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Infection and Immunity, June 1999, p. 2847-2854, Vol. 67, No. 6
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Interruption of Multiple Cellular Processes in HT-29 Epithelial Cells by Pseudomonas aeruginosa Exoenzyme S

Joan C. Olson,1,* Jennifer E. Fraylick,1 Eileen M. McGuffie,2 Katherine M. Dolan,1 Timothy L. Yahr,3 Dara W. Frank,4 and Timothy S. Vincent1

Department of Pathology and Laboratory Medicine1 and Department of Experimental Oncology,2 Medical University of South Carolina, Charleston, South Carolina 29425; Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 037553; and Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 532264

Received 7 December 1998/Returned for modification 29 January 1999/Accepted 3 March 1999

Exoenzyme S (ExoS), an ADP-ribosylating enzyme produced by the opportunistic pathogen Pseudomonas aeruginosa, is directly translocated into eukaryotic cells by bacterial contact. Within the cell, ExoS ADP-ribosylates the cell signaling protein Ras and causes inhibition of DNA synthesis and alterations in cytoskeletal structure. To further understand the interrelationship of the different cellular effects of ExoS, functional analyses were performed on HT-29 epithelial cells after exposure to ExoS-producing P. aeruginosa 388 and the non-ExoS-producing strain 388Delta S. Two different mechanisms of morphological alteration were identified: (i) a more-transient and less-severe cell rounding caused by the non-ExoS-producing strain 388Delta S and (ii) a more-severe, long-term cell rounding caused by ExoS-producing strain 388. Long-term effects of ExoS on cell morphology occurred in conjunction with ExoS-mediated inhibition of DNA synthesis and the ADP-ribosylation of Ras. ExoS was also found to cause alterations in HT-29 cell function, leading to the loss of cell adhesion and microvillus effacement. Nonadherent ExoS-treated cells remained viable but had a high proportion of modified Ras. While microvillus effacement was detected in both 388- and 388Delta S-treated cells, effacement was more prevalent and rapid in cells exposed to strain 388. We conclude from these studies that ExoS can have multiple effects on epithelial cell function, with more severe cellular alterations associated with the enzymatic modification of Ras. The finding that ExoS had greater effects on cell growth and adherence than on cell viability suggests that ExoS may contribute to the P. aeruginosa infectious process by rendering cells nonfunctional.

* Corresponding author. Mailing address: Medical University of South Carolina, Department of Pathology and Laboratory Medicine, 165 Ashley Ave., Charleston, SC 29425. Phone: (843) 792-7761. Fax: (843) 792-4157. E-mail: olsonj{at}musc.edu.

Infection and Immunity, June 1999, p. 2847-2854, Vol. 67, No. 6
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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