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ORIGINAL RESEARCH article
Front. Microbiol.
Sec. Microbial Physiology and Metabolism
Volume 16 - 2025 | doi: 10.3389/fmicb.2025.1569627
The Sigma Factor σ 54 (rpoN) functions as a Global Regulator of Antibiotic Resistance, Motility, Metabolism, and Virulence in Clostridioides difficile
Provisionally accepted
Ying YangTingyu HuangJunyi YangRuirui ShaoLuhong ShuPing LingYingjun LuWeihao MaJian LiaoZhizhong Guan
Xiaolan Qi
Guzhen Cui
Wei Hong*- Guizhou Medical University, Guiyang, China
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Clostridioides difficile, a major cause of antibiotic-associated diarrhea and pseudomembranous colitis, is increasingly resistant to antibiotics and poses a significant threat due to its regulated virulence. The alternative sigma factor σ 54 (rpoN) is known to regulate gene expression broadly, affecting microbial adaptation. Our study investigates how rpoN influences gene expression, physiology, and virulence in C. difficile. We used a modified CRISPR-Cpf1 system to create a rpoN deletion strain (∆rpoN) and a complemented strain (::rpoN) in the CD630 background, comparing their phenotypes and transcriptomes with the wild type. The ∆rpoN strain showed reduced motility and increased susceptibility to seven antibiotics, including β-lactams (amoxicillin, ampicillin, cefoxitin), nitroimidazoles (metronidazole), glycopeptides (vancomycin), fluoroquinolones (norfloxacin), and aminoglycosides (kanamycin). It also exhibited increased toxin gene expression, higher autolysis rates, and enhanced cytotoxicity and virulence in animal models. Additionally, rpoN deletion led to a decrease in glucose metabolic rate, which we attribute to the downregulation of glycolytic enzymes. Transcriptomic analysis indicated that reduced motility in ∆rpoN is due to downregulation of flagellar biosynthesis genes, while increased autolysis is linked to upregulation of autolysin genes like cwp19 and acd. The enhanced release of toxins due to higher autolysis rates contributes to the increased virulence of ∆rpoN. Our findings establish rpoN as a global regulator critical for antibiotic resistance, motility, metabolism, toxin production, and pathogenicity in C. difficile, suggesting its potential as a therapeutic target to mitigate virulence and resistance.
Keywords: Clostridioides difficile, sigma-54, RpoN, motility, toxin production, Pathogenesis
Received: 01 Feb 2025; Accepted: 09 Apr 2025.
Copyright: © 2025 Yang, Huang, Yang, Shao, Shu, Ling, Lu, Ma, Liao, Guan, Qi, Cui and Hong. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Wei Hong, Guizhou Medical University, Guiyang, China
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