No abstract

The actin crosslinking domain (ACD) is an actin-specific toxin produced by several pathogens, including life-threatening spp. of Vibrio cholerae, Vibrio vulnificus, and Aeromonas hydrophila. Actin crosslinking by ACD is thought to lead to slow cytoskeleton failure owing to a gradual sequestration of actin in the form of nonfunctional oligomers. Here we found that ACD converted cytoplasmic actin into highly toxic oligomers that potently "poisoned" the ability of major actin assembly proteins, formins, to sustain actin polymerization. Thus, ACD can target the most abundant cellular protein by employing actin oligomers as secondary toxins to efficiently subvert cellular functions of actin while functioning at very low doses. Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptBacterial toxins are the deadliest compounds on the planet. As little as a single molecule of a delivered toxin can compromise vital functions or even kill an affected host cell (1, 2). This is achieved by amplification of a toxin enzymatic activity via signaling cascades (e.g. by cholera, pertussis, and anthrax toxins) or via enzymatic inhibition of vital host complexes present in relatively few copies (e.g. Shiga and diphtheria toxins acting on ribosomes). Such efficiency is crucial because i) the amount of a toxin produced early upon infection is limited by an initially small number of bacterial cells; ii) the host is protected by commensal bacteria; and iii) the host immune system efficiently neutralizes toxins by means of adaptive (antibodies) and innate (e.g. defensins) (3) humoral defense factors.Owing to its importance in multiple cellular processes, actin is a common target for bacterium-and parasite-produced toxins. Upon delivery to the cytoplasm of host cells via Type I (as part of MARTX toxin) (4) or Type VI (within VgrG1 toxin) (5) secretion systems, the actin crosslinking domain toxin (ACD) catalyzes the covalent crosslinking of K50 in subdomain 2 of one actin monomer with E270 in subdomain 3 of another actin monomer via an amide bond, resulting in the formation of actin oligomers (6, 7). The actin subunits in the oligomers are oriented similar to short-pitch subunits in the filament, except that a major twist of the subdomain-2, required to accommodate such orientation, disrupts the normal inter-subunit interface and precludes polymerization (6).The currently accepted mechanism of ACD toxicity, via sequestering of bulk amounts of actin as non-functional oligomers, is compromised owing to the high concentration (hundreds of micromolar) of actin in a typical animal cell. Extrapolation of in vitro determined rates of the ACD activity (7) to cellular conditions suggests that a single ACD molecule per cell (i.e. ~ 1 pM) would require over six months to covalently crosslink half of all cytoplasmic actin.In contrast to these estimations, the integrity of the intestinal cell monolayers was disrupted when only a small fraction of cellular actin (2-6%) was crosslinked by ACD ( Fig. 1A-C; fig. S1). To account fo...

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.

MARTX (multifunctional autoprocessing repeats-in-toxin) family toxins are produced by Vibrio cholerae, Vibrio vulnificus, Aeromonas hydrophila and other Gram-negative bacteria. Effector domains of MARTX toxins cross the cytoplasmic membrane of a host cell through a putative pore formed by the toxin's glycine-rich repeats. The structure of the pore is unknown and the translocation mechanism of the effector domains is poorly understood. We examined the thermodynamic stability of the effector domains of V. cholerae and A. hydrophila MARTX toxins to elucidate the mechanism of their translocation. We found that all but one domain in each toxin are thermodynamically unstable and several acquire a molten globule state near human physiological temperatures. Fusion of the most stable cysteine protease domain to the adjacent effector domain reduces its thermodynamic stability ∼ 1.4-fold (from D G H 2 O 21.8 to 16.1 kJ mol(-1) ). Precipitation of several individual domains due to thermal denaturation is reduced upon their fusion into multi-domain constructs. We speculate that low thermostability of the MARTX effector domains correlates with that of many other membrane-penetrating toxins and implies their unfolding for cell entry. This study extends the list of thermolabile bacterial toxins, suggesting that this quality is essential and could be susceptible for selective targeting of pathogenic toxins.

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)].