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Pseudomonas aeruginosautilizes a type III secretion system (T3SS) to directly inject effector proteins into host cells, leading to severe acute infections. Here, we present a protocol for detecting the T3SS-mediated cytotoxicity of P. aeruginosausing the A549 cell line. We describe the steps for the preparation of the A549 cell line and P. aeruginosastrains, cell seeding, bacterial culture, infection, and cytotoxicity assay. Additionally, we provide detailed procedures for data analysis.

Quorum sensing (QS) is a cell-to-cell communication mechanism mediated by small diffusible signaling molecules. Previous studies showed that RpfR controls Burkholderia cenocepaciavirulence as a cis-2-dodecenoic acid (BDSF) QS signal receptor. Here, we report that the fatty acyl-CoA ligase DsfR (BCAM2136), which efficiently catalyzes in vitrosynthesis of lauryl-CoA and oleoyl-CoA from lauric acid and oleic acid, respectively, acts as a global transcriptional regulator to control B. cenocepaciavirulence by sensing BDSF. We show that BDSF binds to DsfR with high affinity and enhances the binding of DsfR to the promoter DNA regions of target genes. Furthermore, we demonstrate that the homolog of DsfR in B. lata, RS02960, binds to the target gene promoter, and perception of BDSF enhances the binding activity of RS02960. Together, these results provide insights into the evolved unusual functions of DsfR that control bacterial virulence as a response regulator of QS signal.