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Published on: October 28, 2022
Characterization of an actin-targeting ADP-ribosyltransferase from Aeromonas hydrophila
Adin Shniffer1, Danielle D Visschedyk, Ravikiran Ravulapalli
1Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
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.
Insights
Mono-ADP-ribosyltransferase (mART) toxins, like VahC, cause disease by targeting actin. Researchers identified key residues and found broad-spectrum inhibitors, including suramin, offering new therapeutic avenues.
Area of Science:
- Microbiology
- Biochemistry
- Toxicology
Background:
- Mono-ADP-ribosyltransferase (mART) toxins are linked to various human diseases.
- VahC, an actin-targeting mART from Aeromonas hydrophila, causes gastrointestinal and extraintestinal infections in humans.
Purpose of the Study:
- To characterize the VahC toxin's enzymatic activity, mechanism, and cytotoxicity.
- To identify potential inhibitors of VahC and related actin-targeting mART toxins.
Main Methods:
- Enzymatic assays to determine kinetic parameters (K(m), k(cat)).
- Cell-based assays (caspase-mediated apoptosis) and alanine-scanning mutagenesis.
- X-ray crystallography to elucidate the active site structure.
- In vitro inhibition assays with small molecules.
Main Results:
- VahC ADP-ribosylates actin at Arg-177 with specific kinetic parameters.
- VahC induces actin depolymerization and caspase-mediated apoptosis in HeLa cells.
- Key catalytic residues and active site loop residues were identified.
- Suramin emerged as a potent inhibitor (IC(50) = 20 μM) of VahC and other actin-targeting mART toxins.
Conclusions:
- VahC's mechanism involves ADP-ribosylation of actin, leading to cytotoxicity.
- Structural and biochemical analyses revealed critical residues for VahC catalysis.
- The identified inhibitors represent the first broad-spectrum agents against actin-targeting mART toxins, offering therapeutic potential.
