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Infection and Immunity, September 2003, p. 5296-5305, Vol. 71, No. 9
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.9.5296-5305.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Characterization of Pseudomonas aeruginosa Exoenzyme S as a Bifunctional Enzyme in J774A.1 Macrophages

Claudia L. Rocha,1 Jenifer Coburn,2 Elizabeth A. Rucks,1,{dagger} and Joan C. Olson1*

Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, South Carolina 29425,1 Division of Geographic Medicine and Infectious Diseases, Tufts-New England Medical Center, Boston, Massachusetts 021112

Received 24 February 2003/ Returned for modification 1 April 2003/ Accepted 4 June 2003

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.

* Corresponding author. Present address: Department of Microbiology, Immunology and Cell Biology, West Virginia University, 2095 Health Sciences North, Morgantown, WV 26506-9177. Phone: (304) 293-2649. Fax: (304) 293-5843. E-mail: jolson{at}hsc.wvu.edu.

Editor: D. L. Burns

{dagger} Present address: Department of Microbiology, Immunology and Cell Biology, West Virginia University, Morgantown, WV 26506-9177.

Infection and Immunity, September 2003, p. 5296-5305, Vol. 71, No. 9
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.9.5296-5305.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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