ADP-ribosylation of oncogenic Ras proteins by Pseudomonas aeruginosa exoenzyme S in vivo

The exoenzyme S (ExoS)-producing Pseudomonas aeruginosa strain, 388, and co rresponding ExoS knock-out strain, 388 Delta exoS, were used in a bacterial and mammalian co-culture system as a model for the contact-dependent deliv ery of ExoS into host cells. Examination of DNA synthesis and Ras ADP-ribos ylation in tumour cell lines expressing normal and mutant Ras revealed a de crease in DNA synthesis concomitant with ADP-ribosylation of Ras proteins a fter exposure to ExoS-producing bacteria, but not after exposure to non-Exo S-producing bacteria. Examination of normal H-Ras, K-Ras and N-Ras by two-d imensional electrophoresis after exposure to bacteria revealed differences in the degree of ADP-ribosylation by ExoS, with H-Ras being modified most e xtensively. ADP-ribosylation of oncogenic forms of Ras was examined in vivo using cancer lines expressing mutant forms of H-, N- or K-Ras. The mutant Ras proteins were modified in a manner qualitatively similar to their norma l counterparts. Using Ras/Raf-1 co-immunoprecipitation after co-culture, it was found that exposure to ExoS-producing;ng bacteria caused a decrease in the amount of Raf-l associated with EGF-activated Ras and oncogenic Res. T he results from this study indicate that ExoS ADP-ribosylates both normal a nd mutant Ras proteins in vivo and inhibits signalling through Ras.