Bacterial single cell RNA-seq unveils cyclic-di-GMP as an antitoxin critical for antibiotic tolerance in biofilm [ChIP-seq]

Biofilms are heterogeneous bacterial communities featured by high persister prevalence, responsible for antibiotic tolerance. However, the mechanisms underlying persister formation within biofilms remained ambiguous. Here, by developing and utilizing a ribosomal RNA depleted bacterial single-cell RNA-seq method, RiboD-mSPLiT, we resolved biofilm heterogeneity and discovered pdeI as a marker gene for persister subgroup within biofilms. Remarkably, our findings elucidated that PdeI upregulates cellular levels of c-di-GMP, which acts as an antitoxin to modulate the toxicity of toxin protein HipH. HipH localizes on nucleoid and functions as a potent DNase, inducing cells into a viable but non-culturable state. Conversely, c-di-GMP interacts with HipH, reducing its genotoxic effects and enabling cells to enter a persister state, resulting in drug tolerance. Importantly, by targeting this toxin-antitoxin system, we repressed drug tolerance in Uropathogenic Escherichia coli infections, offering promising therapeutic strategies against chronic and relapsing infections. Chromatin Immunoprecipitation DNA-sequencing (Chip-seq) is performed in overexpression of HipH in E.coli with or without C-di-Gmp(CDG) ** The submitter states that the study's data have undergone significant changes and are no longer accurately reflected in SuperSeries GSE239505 [Feb 20, 2024]. The correct data are in SuperSeries GSE260458. GSE260458 does not include Chip-seq data. **