Protocol for examining the T3SS-mediated cytotoxicity of Pseudomonas aeruginosa using the A549 cell line

Summary Pseudomonas aeruginosa utilizes 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. aeruginosa using the A549 cell line. We describe the steps for the preparation of the A549 cell line and P. aeruginosa strains, cell seeding, bacterial culture, infection, and cytotoxicity assay. Additionally, we provide detailed procedures for data analysis. For complete details on the use and execution of this protocol, please refer to Huang et al. 1