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

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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 ΔN222), and a point mutant, ΔN222‐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 ΔN222 expressed ADP‐ribosyltransferase activity. Co‐transfection of ΔN222, but not ΔN222‐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 ΔN222 resulted in a greater percentage of cells staining with trypan blue than when cells were transfected with either ΔN222‐E381A or control plasmid. Together, these data indicate that expression of the ADP‐ribosyltransferase domain of exoenzyme S is cytotoxic to eukaryotic cells.