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

Abstract

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.

Oligomerization Affects the Ability of Human Cyclase-Associated Proteins 1 and 2 to Promote Actin Severing by Cofilins

“…Actin was purified from skeletal muscle acetone powder: either of rabbit origin purchased from Pel-Freez Biologicals (Rogers, AR, USA) or of chicken origin prepared in-house from flash-frozen chicken breast (Trader Joe’s, Columbus, OH, USA), as previously described [75,76,77].…”

“…TIRF microscopy was conducted as described previously [76,77]. Briefly, immediately before the experiment, flow chambers were functionalized by incubation with 0.1 mg/ml streptavidin in phosphate-buffered saline (PBS) and blocked for 3 min in blocking buffer (1% ( w / v ) bovine serum albumin (BSA) in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl), followed by successive washes with the blocking buffer and 1× TIRF buffer (10 mM imidazole, pH 7.0, 50 mM KCl, 50 mM DTT, 1 mM MgCl 2 , 1 mM EGTA, 0.2 mM ATP, 50 µM CaCl 2 , 15 mM glucose, 20 µg/mL catalase, 100 µg/mL glucose oxidase, 15% glycerol, and 0.5% methylcellulose-400cP (Sigma Aldrich, St. Louis, MO, USA)).…”

Guardians of the Cell: Effector-Triggered Immunity Steers Mammalian Immune Defense

“…In contrast, ADP ribosylation of actin at Thr148 by the Photorhabdus luminescens toxin (TccC3) induces aggregation and bundling of cellular actin filaments in human HeLa cells, which are largely resistant to depolymerization [113]. Another family of toxins, which are present in species including Vibrio cholerae and Aeromonas hydrophila, are capable of crosslinking actin monomers into dimers, trimers, and higher order oligomers that are incapable of incorporation into filaments, and furthermore, serve to sequester and potently inhibit many actin assembly factors, including the ARP2/3 complex, Spire, and Formins [114]. Alternative strategies to PTM are also used by bacteria to target the host's actin cytoskeleton, including the direct processing of actin by bacterial metalloproteases, such as ECP32 from Serratia proteamaculans and RavK from Legionella pneumophila which directly cleave actin, abolishing its ability to be incorporated into polymers in human epithelial cell lines and COS-1 cells [115,116].…”

The Vibrio cholerae MARTX toxin simultaneously induces actin collapse while silencing the inflammatory response to cytoskeletal damage

“…ACD has two putative cytotoxic functions. The first is the inhibitor action of toxic actin oligomers (dimers and trimers) formed when only 2-4% of total actin has been cross-linked (19,36). The second is ACD will eventually cross-link nearly 100% of cellular actin into higher order oligomers (10 to 15-mers) to sequester bulk actin and induce cytoskeletal collapse (16).…”