title: Intracellular expression of the ADP-ribosyltransferase domain of Pseudomonas exoenzyme S is cytotoxic to eukaryotic cells.
abstract: Exoenzyme S of Pseudomonas aeruginosa is an ADP-ribosyltransferase, which is secreted via a type III-dependent secretion mechanism and has been demonstrated to exert cytotoxic effects on eukaryotic cells. Alignment studies predict that the amino-terminus of exoenzyme S has limited primary amino acid homology with the YopE cytotoxin of Yersinia, while biochemical studies have localized the FAS-dependent ADP-ribosyltransferase activity to the carboxyl-terminus. Thus, exoenzyme S could interfere with host cell physiology via several independent mechanisms. The goal of this study was to define the role of the ADP-ribosyltransferase domain in the modulation of eukaryotic cell physiology. The carboxyl-terminal 222 amino acids of exoenzyme S, which represent the FAS-dependent ADP-ribosyltransferase domain (termed deltaN222), and a point mutant, deltaN222-E381A, which possesses a 2000-fold reduction in the capacity to ADP-ribosylate, were transiently expressed in eukaryotic cells under the control of the immediate early CMV promoter. Lysates from cells transfected with deltaN222 expressed ADP-ribosyltransferase activity. Co-transfection of deltaN222, but not deltaN222-E381A, resulted in a decrease in the steady-state levels of two reporter proteins, green fluorescent protein and luciferase, in both CHO and Vero cells. In addition, transfection with deltaN222 resulted in a greater percentage of cells staining with trypan blue than when cells were transfected with either deltaN222-E381A or control plasmid. Together, these data indicate that expression of the ADP-ribosyltransferase domain of exoenzyme S is cytotoxic to eukaryotic cells.
authors: Medical College of Wisconsin, Department of Microbiology and Molecular Genetics, Milwaukee 53226, USA.
authors: K J
authors: KJ
authors: Pederson
authors: J T
authors: JT
authors: Barbieri
mesh_terms: D036002:ADP Ribose Transferases / Q000235:genetics / Q000633:toxicity*; D000818:Animals; D001427:Bacterial Toxins; D016466:CHO Cells; D006224:Cricetinae; D005057:Eukaryotic Cells; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D007424:Intracellular Fluid; D008164:Luminescent Proteins / Q000235:genetics; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000633:toxicity*; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D014162:Transfection
chemical_list: D001427:Bacterial Toxins; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D036002:ADP Ribose Transferases; D011065:Poly(ADP-ribose) Polymerases; C444820:exoenzyme S