Rounding Out the Understanding of ACD Toxicity with the Discovery of Cyclic Forms of Actin Oligomers

Abstract

Actin is an essential element of both innate and adaptive immune systems and can aid in motility and translocation of bacterial pathogens, making it an attractive target for bacterial toxins. Pathogenic Vibrio and Aeromonas genera deliver actin cross-linking domain (ACD) toxin into the cytoplasm of the host cell to poison actin regulation and promptly induce cell rounding. At early stages of toxicity, ACD covalently cross-links actin monomers into oligomers (AOs) that bind through multivalent interactions and potently inhibit several families of actin assembly proteins. At advanced toxicity stages, we found that the terminal protomers of linear AOs can get linked together by ACD to produce cyclic AOs. When tested against formins and Ena/VASP, linear and cyclic AOs exhibit similar inhibitory potential, which for the cyclic AOs is reduced in the presence of profilin. In coarse-grained molecular dynamics simulations, profilin and WH2-motif binding sites on actin subunits remain exposed in modeled AOs of both geometries. We speculate, therefore, that the reduced toxicity of cyclic AOs is due to their reduced configurational entropy. A characteristic feature of cyclic AOs is that, in contrast to the linear forms, they cannot be straightened to form filaments (e.g., through stabilization by cofilin), which makes them less susceptible to neutralization by the host cell.

Steps of actin filament branch formation by Arp2/3 complex investigated with coarse-grained molecular dynamics

“…We apply the Kim and Hummer (2008) Model A (KH-A), where each residue is represented by its C α atom, as described in Horan et al (2020) ; Smith et al (2021) . In the KH-A model, pairs of non-bonded residues interact through a Lennard-Jones (LJ)-type potential, either attractive or repulsive, based on the Miyazawa and Jernigan (1996) pairwise interaction matrix.…”

“…Its activation also includes multiple binding events (of VCA, actin monomers, and mother filament), conformational changes, and flexible regions such as the VCA domain and the D-loop of actin. Motivated by recent coarse-grained (CG) MD studies of pure actin and formin-actin polymerization ( Horan et al, 2018 ; Horan et al, 2020 ; Smith et al, 2021 ), here we explore the use of these methods to study Arp2/3 complex activation. We used the model A of Kim and Hummer (2008) (KH-A), which has much reduced computational cost compared to all atom simulations but still retains aminoacid specificity.…”

Bacterial Nucleotidyl Cyclases Activated by Calmodulin or Actin in Host Cells: Enzyme Specificities and Cytotoxicity Mechanisms Identified to Date

“…ExoY may also impact actin self-assembly [ 17 , 99 ]. ACD induces the formation of toxic actin monomers [ 23 , 100 ] that will lead, together with RID, to a disruption of tight junctions (TJ).…”

“…They use a cofactor that is original in this family, namely monomeric or polymerized actin [ 17 , 21 ]. Actin is a frequent target of bacterial and viral effector proteins, which can affect the polymerization state of actin in different ways to the advantage of the pathogen by introducing chemical modifications on actin, such as ADP-ribosylation [ 22 ] or crosslinking [ 23 ]. Its use as a cofactor that strongly stimulates the catalytic activity of the NC enzyme represents an original way to target actin among cytoskeletoxins, i.e., effector proteins of pathogens that target the host cell cytoskeleton.…”

Allosteric Regulation of Human Plastins

“…Intermolecular covalent cross-linking of actin monomers by actin crosslinking domain (ACD) toxin brings actin subunits into oligomers that do not polymerize as their longitudinal contacts are disturbed by artificial covalent bonds 60 . The polymerization of ACD-crosslinked actin oligomers can be rescued, however, if the perturbed contacts are compensated by enforcing the remaining contacts by phalloidin or reshaping the contacts by cofilin 60,61 . Accordingly, in agreement with its ability to staple adjacent subunits 37 by the strong ABD2-actin contacts, ABD2PLS2 effectively rescued polymerization of ACD-cross-linked actin oligomers (Supplementary Fig.…”