Characterization of an actin-targeting ADP-ribosyltransferase from Aeromonas hydrophila.

The mono-ADP-ribosyltransferase (mART) toxins are contributing factors to a number of human diseases, including cholera, diphtheria, traveler's diarrhea, and whooping cough. VahC is a cytotoxic, actin-targeting mART from Aeromonas hydrophila PPD134/91. This bacterium is implicated primarily in diseases among freshwater fish species but also contributes to gastrointestinal and extraintestinal infections in humans. VahC was shown to ADP-ribosylate Arg-177 of actin, and the kinetic parameters were K(m)(NAD(+)) = 6 µM, K(m)(actin) = 24 µM, and k(cat) = 22 s(-1). VahC activity caused depolymerization of actin filaments, which induced caspase-mediated apoptosis in HeLa Tet-Off cells. Alanine-scanning mutagenesis of predicted catalytic residues showed the predicted loss of in vitro mART activity and cytotoxicity. Bioinformatic and kinetic analysis also identified three residues in the active site loop that were critical for the catalytic mechanism. A 1.9 Å crystal structure supported the proposed roles of these residues and their conserved nature among toxin homologues. Several small molecules were characterized as inhibitors of in vitro VahC mART activity and suramin was the best inhibitor (IC(50) = 20 µM). Inhibitor activity was also characterized against two other actin-targeting mART toxins. Notably, these inhibitors represent the first report of broad spectrum inhibition of actin-targeting mART toxins.

Photox, a novel actin-targeting mono-ADP-ribosyltransferase from Photorhabdus luminescens.

Photorhabdus luminescens is a pathogenic bacterium that produces many toxic proteins. The mono-ADP-ribosyltransferases (mARTs) are an enzyme class produced by numerous pathogenic bacteria and participate in disease in plants and animals, including humans. Herein we report a novel mART from P. luminescens called Photox. This 46-kDa toxin shows high homology to other actin-targeting mARTs in hallmark catalytic regions and a similar core catalytic fold. Furthermore, Photox shows in vivo cytotoxic activity against yeast, with protection occurring when catalytic residues are substituted with alanine. In vitro, enzymatic activity (k(cat), 1680 +/- 75 min(-1)) is higher than that of the related iota toxin, and diminishes by nearly 14,000-fold following substitution of the catalytic Glu (E355A). This toxin specifically ADP-ribosylates monomeric alpha-skeletal actin and nonmuscle beta- and gamma-actin at Arg(177), inhibiting regular polymerization of actin filaments. These results indicate that Photox is indeed an ADP-ribosyltransferase, making it the newest member of the actin-targeting mART family.