Intracellular localization and processing of Pseudomonas aeruginosa ExoS in eukaryotic cells

ExoS is a type III cytotoxin of Pseudomonas aeruginosa, which modulates two eukaryotic signalling pathways. The N-terminus (residues 1-234) is a GTPas e activating protein (GAP) for RhoGTPases, while the C-terminus (residues 2 32-453) encodes an ADP-ribosyltransferase. Utilizing a series of N-terminal deletion peptides of ExoS and an epitope-tagged full-length ExoS, two inde pendent domains have been identified within the N-terminus of ExoS that are involved in intracellular localization and expression of GAP activity. N-t erminal peptides of ExoS localized to the perinuclear region of CHO cells, and a membrane localization domain was localized between residues 36 and 78 of ExoS. The capacity to elicit CHO cell rounding and express GAP activity resided within residues 90-234 of ExoS, which showed that membrane localiz ation was not required to elicit actin reorganization. ExoS was present in CHO cells as a full-length form, which fractionated with membranes, and as an N-terminally processed fragment, which localized to the cytosol. Thus, E xoS localizes in eukaryotic cells to the perinuclear region and is processe d to a soluble fragment, which possesses both the GAP and ADP-ribosyltransf erase activities.