Bacterial single cell RNA-seq unveils cyclic-di-GMP controlled toxin activity critical for drug tolerance in chronic infections

Abstract Biofilms are heterogeneous bacterial communities featured by a high persister prevalence, responsible for antibiotic tolerance and chronic infections. However, the mechanisms underlying persister formation within biofilms remained unclear. Here, by developing and utilizing a ribosomal RNA depleted bacterial single-cell RNA-seq method, RiboD-PETRI, we resolved biofilm heterogeneity and discovered pdeI as a distinctive marker for persister subpopulation. Remarkably, we elucidated that PdeI upregulates cellular levels of cyclic-di-GMP (c-di-GMP), which controls both the expression and toxicity of HipH (YjjJ). Specifically, HipH localizes on nucleoid and functions as a potent deoxyribonuclease, inducing cells into a viable but non-culturable (VBNC) state. c-di-GMP counteracts the genotoxic impact of HipH through a physical interaction, thereby facilitating the transition of cells into a persister state that concurrently fosters drug tolerance. Moreover, by targeting this toxin-antitoxin system, we inhibited drug tolerance in Uropathogenic Escherichia coli infections, offering promising therapeutic strategies against chronic and relapsing infections.