MODIFICATION OF RAS IN EUKARYOTIC CELLS BY PSEUDOMONAS-AERUGINOSA EXOENZYME-S

Genetic and functional data suggest that Pseudomonas aeruginosa exoenz yme S (ExoS), an ADP-ribosyltransferase, is translocated into eukaryot ic cells by a bacterial type III secretory mechanism activated by cont act between bacteria and host cells. Although purified ExoS is not tox ic to eukaryotic cells, ExoS-producing bacteria cause reduced prolifer ation and viability, possibly mediated by bacterially translocated Exo S. To investigate the activity of translocated ExoS, we examined in vi vo modification of Ras, a preferred in vitro substrate. The ExoS-produ cing strain P. aeruginosa 388 and an isogenic mutant strain, 388 Delta exoS, which fails to produce ExoS, were cocultured with HT29 colon ca rcinoma cells. Ras was found to be ADP-ribosylated during coculture wi th 388 bat not with 388 Delta exoS, and Ras modification by 388 corres ponded 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 Delta exoS, was unable to modify intracellular Ras. Other ExoS-producing strains caused modification of Ras, indicati ng that this is not a strain-specific event. ADP-ribosylation of Rap1, an additional Ras family substrate for ExoS in vitro, was not detecta ble in vivo under conditions sufficient for Ras modification, suggesti ng possible ExoS substrate preference among Ras-related proteins. Thes e results confirm that intracellular Ras is modified by bacterially tr anslocated ExoS and that the inhibition of target cell proliferation c orrelates with the efficiency of Ras modification.