The Nucleoid Proteins Fis and IHF Positively Regulate the Gene Expression of Operons Responsible for Producing the Cytotoxins Tilimycin and Tilivalline in Klebsiella oxytoca

Introduction Klebsiella oxytoca causes antibiotic‐associated hemorrhagic colitis due to the production of the enterotoxins tilimycin and tilivalline. These toxins are synthesized by enzymes encoded in the aroX and NRPS operons, which are expressed divergently. This study investigated how the nucleoid‐associated proteins Fis and IHF regulate these operons and influence the production of enterotoxins. Methods We used reverse transcription quantitative PCR (RT‐qPCR) to assess the role of Fis and IHF in the transcription of the aroX and NRPS operons. Electrophoretic mobility shift assays (EMSAs) were used to examine the binding of Fis and IHF to the regulatory region. Additionally, Caco‐2 viability assays were performed using cells infected with WT, mutant, and complemented strains. Results RT‐qPCR demonstrated that deletions of fis or ihfA / ihfB significantly reduced operon expression. EMSA confirmed that Fis and IHF bind specifically to the regulatory region between the aroX and NRPS operons. Viability assays in Caco‐2 epithelial cells indicated increased host cell survival when exposed to the deletion mutants. Genetic complementation restored both transcription levels and cytotoxicity. Conclusions Fis and IHF are positive regulators of the aroX and NRPS operons, enhancing the production of tilimycin and tilivalline. These findings highlight the potential of targeting Fis and IHF for therapeutic intervention in antibiotic‐associated hemorrhagic colitis.