Towards a safer probiotic: clbD deletion attenuates EcN-induced DNA damage responses and epithelial inflammatory transcriptional responses.

Escherichia coli Nissle 1917 (EcN) is widely used as a probiotic and engineering chassis, but its carriage of the pks genomic island raises safety concerns related to the genotoxin colibactin. Here, we constructed a clbD deletion mutant (EcNΔclbD) and a chromosomally complemented strain (EcNΔclbD::clbD) using a CRISPR/Cas9-based genome-editing strategy. Locus-specific PCR confirmed the expected clbD deletion and restoration patterns. DNA damage responses were evaluated by γH2AX immunofluorescence and alkaline comet assay after bacterial co-culture. Compared with EcN, EcNΔclbD markedly reduced γH2AX positivity and comet-positive cells, whereas clbD complementation restored both phenotypes to levels comparable to EcN. In NCM460 colonic epithelial cells, EcNΔclbD induced lower mRNA expression of pro-inflammatory cytokines and TLR4-MYD88-NFKB1-related transcripts than EcN. Additional CCK-8 and cell-associated bacterial load assays showed no significant differences between EcN- and EcNΔclbD-treated groups, suggesting that the reduced inflammatory transcription was not primarily attributable to altered host cell CCK-8 readout or bacterial exposure. qPCR analysis of neighboring pks genes showed detectable local transcriptional changes after clbD deletion that were restored toward the parental EcN pattern by complementation. Transmission electron microscopy revealed no detectable morphological or ultrastructural disruption after clbD deletion. Exploratory untargeted metabolomics suggested a metabolic shift associated with clbD deletion, while gastrointestinal stress assays revealed condition-dependent fitness effects without a generalized growth defect in LB medium. Together, these findings indicate that clbD deletion attenuates EcN-induced DNA damage responses and epithelial inflammatory transcription in vitro. EcNΔclbD may represent a candidate safety-optimized EcN chassis requiring further validation in additional epithelial models and in vivo systems.