Abstract:[Objective] To study the effect of type III secretion system (TTSS) inhibitors on virulence factors, TTSS, bacterial flagella and pilin, of Pseudomonas aeruginosa PAO1. [Methods] The transcriptional reporters of exoT and exoY genes were used to monitor the exoT and exoY expression in the wild type strain PAO1 supplemented with TTSS inhibitors. The protein level of FliC was analyzed by SDS-PAGE. The twitching motility mediated by bacterial pilin was examined on the BM2 medium. [Results] Transcriptional fusion assay indicated that the expression of exoT and exoY was decreased when the TTSS inhibitors were supplemented. Although the production of PcrV was not changed in the cells when compounds TS52, TS53 and TS94 were added in the medium, the translocation of PcrV protein in the medium was decreased. The level of flagella structural protein FliC was decreased when the compound TS53 was supplemented in the medium. In addition, compounds TS52, TS53 and TS88 inhibited the ability of twitching motility, whereas TS94 increased the twitching motility. [Conclusion] Besides the TTSS effector genes, TTSS inhibitors influence other virulence factors, such as flagella and type IV pilus-dependent twitching motility. These data will provide the theoretical basis for further clinical trials.