Pseudomonas aeruginosa injects type III effector ExoS into epithelial cells through the function of type IV pili

•P. aeruginosa penetrates Caco-2 cell layers through the function of type IV pili (TFP).•TFP are not required for association of P. aeruginosa with Caco-2 cells.•TFP are required for disruption of Caco-2 cell layers by P. aeruginosa.•TFP are required for injection of ExoS into host cell by type III secretion system. Translocation of Pseudomonas aeruginosa through epithelial tissues can cause sepsis. Here, we examined whether P. aeruginosa penetrates epithelial cell layers using type IV pili (TFP). Deletion of TFP (pilA) did not affect association with Caco-2 cells, although it decreased penetration through, and disruption of, Caco-2 cell monolayers. We found that TFP are necessary for injection of the type III effector ExoS, which impairs defense against P. aeruginosa penetration, into host cells. Deletion of pilA attenuated oral infection in silkworms. We conclude that P. aeruginosa injects ExoS into cells through the function of TFP, enabling penetration of epithelial barriers.