Intracellular expression of the ADP-ribosyltransferase domain of Pseudomonas exoenzyme S is cytotoxic to eukaryotic cells

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 re present the FAS-dependent ADP-ribosyltransferase domain (termed Delta N222), and a point mutant, Delta N222-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 Delta N222 expressed ADP-ribosyltransferase activity. Co-transfection of Delta N222, but not Delta N222-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 Delta N222 resulted in a greater percentage of cells staining with trypan blue than when cells were transfected with either Delta N222-E381A or control plasmid. Together, these data indicate that expression of the ADP-ribosyltransferase domain of exoenzyme S is cytotoxic to eukaryotic cells.