ACD toxin–produced actin oligomers poison formin-controlled actin polymerization

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...

ADP-Ribosylation and Cross-Linking of Actin by Bacterial Protein Toxins

“…Moreover, only a small fraction of actin modification by ACD appears to be sufficient to induce major cellular effects. The answer to these questions may be the recent finding of the role of cross-linked actin on formin-regulated actin polymerization (Heisler et al 2015) (Fig. 8).…”

“…For host-pathogen interactions, this means inhibition of migration of immune cells or blockade of phagocytosis. At least in vitro, the rate of actin cross-linking induced by ACD is rather low (Heisler et al 2015). Considering the large pool of actin in cells, the rapid effects of ACD on cell morphology are difficult to explain.…”

Targeting and inactivation of bacterial toxins by human defensins

“…The toxin is comprised of several well-defined components: i) a variable number (from one to five) of effector domains with distinct toxicities found in different combinations; ii) a cysteine protease domain (CPD) always positioned C-terminally to the effector domains and responsible for domain liberation upon delivery into a host cell (Prochazkova et al, 2009; Shen et al, 2009); and iii) the N- and C-terminal glycine-rich Ca 2+ -binding repeats that serve for membrane integration and translocation across the membrane (Kim et al, 2015). The specific enzymatic activity of one of the MARTX effector domains, actin cross-linking domain (ACD; (Heisler et al, 2015; Kudryashova et al, 2016a)) produced by V. cholerae and A. hydrophila , was tested in vitro and found to be potently inhibited by α- (HNP1, HD5; (Kudryashova et al, 2014b)) and θ-defensins (RC1 and RC101; (Kudryashova et al, 2015)). Cytotoxicity of both ACD and Rho inhibitory effector domain (RID; (Ahrens et al, 2013)) was prevented by HNP1 in cell culture experiments (Kudryashova et al, 2014b).…”

How Bacteria Subvert Animal Cell Structure and Function

“…Exciting new studies suggest that actin can be converted into a second messenger that amplifies low abundant toxin function (Heisler et al 2015). The actin cross-linking domain (ACD) found in toxins such as the multifunctional autoprocessing repeats-in-toxin (MARTX)-containing toxins covalently cross-links actin into oligomers that are structurally incompatible for polymerization, resulting in rapid cell rounding with loss of all polymerized actin (Fullner & Mekalanos 2000, Lin et al 1999).…”

“…It was previously thought that ACD toxicity was a consequence of cross-linking the G-actin pool into polymerization-deficient oligomers (Cordero et al 2006, Fullner & Mekalanos 2000, Lin et al 1999). However, due to the inconsistencies between in vitro ACD activity (which is low) and cell G-actin concentrations (which are high), Heisler et al (2015) proposed that toxicity is caused by ACD cross-linked actin oligomers targeting upstream actin regulator proteins. Indeed, they found that actin oligomers poison the formin family of actin nucleators.…”