Indispensable role of AcrEF in modulating Salmonella virulence and disrupting host tight junctions to facilitate paracellular entry and invasion

Background: The Salmonella stress response regulator CpxR controls the AcrEF drug efflux pump, which is crucial for key virulence traits such as invasiveness and disruption of the host paracellular pathway. Understanding the role of acrEF and its regulation by CpxR can provide new insights into Salmonella pathogenesis and future therapeutic development. Methods: We performed differential gene expression analysis using Salmonella enterica serovar Typhimurium (ST) wild-type and cpxR mutants to identify virulence-associated genes affected by the stress response system. The candidate genes acrE and acrF were deleted and used to evaluate virulence-related phenotypes, including adhesion, invasion, and epithelial barrier disruption both in vitro and in vivo, in wild-type and mutant strains. Results: Deletion of acrEF in both ST wild-type and cpxR mutants significantly reduced Salmonella adhesion, invasion of multiple epithelial cell lines, and expression of virulence genes. It also led to enhanced tight junction integrity in epithelial cells, potentially via upregulation of genes like ZO-1, suggesting a novel invasion mechanism. The loss of acrEF function impaired the bacteria’s ability to breach host cell tight junctions, which directly correlated with attenuated invasion and survival in vivo. These effects were similarly observed in both wild-type and cpxR mutants, indicating a central role for acrEF in Salmonella virulence. Conclusion: The AcrEF efflux pump plays a key role in regulating Salmonella virulence, particularly in modulating tight junction disruption and epithelial invasion. Although CpxR may regulate acrEF expression, the loss of acrEF function independently results in significant attenuation of virulence. These findings reveal a critical pathway of Salmonella epithelial invasion mediated by the AcrEF system and regulated, in part, by the CpxR stress response regulator. Clinical trial number: Not applicable.