Intracellular localization of type III-delivered Pseudomonas ExoS with endosome vesicles.
ExoS (453 amino acids) is a bi-functional type III cytotoxin produced by Pseudomonas aeruginosa. Residues 96-219 include the Rho GTPase-activating protein (RhoGAP) domain, and residues 234-453 include the 14-3-3-dependent ADP-ribosyltransferase domain. Earlier studies also identified an N-terminal domain (termed the membrane localization domain) that comprises residues 51-77 and includes a novel leucine-rich motif that targets ExoS to the perinuclear region of cultured cells. There is limited information on how ExoS or other type III cytotoxins enter and target intracellular host proteins. Type III-delivered ExoS localized to both plasma membrane and perinuclear region, whereas ExoS(DeltaMLD) was localized to the cytosol. Plasma membrane localization of ExoS was transient and had a half-life of approximately 20 min. Type III-delivered ExoS co-immunoprecipitated 14-3-3 proteins and Rab9, Rab6, and Rab5. Immunofluorescence experiments showed that ExoS colocalized with Rab9, Rab6, and Rab5. Fluorescent energy transfer was detected between ExoS and 14-3-3 proteins but not between ExoS and Rabs proteins. Together, these results indicate that type III-delivered ExoS localizes on the host endosomes and utilizes multiple pathways to traffic from the plasma membrane to the perinuclear region of intoxicated host cells.
Zhang
Yue
Y
Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Deng
Qing
Q
Barbieri
Joseph T
JT
D048948:14-3-3 Proteins / Q000378:metabolism; D036002:ADP Ribose Transferases / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D011485:Protein Binding; D021381:Protein Transport; D011550:Pseudomonas aeruginosa / Q000472:pathogenicity*; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism
D048948:14-3-3 Proteins; D001427:Bacterial Toxins; D020690:GTPase-Activating Proteins; C094135:Rab6 protein; C085379:rho GTPase-activating protein; D036002:ADP Ribose Transferases; C444820:exoenzyme S; C079856:RAB9A protein, human; D020691:rab GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins