Characterization of an Actin-targeting ADP-ribosyltransferase from Aeromonas hydrophila

Adin Shniffer,Anthony A Petrie,A.Rod Merrill,Ravikiran Ravulapalli,Ashok K Chopra,Zachari J Turgeon,Giovanni Suarez,Danielle D Visschedyk

doi:10.1074/jbc.m112.397612

Abstract

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.

Highlights

VahC toxin from Aeromonas hydrophila inactivates actin by transferring ADP-ribose from NADϩ
Sequence alignment of Type IV toxins that bind actin, showed that the binding residues in the loop regions are poorly conserved (Fig. 1). Further analysis of these regions based upon crystal structures and from the current high resolution crystal structure of VahC shows that the enzyme-substrate interface for these Type IV mARTs in general possesses a similar pattern for the electrostatic surface potential
ADP-ribosylation of all actin isoforms is consistent with some Type IV mART toxins (Photox, SpvB, iota, Sa, and CDTa), but it is not observed in all cases (30, 39 – 41)

Summary

Background

VahC toxin from Aeromonas hydrophila inactivates actin by transferring ADP-ribose from NADϩ. Results: The crystal structure of VahC reveals new insights into the interaction of the actin substrate with the actin-targeting toxins, and potent inhibitors have been developed. VahC is a cytotoxic, actin-targeting mART from Aeromonas hydrophila PPD134/91. This bacterium is implicated primarily in diseases among freshwater fish species and contributes to gastrointestinal and extraintestinal infections in humans. Inhibitor activity was characterized against two other actin-targeting mART toxins. Virulence factors produced by invading pathogenic microbes play an important role in diseases caused by bacterial infections. We characterize a new actin-targeting toxin as the protein product of the virulence gene (vahC), from A. hydrophila PPD134/91 [17]. We have observed effective in vitro inhibition of VahC and similar Type IV mART toxins by novel inhibitors identified from a virtual screen method [22]

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION