Deciphering the genetic regulation of bacterial heterogeneity in response to antibiotic stress using single-cell transcriptomics

Abstract Bacteria utilize diverse stress responses to survive and adapt under stress conditions. These responses contribute to the development of antibiotic resistance (mutation acquisition) and bacterial persistence (transient phenotypic adaptation), posing a significant threat to cause antibiotic treatment failure. Currently, most efforts to understand bacterial responses to stress primarily focus on characterizing entire populations, assuming uniform stress-response pathways across all cells. However, using a high throughput bacterial single-cell RNA sequencing (scRNA-seq) technology I developed over the past three years, we discovered that heterogeneous subpopulations of bacteria are prevalent during antibiotic treatment, including unique subpopulations enriched with antibiotic-resistant and persistent cells. For the first time, we can map the full picture of genetic factors driving heterogeneity and elucidate the underlying molecular mechanisms. The long-term goal of my research program is to comprehensively characterize bacterial heterogeneity in response to antibiotic stress and identify the underlying genetic regulators that can be potentially used as drug targets to prevent the development of antibiotic resistance and persistence. Using bacterial single-cell transcriptomics, we have recognized several candidate genes encoding the potential regulators for heterogeneous molecular responses under antibiotic stress. Among these candidates, we will focus on two genes: one that encodes a protein that stabilizes specific mRNAs, and a second uncharacterized protein that may impact cell-envelope permeability. Over the next five years, we will employ a synergistic approach of bacterial single-cell transcriptomics and molecular genetics to rigorously characterize the functions of these candidate genes and their corresponding pathways and uncover how they mediate heterogeneous bacterial responses under stress. Further application of this innovative approach over time will build a library of gene targets, thus opening a new direction that will expand our understanding of bacterial heterogeneity and reveal effective strategies to combat currently hidden bacterial adaptation mechanisms that threaten to cause treatment failure.