Characterization of Pseudomonas aeruginosa exoenzyme S as a bifunctional enzyme in J774A.1 macrophages.
Pseudomonas aeruginosa exoenzyme S (ExoS) is a type III secretion (TTS) effector, which includes both a GTPase-activating protein (GAP) activity toward the Rho family of low-molecular-weight G (LMWG) proteins and an ADP-ribosyltransferase (ADPRT) activity that targets LMWG proteins in the Ras, Rab, and Rho families. The coordinate function of both activities of ExoS in J774A.1 macrophages was assessed by using P. aeruginosa strains expressing and translocating wild-type ExoS or ExoS defective in GAP and/or ADPRT activity. Distinct and coordinated functions were identified for both domains. The GAP activity was required for the antiphagocytic effect of ExoS and was linked to interference of lamellopodium and membrane ruffle formation. Alternatively, the ADPRT activity of ExoS altered cellular adherence and morphology and was linked to effects on filopodium formation. The cellular mechanism of ExoS GAP activity included an inactivation of Rac1 function, as determined in p21-activated kinase 1-glutathione S-transferase (GST) pull-down assays. The ADPRT activity of ExoS targeted Ras and RalA but not Rab or Rho proteins, and Ral binding protein 1-GST pull-down assays identified an effect of ExoS ADPRT activity on RalA activation. The results from these studies confirm the bifunctional nature of ExoS activity within macrophages when translocated by TTS.
Rocha
Claudia L
CL
Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
Coburn
Jenifer
J
Rucks
Elizabeth A
EA
Olson
Joan C
JC
D036002:ADP Ribose Transferases / Q000502:physiology* / Q000633:toxicity; D000818:Animals; D001427:Bacterial Toxins / Q000633:toxicity; D001693:Biological Transport, Active; D002460:Cell Line; D020558:GTP Phosphohydrolases / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000502:physiology / Q000633:toxicity; D008264:Macrophages / Q000187:drug effects / Q000276:immunology* / Q000382:microbiology / Q000648:ultrastructure; D051379:Mice; D008855:Microscopy, Electron, Scanning; D010587:Phagocytosis; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000472:pathogenicity*; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020725:ral GTP-Binding Proteins
D001427:Bacterial Toxins; D020690:GTPase-Activating Proteins; C085379:rho GTPase-activating protein; D036002:ADP Ribose Transferases; C444820:exoenzyme S; D020558:GTP Phosphohydrolases; C118120:Rala protein, mouse; D020830:rac1 GTP-Binding Protein; D020725:ral GTP-Binding Proteins