Inhibition of WH2 domain-containing proteins by actin oligomers produced by ACD toxins
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Date
2017-05
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The Ohio State University
Abstract
The actin crosslinking domain (ACD) is an actin-specific effector domain found in various toxins produced by Vibrio and Aeromonas spp. of bacteria. ACD catalyzes the formation of a covalent bond between actin monomers (G-actin), forming oligomers that are unable to polymerize into filaments (F-actin) but retain their ability to bind to G-actin-binding domains. Recently, our group demonstrated that these oligomers are toxic on their own and able to amplify the ACD toxicity by potently inhibiting formins, which are actin nucleators. Because such potent inhibition is enabled by the abnormally high affinity of oligomers for tandem actin-binding domains of formins, we hypothesized that oligomers may also target other proteins with multiple G-actin-binding domains, thereby increasing ACD toxicity. One conserved G-actin-binding domain commonly found in an array of actin regulatory proteins is the WASP-homology 2 (WH2) domain. WH2 domains are found in Spire, Arp2/3 complex activators (WASP, WAVE, etc.), and bacterial toxins VopL/F. In these proteins, WH2 domains contribute to nucleation of new filaments by promoting proximity of actin monomers, either solitary (tandem WH2 domains of Spire, VopL/F) or in concert with other proteins (Arp2/3 complex). To test the hypothesis that ACD-produced actin oligomers may bind to and inhibit WH2 domain-containing proteins, we assessed the effects of the oligomers on the nucleation activity of these proteins. Using native polyacrylamide gel electrophoresis (native PAGE) we demonstrated that the oligomers form complexes with Spire, N-WASP-VCA, and VopL/F. Next, we employed Total Internal Reflection Fluorescence (TIRF) microscopy and pyrene-actin polymerization assays to assess the effects of the oligomers on the single filament and bulk levels, respectively. We found by TIRF microscopy that low nanomolar concentrations of oligomers efficiently blocked VopL/F- and Spire-mediated nucleation. The dose-dependent inhibition of Spire, Arp2/3 activator N-WASP-VCA domain, and VopL/F-mediated actin nucleation was further confirmed by bulk actin polymerization assays in solution. Both methods revealed that actin oligomers inhibit Spire and VopL/F with low nanomolar affinities, likely owing to the tandem organization of WH2 domains, allowing for multiple high affinity interactions with the actin oligomers. In contrast, the inhibition of Arp2/3 complex activation requires significantly higher concentrations of the oligomers. In conclusion, we have now identified new members of the toxicity cascades that ACD initiates by converting actin monomers into potent secondary toxins. By further understanding the toxicity cascade initiated by the oligomers on a molecular level, we can better prepare against new and emerging strains of pathogenic bacteria that utilize actin-related toxins for infection.
Description
3rd Place at the 2016 ACS (American Chemical Society) Columbus Section and OSU Chemistry and Biochemistry Club Research Forum
Keywords
actin, toxin, Vibrio Cholerae, Vibrio Parahaemolyticus, ACD, Actin Crosslinking Domain