A spatially resolved single-cell transcriptomic technique for microbial pathogenesis

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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.