达图谱，我们将创造有希望的新见解，局部重塑病原体，微生物组，和， 最后，主持人。凭借其空间分辨率，灵敏度和转录组范围的多重功能，我们 预计细菌MERFISH将立即用于广泛的细菌系统的研究。

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.