Abstract:[Objective] In view of the enhanced cell-to-cell spread ability of the dsbA-deleted strain (ΔdsbA) of Listeria monocytogenes, this study aims to elucidate the mechanism that how the disulfide bond formation protein DsbA mediates this biological process. [Methods] The mRNA and protein levels of virulence factors in the wild type and ΔdsbA were compared by RT-qPCR and Western blotting, respectively. The immunofluorescence co-localization analysis method was employed to observe the impact of DsbA deficiency on the actin recruitment by the virulence factor ActA in the cell-to-cell spread of L. monocytogenes (analyzing the length and quantity of the comet tails formed on one side of the bacteria by co-localization of ActA and actin). The presence or absence of interaction between DsbA and ActA was determined by isothermal titration calorimetry (ITC). [Results] Compared with the wild type, ΔdsbA showed no significant changes in the mRNA levels of virulence factors, downregulated protein levels of InlA, InlB, PlcA, and PlcB, and upregulated protein levels of ActA and LLO. In addition, ΔdsbA showed increased number and average length of comet tails, which indicated that the actin recruitment of ΔdsbA was enhanced. The ITC results revealed that DsbA bound to ActA, which gradually showed endothermic reactions, suggesting the presence of interaction between DsbA and ActA. [Conclusion] This study proved for that DsbA attenuated the recruitment ability of actin by regulating virulence proteins, thus affecting the cell-to-cell spread of L. monocytogenes. The findings help to further dissect the virulence regulatory mechanisms of L. monocytogenes during host infection, which is of great importance for controlling the contamination of zoonotic intracellular pathogens threatening public health.