Infect Immun, June 1998, p. 2607-2613, Vol. 66, No. 6
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, South Carolina 29425,1 and Department of Microbiology, Medical College of Wisconsin, Milwaukee, Wisconsin 532262
Received 4 December 1997/Returned for modification 20 February 1998/Accepted 31 March 1998
Genetic and functional data suggest that Pseudomonas
aeruginosa exoenzyme S (ExoS), an ADP-ribosyltransferase, is
translocated into eukaryotic cells by a bacterial type III secretory
mechanism activated by contact between bacteria and host cells.
Although purified ExoS is not toxic to eukaryotic cells, ExoS-producing bacteria cause reduced proliferation and viability, possibly mediated by bacterially translocated ExoS. To investigate the activity of
translocated ExoS, we examined in vivo modification of Ras, a preferred
in vitro substrate. The ExoS-producing strain P. aeruginosa 388 and an isogenic mutant strain, 388
exoS, which fails
to produce ExoS, were cocultured with HT29 colon carcinoma cells. Ras
was found to be ADP-ribosylated during coculture with 388 but not with
388
exoS, and Ras modification by 388 corresponded with
reduction in HT29 cell DNA synthesis. Active translocation by bacteria
was found to be required, since exogenous ExoS, alone or in the
presence of 388
exoS, was unable to modify intracellular
Ras. Other ExoS-producing strains caused modification of Ras,
indicating that this is not a strain-specific event. ADP-ribosylation
of Rap1, an additional Ras family substrate for ExoS in vitro, was not
detectable in vivo under conditions sufficient for Ras modification,
suggesting possible ExoS substrate preference among Ras-related
proteins. These results confirm that intracellular Ras is modified by
bacterially translocated ExoS and that the inhibition of target cell
proliferation correlates with the efficiency of Ras modification.
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