Actin Cross-Linking Toxin Is a Universal Inhibitor of Tandem-Organized and Oligomeric G-Actin Binding Proteins

• Published in: Current biology Vol. 28; no. 10; pp. 1536 - 1547.e9
• Main Authors: Kudryashova, Elena, Heisler, David B., Williams, Blake, Harker, Alyssa J., Shafer, Kyle, Quinlan, Margot E., Kovar, David R., Vavylonis, Dimitrios, Kudryashov, Dmitri S.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 21-05-2018
• Subjects: actin cytoskeleton; actin-binding proteins; bacterial toxins; cross-linking; Ena/VASP; multivalent interaction; nucleation promoting factors; single-molecule speckle live-cell microscopy; toxicity amplification; WH2-domain; actin cytoskeleton; nucleation promoting factors; toxicity amplification; cross-linking; single-molecule speckle live-cell microscopy; actin-binding proteins; multivalent interaction; WH2-domain; bacterial toxins; Ena/VASP
• ISSN: 0960-9822; 1879-0445
• DOI: 10.1016/j.cub.2018.03.065

Delivery of bacterial toxins to host cells is hindered by host protective barriers. This obstruction dictates a remarkable efficiency of toxins, a single copy of which may kill a host cell. Efficiency of actin-targeting toxins is further hampered by an overwhelming abundance of their target. The actin cross-linking domain (ACD) toxins of Vibrio species and related bacterial genera catalyze the formation of covalently cross-linked actin oligomers. Recently, we reported that the ACD toxicity can be amplified via a multivalent inhibitory association of actin oligomers with actin assembly factors formins, suggesting that the oligomers may act as secondary toxins. Importantly, many proteins involved in nucleation, elongation, severing, branching, and bundling of actin filaments contain G-actin-binding Wiskott-Aldrich syndrome protein (WASP)-homology motifs 2 (WH2) organized in tandem and therefore may act as a multivalent platform for high-affinity interaction with the ACD-cross-linked actin oligomers. Using live-cell single-molecule speckle (SiMS) microscopy, total internal reflection fluorescence (TIRF) microscopy, and actin polymerization assays, we show that, in addition to formins, the oligomers bind with high affinity and potently inhibit several families of actin assembly factors: Ena/vasodilator-stimulated phosphorprotein (VASP); Spire; and the Arp2/3 complex, both in vitro and in live cells. As a result, ACD blocks the actin retrograde flow and membrane dynamics and disrupts association of Ena/VASP with adhesion complexes. This study defines ACD as a universal inhibitor of tandem-organized G-actin binding proteins that overcomes the abundance of actin by redirecting the toxicity cascade toward less abundant targets and thus leading to profound disorganization of the actin cytoskeleton and disruption of actin-dependent cellular functions.
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•ACD toxin is a potent universal inhibitor of various actin assembly factors•In live cells, ACD toxin stalls dynamics of formins, Ena/VASP, Spire, and NPFs•ACD toxicity is amplified by redirecting from actin to less abundant targets
The shared ability of actin assembly factors to bind several actin molecules aids actin filament nucleation and growth. Kudryashova, Heisler, et al. show that bacterial toxin ACD targets this common property by producing covalent actin oligomers, which potently inhibit many actin assembly factors, leading to disruption of cellular actin dynamics.