Interruption of multiple cellular processes in HT-29 epithelial cells by Pseudomonas aeruginosa exoenzyme S

Exoenzyme S (ExoS), an ADP-ribosylating enzyme produced by the opportunisti c 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 alteratio ns in cytoskeletal structure. To further understand the interrelationship o f the different cellular effects of ExoS, functional analyses were performe d on HT-29 epithelial cells after exposure to ExoS-producing P. aeruginosa 388 and the non-ExoS-producing strain 388 Delta 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 388 Delta S a nd (ii) a more-severe, long-term cell rounding caused by ExoS-producing str ain 388. Long-term effects of ExoS on cell morphology occurred in conjuncti on with ExoS-mediated inhibition of DNA synthesis and the ADP-ribosylation of pas. ExoS was also found to cause alterations in HT-29 cell function, le ading to the loss of cell adhesion and microvillus effacement. Nonadherent ExoS-treated cells remained viable but had a high proportion of modified Ra s. While microvillus effacement was detected in both 388- and 388 Delta S-t reated cells, effacement was more prevalent and rapid in cells exposed to s train 388. We conclude from these studies that ExoS can have multiple effec ts on epithelial cell function, with more severe cellular alterations assoc iated with the enzymatic modification of Ras. The finding that ExoS had gre ater effects on cell growth and adherence than on cell viability suggests t hat ExoS may contribute to the P. aeruginosa infectious process by renderin g cells nonfunctional.