Auto-ADP-ribosylation of Pseudomonas aeruginosa ExoS.
Pseudomonas aeruginosa Exoenzyme S (ExoS) is a bifunctional type-III cytotoxin. The N terminus possesses a Rho GTPase-activating protein (GAP) activity, whereas the C terminus comprises an ADP-ribosyltransferase domain. We investigated whether the ADP-ribosyltransferase activity of ExoS influences its GAP activity. Although the ADP-ribosyltransferase activity of ExoS is dependent upon FAS, a 14-3-3 family protein, factor-activating ExoS (FAS) had no influence on the activity of the GAP domain of ExoS (ExoS-GAP). In the presence of NAD and FAS, the GAP activity of full-length ExoS was reduced about 10-fold, whereas NAD and FAS did not affect the activity of the ExoS-GAP fragment. Using [(32)P]NAD, ExoS-GAP was identified as a substrate of the ADP-ribosyltransferase activity of ExoS. Site-directed mutagenesis revealed that auto-ADP-ribosylation of Arg-146 of ExoS was crucial for inhibition of GAP activity in vitro. To reveal the auto-ADP-ribosylation of ExoS in intact cells, tetanolysin was used to produce pores in the plasma membrane of Chinese hamster ovary (CHO) cells to allow the intracellular entry of [(32)P]NAD, the substrate for ADP-ribosylation. After a 3-h infection of CHO cells with Pseudomonas aeruginosa, proteins of 50 and 25 kDa were preferentially ADP-ribosylated. The 50-kDa protein was determined to be auto-ADP-ribosylated ExoS, whereas the 25-kDa protein appeared to represent a group of proteins that included Ras.
Riese
Matthew J
MJ
Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Goehring
Udo-Michael
UM
Ehrmantraut
Mary E
ME
Moss
Joel
J
Barbieri
Joseph T
JT
Aktories
Klaus
K
Schmidt
Gudula
G
D036002:ADP Ribose Transferases / Q000378:metabolism*; D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001427:Bacterial Toxins; D015153:Blotting, Western; D016466:CHO Cells; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005091:Exons; D008168:Lung / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013744:Tetanus Toxin / Q000494:pharmacology; D013997:Time Factors; D018631:ras Proteins / Q000378:metabolism
D001427:Bacterial Toxins; D011994:Recombinant Proteins; D013744:Tetanus Toxin; D000246:Adenosine Diphosphate Ribose; C026167:tetanolysin; D001120:Arginine; D036002:ADP Ribose Transferases; D011065:Poly(ADP-ribose) Polymerases; C444820:exoenzyme S; D020727:ADP-Ribosylation Factors; D018631:ras Proteins