ADP-ribosylation and subcellular redistribution of Rac1 by Exoenzyme S. (South Carolina Academy of Sciences Abstracts).

Exoenzyme S (ExoS) is a bifunctional protein secreted by Pseudomonas aeruginosa via the bacterial type III secretory (TTS) process. ExoS includes a GTPase activating (GAP) domain located in the amino-terminus that affects Rho GTPases involved in cytoskeletal regulation. The carboxy-terminus of ExoS functions as an ADP-ribosyltransferase (ADPRT), which has a somewhat diverse specificity, but preferentially targets proteins in the Ras and Rab families. The TTS mediated translocation of ExoS into human epithelial cells affects cell growth, morphology, and adherence. The question being asked in these studies is whether Rho family proteins can function as targets of US translocated ExoS ADPRT activity, contributing to the permanent effects of ExoS on cell morphology. ADPRT substrate modification can be assessed in eukaryotic cells based on a differential shift in protein mass following the co-culture of cells with ExoS producing strain 388 or the ExoS null strain, 388[DELTA]S. No evidence was obtained in these analyses that RhoA, B and C were substrates of type III translocated ExoS ADPRT activity. However, a shift in the mobility of Raci was detected following exposure to ExoS producing bacteria, which was confirmed by two-dimensional electrophoresis to be consistent with ADP-ribosylation. Raci was also found to localize to the membrane compartment in response to ExoS, suggesting that Rac1 is activated following ADP-ribosylation by ExoS. Bacterial translocation of ExoS having mutated GAP or ADPRT activities, confirmed the requirement of ExoS ADPRT activity for Rac1 modification and membrane localization. To further assess whether Rac1 was activated in response to ExoS, Raci interaction with its downstream effector, p21 activated kinase (PAK), was determined using in vitro pulldown assays along with in vivo co-precipitation (pulldown) assays which confirmed the ability of ADPribosylated Rac1 to interact with PAK. Following co-culture of epithelial cells with ExoS producing bacteria, an increase in active Rac1 was detected confirming the ability of type III translocated ExoS to cause Rac1 activation. Arg66, Arg68 and Arg163 in Rac1 were identified as potential sites of ADP-ribosylation by ExoS through the sequential and/or combined mutation of the nine arginine residues in Rac 1, coupled with in vitro ExoS ADPRT reactions. Transfection of HA tagged R66K and R68K into epithelial cells identified Arg66, in the GAP domain of Rac1, as the preferred site of ADP-ribosylation by bacterially translocated ExoS. We conclude from these studies that bacterially translocated ExoS causes ADP-ribosylation mediated activation of Rac1 and appears to supemede ExoS GAP mediated inactivation of Rac1.

Deanne M. Greene, Timothy S. Vincent, Joan C. Olson
Department of Microbiology, Immunology, and Cell Biology
West Virginia University Morgantown, WV, EPSCoR