Understanding Bacterial Persistence using Single-Cell Transcriptomics

Abstract

Persistence is a bacterial phenomenon where a small fraction of bacterial cells, called persisters, survive even a supralethal dose of antibiotic treatment and resume dividing after the treatment is complete to form colonies when on a surface. Persisters are genetically identical to the dead cells, and progenies of persisters behave like untreated parent cells. Persisters arise from all antibiotic treatments and have been implicated in the relapse of infections in clinical settings, and the emergence of antibiotic resistance. I study persistence against fluoroquinolone (FQ) antibiotics as these antibiotics are effective in killing the non-growing bacteria inhabiting bacterial infections, along with the growing ones, unlike most other classes of antibiotics. Nonetheless, persisters emerge from FQ treatments against non-growing bacterial populations and lead to treatment failures. The studies so far indicate a cell-cell heterogeneity in the mechanisms and genetic pathways that cause the formation of persisters during the period of recovery following an FQ treatment. In this thesis, I use bacterial single-cell transcriptomics to capture both the phenotypic and mechanistic heterogeneity of FQ persisters. I identify the genetic pathways that promote persistence and the cell states primed to divide as persisters after FQ treatment. In addition to DNA repair and protein disaggregation, I discover type I fimbriation to also promote persistence and be active in a small subpopulation of FQ-treated cells during recovery. I find that FQ persistence is strongly dependent on fimbrial and flagellar genes but in a functionally redundant manner. I further discover that FQ treatment induces the intercellular signaling of Autoinducer-2 (AI-2) during FQ recovery, and this signaling promotes persister formation by activating fimbrial or flagellar genes during recovery. FQ treatments also stimulate the formation of extracellular vesicles during recovery. Using a genetically engineered Cre-loxP system of donor and recipient bacterial cells and single-cell transcriptomics, I further identify and characterize a tiny subpopulation of transcriptionally and translationally deficient cells that get primed to become persisters by receiving functional proteins via extracellular vesicles. Intercellular exchange of both signaling molecules and vesicle-mediated functional proteins thus emerge as previously unknown mechanisms of persister formation; they reveal the collective and cooperative nature of bacterial persistence against antibiotic treatments.