A spatially resolved single-cell transcriptomic technique for microbial pathogenesis

基于图像的单细胞转录组技术是一种新兴的技术套件，它可以使 转录组的部分可在单个细胞内直接成像和定量。一种这样的方法-- MerFish-凭借其高空间分辨率、高探测能力的独特组合，已成为领导者 效率、单分子敏感性、转录组范围的多路传输、大吞吐量以及经证实的能力 在完整的哺乳动物组织中发现、识别、功能注释和绘制不同类型的细胞。 这些方法为细菌系统的研究提供了巨大的希望。他们可以发现并分析 罕见但临床相关的抗生素耐药细胞群体，定义和表征了新的抗生素耐药机制 毒力因子从基因表达的相关性来调控，并将细菌的内部组织联系起来 转录组有助于我们对其调节能力的日益了解。此外，这些方法保证了 能够绘制细菌基因在自然环境中的表达图，揭示细菌行为的空间梯度 微生物群落，生物膜中的细胞专化性，感染部位的宿主-病原体相互作用，以及 不可培养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，目前还没有针对细菌的空间可分辨的单细胞转录方法。因此，要 针对这一需求，我们将创造细菌-美洲鱼。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密度和 探索、优化和验证一套膨胀化学物质和凝胶锚定方法 细菌的体积膨胀100到10,000倍。我们将开发细菌-美人鱼并对其进行基准测试 两种模式菌，大肠杆菌和枯草杆菌，在两个尺度上有约200个基因和转录组(约2000个基因)。 然后，我们将通过对细菌-美人鱼的两项重点研究来展示细菌-美人鱼的发现潜力 小鼠肠道病原体--一种人类肠道致病性大肠杆菌感染模型。首先，我们将 利用单分子敏感性和单细胞分辨率来探索C。 齿状菌培养，目的是描述多个致病方面，包括最近描述的 VF抑制条件下致病的“活性”EPEC的亚群。第二，我们将探索基因 小鼠肠道感染过程中轮状芽胞杆菌及其周围微生物群的表达。我们会 感染含有特定微生物区系的小鼠--改变的舍德勒菌群(ASF)--并描述整个转录组 轮状突起的基因表达和ASF所有8个成员的关键应激和代谢途径的表达 在感染期间的特定时间点，在小鼠盲肠和结肠的切片中。单细胞，空间基因- 我们将创建的表达图谱有望为病原体、微生物组和 最终，主持人。凭借其空间分辨率、敏感度和转录组范围的多路复用，我们 预计细菌鱼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.