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Role of Actin and Actin-binding Proteins in the Pathogenesis of Actin-targeting Bacterial Toxins

Heisler, David Bruce
http://rave.ohiolink.edu/etdc/view?acc_num=osu1501519777175964

Abstract Details

2017, Doctor of Philosophy, Ohio State University, Biochemistry Program, Ohio State.
Proteinaceous bacterial toxins are among the deadliest compounds on earth; as little as a single copy of a toxin can be sufficient to compromise a host cell. Most toxins amplify their toxicity by targeting low abundant, essential host proteins or by altering signaling cascades. However, toxins that directly target actin, one of the most abundant proteins in the cell, seem doomed to inefficacy. Upon delivery to the cytoplasm of host cells, the actin cross-linking domain, from pathogenic strains of Vibrio and Aeromonas spp., catalyzes the covalent cross-linking of actin monomers into non-functional, non-polymerizable actin oligomers. The original hypothesis of ACD pathogenicity required the passive cross-linking of a bulk population of the actin cytoskeleton and to cause a subsequent morphological change in cell shape. In contrast to this hypothesis, we found that low doses of the actin oligomers bind to monomeric actin binding domains to potently inhibit actin regulatory proteins. Through a multivalent interaction with the formin homology 1 domain’s polyproline rich stretches and the formin homology 2 domains, the actin oligomers bind with low nanomolar affinity, preventing the nucleation and elongation of actin by formins. This novel “gain-of-function” mechanism of toxicity exerted by ACD converts actin monomers into potent secondary toxins to disrupt the actin homeostasis of a host cell. In addition to formins, we predicted the existence of other targets of the ACD-produced oligomers due to the organization of many actin binding proteins, which consist of several closely positioned G-actin-binding domains. We identified a diverse pool of actin-binding proteins that contain a short, G-actin-binding Wiskott-Aldrich syndrome homology motifs 2 (WH2) as additional targets of the oligomers: Ena/VASP, Spire, and activator of the Arp2/3 complex N-WASP. We predict that other nucleation promoting factors the Arp2/3 complex will also serve as targets. Herein, we identify the oligomers as universal inhibitors against WH2 domain-containing proteins. Next, we identified a bacterial effector protein acting on the host cytoplasm that share properties with eukaryotic proteins and is potently inhibited by the oligomers. The Vibrio effector VopF/L is a dimeric protein that brings six WH2 domains in close proximity to nucleate filaments in vitro. We have tested and confirmed the inhibition of VopF/L in vitro and used single-molecule speckle (SiMS) microscopy to demonstrate a potential mechanism of effector domain regulation utilized by bacteria during the infection of host cells. Additionally, we provide the first evidence of the dynamic behavior of VopF/L in living cells, raising questions concerning the reorganizing functional activities of VopF/L. Finally, similar to ACD, actin-targeting ADP-ribosylating toxins (ART) would have to overcome problems associated with targeting a highly abundant substrate. Despite low sequence homology, all but one actin-specific ARTs covalently ADP-ribosylate arginine-177 on monomeric actin, illustrating its strategic, but likely underappreciated role in toxicity. We found that profilin, a monomeric actin binding protein, enhances the modification rate of some but not all ARTs via a profilin binding sequence in the ART. Altogether, these results demonstrate a previously unappreciated connection between the pathogenic mechanisms of bacteria and the proteins that regulate the actin cytoskeleton.
Dmitri Kudryashov (Advisor)
Comert Kural (Committee Member)
Jeffrey Parvin (Committee Member)
Jian-Qiu Wu (Committee Member)
187 p.
Biochemistry; Biology; Microbiology
bacterial toxins; ACD; actin;

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Citations

  • Heisler, D. B. (2017). Role of Actin and Actin-binding Proteins in the Pathogenesis of Actin-targeting Bacterial Toxins [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1501519777175964

    APA Style (7th edition)

  • Heisler, David. Role of Actin and Actin-binding Proteins in the Pathogenesis of Actin-targeting Bacterial Toxins. 2017. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1501519777175964.

    MLA Style (8th edition)

  • Heisler, David. "Role of Actin and Actin-binding Proteins in the Pathogenesis of Actin-targeting Bacterial Toxins." Doctoral dissertation, Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1501519777175964

    Chicago Manual of Style (17th edition)

osu1501519777175964
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© 2017, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.