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Objective Pseudomonas aeruginosa is a ubiquitous and opportunistic pathogen that uses the type III secretion system (TTSS)to inject effector proteins directly into the cytosol of target cells to subvert the host cells functions.Specialized bacterial chaperones are required for effective secretion of some effectors.To identify the chaperone of ExoS,the representative effector secreted by the TTSS of P. aeruginosa,we analyzed the role of a postulated chaperone termed Orfl.Methods By allelic exchange,we constructed the mutant with the deletion of gene Orfl.Analysis of secreted and cell-associated fractions was performed by SDS-PAGE and Western blotting.Using strain expressing in trans Orfl,tagged by V5 polypeptide and histidine,protein-protein interaction Was determined by affinity resin pull-down assay in combination with MALDI-TOF. The role of Orfl in the expression of eroS was evaluated by genel reporter analysis.Results Pull-down assay showed that Orfl binds to ExoS and ExoT.Secretion profile analysis showedthat Orfl was necessary for the optimal secretion of ExoS and ExoT.However,Orfl had no effect on the expression of eroS.Conclusion Orfl is important for the secretion of ExoS probablY by mamtauung ExoS in a secretion-competent conformation.We propose to name Orfl as SpcS for "specific Pseudomonas chaperone for ExoS".