The EnvZ/OmpR two-component regulatory system regulates biofilm formation in <i>Salmonella pullorum</i> via interaction with the LuxS/AI-2 quorum sensing system and activation of the SoxR-AcrAB-TolC pathway

To investigate the mechanisms of biofilm (BF) formation in <i>Salmonella pullorum</i> (SP), a strong BF-forming strain designated 1904D10 was selected. Differential proteomics based on tandem mass tag (TMT) labeling was performed to compare the whole bacterial proteome between planktonic and biofilm states. A total of 219 differentially expressed proteins were identified (96 up-regulated, 123 down-regulated in biofilm state). Key up-regulated proteins included those involved in the EnvZ/OmpR two-component system (TCS), LuxS/AI-2 quorum sensing (QS) system, and AcrAB-TolC efflux pump. An <i>ompR</i> deletion mutant was constructed using Red homologous recombination. Deletion of <i>ompR</i> significantly reduced biofilm formation and significantly increased susceptibility to all tested antibiotics. Electrophoretic mobility shift assay (EMSA) demonstrated that OmpR protein directly binds to the promoter region of <i>soxR</i> (a transcriptional activator of AcrAB-TolC), but not to <i>soxS</i>. Molecular docking predicted a potential interaction between the AI-2 QS signaling molecule and EnvZ protein (binding energy: -23.99 kJ/mol), with hydrogen bonds forming at five amino acid residues. This study provides evidence that OmpR directly binds to the <i>soxR</i> promoter and that AI-2 potentially interacts with EnvZ. These findings offer new mechanistic insights specific to <i>Salmonella pullorum</i>-biofilm (SP-BF) regulation and provide potential targets for biofilm control strategies.