{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,7]],"date-time":"2026-08-07T15:14:15Z","timestamp":1786115655645,"version":"build-2736575974"},"reference-count":35,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,8,6]],"date-time":"2026-08-06T00:00:00Z","timestamp":1785974400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Cell. Infect. Microbiol."],"abstract":"<jats:p>\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    Nissle 1917 (EcN) is widely used as a probiotic and engineering chassis, but its carriage of the\n                    <jats:italic>pks<\/jats:italic>\n                    genomic island raises safety concerns related to the genotoxin colibactin. Here, we constructed a\n                    <jats:italic>clbD<\/jats:italic>\n                    deletion mutant (EcN\u0394\n                    <jats:italic>clbD<\/jats:italic>\n                    ) and a chromosomally complemented strain (EcN\u0394\n                    <jats:italic>clbD<\/jats:italic>\n                    ::\n                    <jats:italic>clbD<\/jats:italic>\n                    ) using a CRISPR\/Cas9-based genome-editing strategy. Locus-specific PCR confirmed the expected\n                    <jats:italic>clbD<\/jats:italic>\n                    deletion and restoration patterns. DNA damage responses were evaluated by \u03b3H2AX immunofluorescence and alkaline comet assay after bacterial co-culture. Compared with EcN, EcN\u0394\n                    <jats:italic>clbD<\/jats:italic>\n                    markedly reduced \u03b3H2AX positivity and comet-positive cells, whereas\n                    <jats:italic>clbD<\/jats:italic>\n                    complementation restored both phenotypes to levels comparable to EcN. In NCM460 colonic epithelial cells, EcN\u0394\n                    <jats:italic>clbD<\/jats:italic>\n                    induced lower mRNA expression of pro-inflammatory cytokines and TLR4\u2013MYD88\u2013NFKB1-related transcripts than EcN. Additional CCK-8 and cell-associated bacterial load assays showed no significant differences between EcN- and EcN\u0394\n                    <jats:italic>clbD<\/jats:italic>\n                    -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\n                    <jats:italic>pks<\/jats:italic>\n                    genes showed detectable local transcriptional changes after\n                    <jats:italic>clbD<\/jats:italic>\n                    deletion that were restored toward the parental EcN pattern by complementation. Transmission electron microscopy revealed no detectable morphological or ultrastructural disruption after\n                    <jats:italic>clbD<\/jats:italic>\n                    deletion. Exploratory untargeted metabolomics suggested a metabolic shift associated with\n                    <jats:italic>clbD<\/jats:italic>\n                    deletion, while gastrointestinal stress assays revealed condition-dependent fitness effects without a generalized growth defect in LB medium. Together, these findings indicate that\n                    <jats:italic>clbD<\/jats:italic>\n                    deletion attenuates EcN-induced DNA damage responses and epithelial inflammatory transcription\n                    <jats:italic>in vitro<\/jats:italic>\n                    . EcN\u0394\n                    <jats:italic>clbD<\/jats:italic>\n                    may represent a candidate safety-optimized EcN chassis requiring further validation in additional epithelial models and\n                    <jats:italic>in vivo<\/jats:italic>\n                    systems.\n                  <\/jats:p>","DOI":"10.3389\/fcimb.2026.1812973","type":"journal-article","created":{"date-parts":[[2026,8,6]],"date-time":"2026-08-06T04:24:36Z","timestamp":1785990276000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Towards a safer probiotic: clbD deletion attenuates EcN-induced DNA damage responses and epithelial inflammatory transcriptional responses"],"prefix":"10.3389","volume":"16","author":[{"given":"Wei-Ming","family":"Chen","sequence":"first","affiliation":[{"name":"Department of Gastroenterology, The National Key Clinical Specialty, Zhongshan Hospital of Xiamen University, School of Medicine, Xiamen University","place":["Fujian, China"]},{"name":"Clinical Research Center for Gut Microbiota and Digestive Diseases of Fujian Province, Xiamen Key Laboratory of Intestinal Microbiome and Human Health","place":["Xiamen, Fujian, China"]},{"name":"Department of Digestive Disease, Institute for Microbial Ecology, School of Medicine, Xiamen University","place":["Xiamen, Fujian, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ming-Yang","family":"Sun","sequence":"additional","affiliation":[{"name":"Department of Gastroenterology, The National Key Clinical Specialty, Zhongshan Hospital of Xiamen University, School of Medicine, Xiamen University","place":["Fujian, China"]},{"name":"Clinical Research Center for Gut Microbiota and Digestive Diseases of Fujian Province, Xiamen Key Laboratory of Intestinal Microbiome and Human Health","place":["Xiamen, Fujian, China"]},{"name":"Department of Digestive Disease, Institute for Microbial Ecology, School of Medicine, Xiamen University","place":["Xiamen, Fujian, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ji-Yuan","family":"Ye","sequence":"additional","affiliation":[{"name":"Department of Gastroenterology, The National Key Clinical Specialty, Zhongshan Hospital of Xiamen University, School of Medicine, Xiamen University","place":["Fujian, China"]},{"name":"Clinical Research Center for Gut Microbiota and Digestive Diseases of Fujian Province, Xiamen Key Laboratory of Intestinal Microbiome and Human Health","place":["Xiamen, Fujian, China"]},{"name":"Department of Digestive Disease, Institute for Microbial Ecology, School of Medicine, Xiamen University","place":["Xiamen, Fujian, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"De-Cheng","family":"Tan","sequence":"additional","affiliation":[{"name":"Department of Gastroenterology, The National Key Clinical Specialty, Zhongshan Hospital of Xiamen University, School of Medicine, Xiamen University","place":["Fujian, China"]},{"name":"Clinical Research Center for Gut Microbiota and Digestive Diseases of Fujian Province, Xiamen Key Laboratory of Intestinal Microbiome and Human Health","place":["Xiamen, Fujian, China"]},{"name":"Department of Digestive Disease, Institute for Microbial Ecology, School of Medicine, Xiamen University","place":["Xiamen, Fujian, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zi-Yi","family":"Zhang","sequence":"additional","affiliation":[{"name":"Department of Gastroenterology, The National Key Clinical Specialty, Zhongshan Hospital of Xiamen University, School of Medicine, Xiamen University","place":["Fujian, China"]},{"name":"Clinical Research Center for Gut Microbiota and Digestive Diseases of Fujian Province, Xiamen Key Laboratory of Intestinal Microbiome and Human Health","place":["Xiamen, Fujian, China"]},{"name":"Department of Digestive Disease, Institute for Microbial Ecology, School of Medicine, Xiamen University","place":["Xiamen, Fujian, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jing-Jing","family":"Liu","sequence":"additional","affiliation":[{"name":"Department of Gastroenterology, The National Key Clinical Specialty, Zhongshan Hospital of Xiamen University, School of Medicine, Xiamen University","place":["Fujian, China"]},{"name":"Clinical Research Center for Gut Microbiota and Digestive Diseases of Fujian Province, Xiamen Key Laboratory of Intestinal Microbiome and Human Health","place":["Xiamen, Fujian, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,8,6]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"207","DOI":"10.1186\/1471-2105-8-207","article-title":"Orthogonal projections to latent structures as a strategy for microarray data normalization","volume":"8","author":"Bylesj\u00f6","year":"2007","journal-title":"BMC 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