The EnvZ/OmpR two-component regulatory system regulates biofilm formation in Salmonella pullorum 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 Salmonella pullorum (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 ompR deletion mutant was constructed using Red homologous recombination. Deletion of ompR 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 soxR (a transcriptional activator of AcrAB-TolC), but not to soxS . 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 soxR promoter and that AI-2 potentially interacts with EnvZ. These findings offer new mechanistic insights specific to Salmonella pullorum -biofilm (SP-BF) regulation and provide potential targets for biofilm control strategies.