Eileen M. McGuffie

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

The exoenzyme S (ExoS)‐producing Pseudomonas aeruginosa strain, 388, and corresponding ExoS knock‐out strain, 388ΔexoS, were used in a bacterial and mammalian co‐culture system as a model for the contact‐dependent delivery of ExoS into host cells. Examination of DNA synthesis and Ras ADP‐ribosylation in tumour cell lines expressing normal and mutant Ras revealed a decrease in DNA synthesis concomitant with ADP‐ribosylation of Ras proteins after exposure to ExoS‐producing bacteria, but not after exposure to non‐ExoS‐producing bacteria. Examination of normal H‐Ras, K‐Ras and N‐Ras by two‐dimensional electrophoresis after exposure to bacteria revealed differences in the degree of ADP‐ribosylation by ExoS, with H‐Ras being modified most extensively. 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 normal counterparts. Using Ras/Raf‐1 co‐immunoprecipitation after co‐culture, it was found that exposure to ExoS‐producing bacteria caused a decrease in the amount of Raf‐1 associated with EGF‐activated Ras and oncogenic Ras. The results from this study indicate that ExoS ADP‐ribosylates both normal and mutant Ras proteins in vivo and inhibits signalling through Ras.