Molecular heterogeneity of a type III cytotoxin, Pseudomonas aeruginosa exoenzyme S.
Pseudomonas aeruginosa ExoS is a bifunctional type III cytotoxin. The N-terminus (residues 1-232) is a Rho GTPase activating protein (GAP) domain, while the C-terminus (residues 233-453) is a FAS-dependent ADP-ribosyltransferase domain that targets Ras and Ras-like GTPases. A membrane localization domain (residues 51-72) localizes ExoS to a perinuclear region within eukaryotic cells. Recent studies observed that ExoS is auto-ADP-ribosylated upon delivery into eukaryotic cells. Auto-ADP-ribosylated ExoS analyzed from eukaryotic cells displayed pI heterogeneity and prompted an analysis of this heterogeneity. Bacterial-associated ExoS and ExoS that had been secreted by P. aeruginosa also showed pI heterogeneity with five charge forms ranging in pI from 5.1 to 5.9. The pI heterogeneity of ExoS was independent of a mass change and thus represented molecular charge conformers. Urea was not required to observe the pI conformers of ExoS; it enhanced the resolution and formation of pI conformers during the focusing component of the analysis. ExoS(E381D), a mutant deficient in ADP-ribosyltransferase activity, isolated from cultured cells showed charge forms that migrated to a more acidic pI than type III secreted ExoS but more basic than auto-ADP-ribosylated ExoS. Incubation of cell lysates with Mn(2+) shifted the pI of ExoS(E381D) to a pI identical to secreted ExoS. This indicates that within the mammalian cells ExoS undergoes a negatively charged modification, in addition to auto-ADP-ribosylation observed for wild-type ExoS. ExoT, ExoU, and YopE also focus into multiple pI forms, suggesting that this is a common property of type III cytotoxins.
Maresso
Anthony W
AW
Microbiology and Molecular Genetics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin 53226, USA.
Riese
Matthew J
MJ
Barbieri
Joseph T
JT
D036002:ADP Ribose Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics; D016466:CHO Cells; D002793:Cholic Acids; D006224:Cricetinae; D003470:Culture Media; D003603:Cytotoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000737:chemistry; D007424:Intracellular Fluid / Q000201:enzymology; D007526:Isoelectric Point; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014508:Urea
D001427:Bacterial Toxins; D002793:Cholic Acids; D003470:Culture Media; D003603:Cytotoxins; D020690:GTPase-Activating Proteins; D010446:Peptide Fragments; D014508:Urea; D036002:ADP Ribose Transferases; C444820:exoenzyme S; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate