Pathogenic Mechanisms of Actin Cross-Linking Toxins: Peeling Away the Layers

Abstract

Actin cross-linking toxins are produced by Gram-negative bacteria from Vibrio and Aeromonas genera. The toxins were named actin cross-linking domains (ACD), since the first and most of the subsequently discovered ACDs were found as effector domains in larger MARTX and VgrG toxins. Among recognized human pathogens, ACD is produced by Vibrio cholerae, Vibrio vulnificus, and Aeromonas hydrophila. Upon delivery to the cytoplasm of a host cell, ACD covalently cross-links actin monomers into non-polymerizable actin oligomers of various lengths. Provided sufficient doses of toxin are delivered, most or all actin can be promptly cross-linked into non-functional oligomers, leading to cell rounding, detachment from the substrate and, in many cases, cell death. Recently, a deeper layer of ACD toxicity with a less obvious but more potent mechanism was discovered. According to this finding, low doses of the ACD-produced actin oligomers can actively disrupt the actin cytoskeleton by potently inhibiting essential actin assembly proteins, formins. The first layer of toxicity is direct (as actin is the immediate and the only target), passive (since ACD-cross-linked actin oligomers are toxic only because they are non-functional), and less potent (as bulk quantities of one of the most abundant cytoplasmic proteins, actin, have to be modified). The second mechanism is indirect (as major targets, formins, are not affected by ACD directly), active (because actin oligomers act as "secondary" toxins), and highly potent [as it affects scarce and essential actin-binding proteins (ABPs)].

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

“…Toxicity amplification is often achieved by targeting essential, low abundant host proteins in signaling or neurotransmission cascades [3-6]; whereas actin, a highly abundant structural protein, is a rare exception to this rule. The actin cross-linking domain (ACD), produced by gram-negative Vibrio, Aeromonas , and other species, is one of the toxins that utilize actin monomers as a substrate [7]. ACD is delivered to the host cytoplasm as one of several effectors of the multifunctional auto-processing repeats-in-toxin (MARTX) toxins [8] or as a single effector domain fused to valine–glycine repeat protein G1 (VgrG1) toxin of the type VI secretion system [9].…”

Coordinated delivery and function of bacterial MARTX toxin effectors

“…Structural prediction of ACD and the later solved structure of the ACD of Type 6 secretion effector VgrG1 revealed ACD has similarities to glutamine synthases with a conserved organization of their active sites (Geissler et al ., ; Durand et al ., ). These studies suggest a catalytic mechanism in which ACD utilizes a glutamic acid residue to coordinate actin, Mg 2+ and ATP and then transfer the γ‐phosphate from the ATP to residues E270 on one actin monomer, which then activates its ligation to the K50 residue of another actin monomer (Geissler et al ., ; Durand et al ., ; Kudryashova et al ., ; Kudryashova et al ., ). The subsequent ACD‐mediated disassembly of the actin cytoskeleton is sufficient to disrupt tight junctions of polarized T84 cell monolayers and inhibit macrophage phagocytosis in vitro (Dolores et al ., ).…”

“…However, these effects on cytoskeleton integrity can occur far ahead of total cross-linking of cellular actin. In fact, the cross-linked actin dimers form early in the cross-linking process themselves can act as secondary toxins by sequestering and inhibiting formins, which are required for actin polymerization and other host cell functions (Heisler et al, 2015;Kudryashova et al, 2017).…”

Intein-mediated cytoplasmic reconstitution of a split toxin enables selective cell ablation in mixed populations and tumor xenografts

“…2). PA/LF N -ACD is a convenient reporter, as the catalytic actin cross-linking activity of ACD (38)(39)(40) leads to effective disruption of the actin cytoskeleton via several mechanisms (41)(42)(43) morphologically manifested as cell rounding (44,45). In contrast to the toxicity of DT that takes days to detect microscopically, the effects of ACD develop promptly and can be reliably monitored within 1 to 2 h. Furthermore, the specific toxicity can be confirmed via the appearance of covalent actin species by Western blot (Fig.…”