Orf1/SpcS chaperones ExoS for type three secretion by Pseudomonas aeruginosa.
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 cell's 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 Orf1.


METHODS
By allelic exchange, we constructed the mutant with the deletion of gene Orf1. Analysis of secreted and cell-associated fractions was performed by SDS-PAGE and Western blotting. Using strain expressing in trans Orf1, 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 Orf1 in the expression of exoS was evaluated by gene reporter analysis.


RESULTS
Pull-down assay showed that Orf1 binds to ExoS and ExoT. Secretion profile analysis showed that Orf1 was necessary for the optimal secretion of ExoS and ExoT. However, Orf1 had no effect on the expression of exoS.


CONCLUSIONS
Orf1 is important for the secretion of ExoS probably by maintaining ExoS in a secretion-competent conformation. We propose to name Orf1 as SpcS for "specific Pseudomonas chaperone for ExoS".
Groupe de Recherche et d'Etude du Processus Inflammatoire, MENRT EA 2938, Centre Hospitalier Universitaire de Grenoble, BP 217, 38043 Grenoble Cedex 9, France.
Da-Kang
DK
Shen
Lauriane
L
Quenee
Mariette
M
Bonnet
Lauriane
L
Kuhn
Madiha
M
Derouazi
Daniele
D
Lamotte
Bertrand
B
Toussaint
Benoit
B
Polack
D036002:ADP Ribose Transferases / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001427:Bacterial Toxins; D017931:DNA Primers; D018832:Molecular Chaperones; D036002:ADP Ribose Transferases; C444820:exoenzyme S