PA5201 represses type III secretion system by binding to the P exsC promoter in Pseudomonas aeruginosa

The type III secretion system (T3SS) is a major virulence determinant in Pseudomonas aeruginosa , which is activated by the master regulator ExsA. exsA is transcribed from two distinct promoters: its operon promoter P exsC and its own promoter P exsA . Here, we identify PA5201, a Tex-family protein, as a novel repressor of T3SS in P. aeruginosa . PA5201 directly binds to the P exsC promoter and represses its activity. Deletion of PA5201 increased T3SS expression, bacterial cytotoxicity, and bacterial load in a mouse acute pneumonia model, whereas overexpression of PA5201 decreased T3SS expression. Reporter assays and western blot analysis further revealed that PA5201-mediated repression of P exsC is independent of ExsA and does not occur at the post-transcriptional level. Electrophoretic mobility shift assay (EMSA) showed that PA5201 binds to the region between −72 and −45 bp relative to the exsC start codon. Collectively, this study identifies PA5201 as a novel T3SS repressor and elucidates its molecular mechanism in P. aeruginosa . IMPORTANCE Pseudomonas aeruginosa is an important opportunistic human pathogen responsible for a wide range of infections, particularly in immunocompromised individuals. The type III secretion system (T3SS) plays a critical role in acute P. aeruginosa infections. Here, we identify PA5201, a previously uncharacterized Tex-family regulator, as a novel transcriptional repressor of T3SS. We demonstrate that PA5201 directly binds to and represses the P exsC promoter. This study reveals a new regulator of T3SS in P. aeruginosa and clarifies the molecular mechanism of PA5201-mediated T3SS regulation.