Wednesday, December 4, 2024
12/4/2024
PROJECT SUMMARY/ABSTRACT Infections caused by the Gram-positive bacterium Staphylococcus aureus are serious and frequently challenging to treat. While its propensity for developing antibiotic resistance has rendered treatment options limited, this problem is exacerbated by phenotypic heterogeneity at infection sites that leads to highly stress- tolerant subpopulations even with antibiotic-sensitive strains. Understanding this heterogeneity and its drivers will aid in effective clearance of S. aureus infection, but capturing the diversity of heterogeneous bacterial states in response to stimuli may additionally help us understand the complex regulatory events triggered, deepening our understanding of the biology of this organism. The central importance of transcriptional regulation in microbial responses to stimuli has made capture of the global transcriptome by RNA sequencing (RNA-seq) an invaluable tool. I previously applied RNA-seq to reveal how genomic structural variations cause antimicrobial resistance in Leishmania parasites, and how different species of Candida fungi adapt to the macrophage phagosome environment. However, capturing the population-averaged transcriptome is of limited utility in understanding variation within populations. For example, why do some bacteria survive an antibiotic treatment while others die? Why do some bacteria proliferate in the host, whereas others remain in a silent, but highly antibiotic-tolerant, state? Single cell RNA- seq (scRNA-seq) has radically expanded our ability to capture heterogeneous regulatory states. I recently applied scRNA-seq to resolve, for the first time, the transcriptional dynamics of the bacterial cell cycle in S. aureus and Escherichia coli, overcoming limitations of bulk methods caused by the difficulty of obtaining fully synchronized, unperturbed populations. This yielded new clues about the changes within proliferating bacteria, and a quantitative description of an important form of intrinsic heterogeneity in these organisms. In this proposal, I move from intrinsic heterogeneity in steady-state populations to heterogeneity in responses to environmental stimuli. In Aim 1, I will explore the relationship between heterogeneous transcriptional responses to the antibiotic vancomycin and tolerance to this drug. In Aim 2, I consider the more complex environmental challenge of intracellular survival within endothelial cells, where S. aureus may enter either a proliferative, lytic state or a persistent one that is highly tolerant to antibiotics. In both cases, the central premise of this work is that bacterial heterogeneity is not only of considerable medical importance, but through innovative analysis strategies, we can use this heterogeneity to develop a more profound understanding of the regulatory biology underlying this behavior. The impact will not only be to provide novel insights into how S. aureus survives and thrives despite complex environmental challenges, but also to develop a conceptual and analytical framework for applying scRNA-seq as a transformative tool for studying bacterial pathogens.
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