A single-cell transcriptomic atlas for the formation and fate of microbial communities

About This Grant

PROJECT SUMMARY/ABSTRACT Microbial communities of unicellular prokaryotes display many features typically observed in more complex multicellular organisms, such as intricate creation of distinct structural features that have varying functionalities. Cells stratified throughout these structures have differential exposure to the environment which leads to vast spatiotemporal heterogeneity, even in genetically identical bacteria. These microbial communities can form across many physiological niches within a host where they are highly recalcitrant to conventional treatments causing a substantial clinical burden. A better understanding of microbial communities is essential for proper management. Although researchers have identified key developmental stages of microbial community formation, many fundamental biological principles surrounding the community lifestyle remain unknown. One major gap in understanding is how bacteria leverage their numerous genetic regulatory systems to coordinate the seemingly stochastic initiation of heterogeneity and structure creation throughout maturation of the community. Another gap in understanding is what subpopulation/portion of cells is responsible for the persistent nature of microbial communities, and specifically how these cells ensure the fate of the community by surviving harsh environmental stressors. These gaps have remained unfilled due to a central crux that the available techniques for studying prokaryotic systems have historically been limited to detecting a large spectrum of genes/transcripts or single cells, but not both. We recently published a prokaryotic single-cell RNA sequencing (scRNA-seq) approach, called BaSSSh-seq, which was the first to be applied to microbial communities. This scRNA-seq technology overcomes the aforementioned crux by allowing us to probe the full transcriptomic space of resolved individual cells. My research program will leverage our scRNA-seq capabilities towards the goal of creating a transcriptomic atlas for the formation and fate of microbial communities, using Staphylococcus aureus as a model bacterium. First, we will perform a fine-grained time course study applying scRNA-seq during community formation to describe when unique subpopulations arise and which regulatory systems are responsible. Imaging studies performed in parallel will be used to correlate the transcriptionally defined subpopulations to spatial stratification within the community. Second, we will isolate and define the profile of a persister cell through strategic depletion of the community and an enhanced, high-resolution scRNA-seq workflow. We will also study community regrowth from a persister population to define mechanisms of community replenishment. Each of the studies will be explored with perturbations to environmental nutrients and stressors to observe how the environment shapes community function. Collectively, this work will generate a transformative view of microbial communities at the single-cell level. While we focus on deducing the regulatory mechanisms coordinating structure and persistence, the datasets that we generate and the techniques that we explore will be an invaluable resource for the broader scientific community for translation to other bacterial species in the ultimate pursuit of improved human health.