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Infection and Immunity, December 2000, p. 7100-7113, Vol. 68, No. 12
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

The Arginine Finger Domain of ExoT Contributes to Actin Cytoskeleton Disruption and Inhibition of Internalization of Pseudomonas aeruginosa by Epithelial Cells and Macrophages

L. Garrity-Ryan, B. Kazmierczak, R. Kowal, J. Comolli,dagger A. Hauser,Dagger and J. N. Engel*

Department of Medicine, Department of Microbiology and Immunology, and the Cardiovascular Research Institute, University of California, San Francisco, San Francisco, California 94143

Received 1 June 2000/Returned for modification 12 July 2000/Accepted 22 September 2000

Pseudomonas aeruginosa, an important nosocomial pathogen of humans, expresses a type III secretion system that is required for virulence. Previous studies demonstrated that the lung-virulent strain PA103 has the capacity to be either cytotoxic or invasive. Analyses of mutants suggest that PA103 delivers a negative regulator of invasion, or anti-internalization factor, to host cells via a type III secretion system. In this work we show that the type III secreted protein ExoT inhibits the internalization of PA103 by polarized epithelial cells (Madin-Darby canine kidney cells) and J774.1 macrophage-like cells. ExoS, which is closely related to ExoT but has additional ADP-ribosylating activity, can substitute for ExoT as an anti-internalization factor. ExoT contains a signature arginine finger domain found in GTPase-activating proteins. Mutation of the conserved arginine in ExoT diminished its anti-internalization activity and altered its ability to disrupt the actin cytoskeleton. Cell fractionation experiments showed that ExoT is translocated into host cells and that mutation of the arginine finger did not disrupt translocation. In a mouse model of acute pneumonia, PA103Delta UDelta T reached the lungs as efficiently as PA103Delta U but showed reduced colonization of the liver. This finding suggests that the ability to resist internalization may be important for virulence in vivo.

* Corresponding author. Mailing address: Division of Infectious Disease, Box 0654, University of California, San Francisco, CA 94143-0654. Phone: (415) 476-7355. Fax: (415) 476-9364. E-mail: Jengel{at}medicine.ucsf.edu.

dagger Present address: Department of Bacteriology, University of Wisconsin---Madison, Madison, WI 53706.

Dagger Present address: Department of Microbiology and Immunology, Northwestern University, Chicago, IL 60611.

Infection and Immunity, December 2000, p. 7100-7113, Vol. 68, No. 12
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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