Characterization of the Enzymatic Activity of the Actin Cross-linking Domain from the Vibrio cholerae MARTX  Toxin

Kudryashov, Dmitri S.; Cordero, Christina L.; Reisler, Emil; Satchell, K.J.F.

doi:10.1074/jbc.m703910200

Cited by 41 publications

(44 citation statements)

References 48 publications

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“…Both loops are distant from the actin binding sites of major G-actin binding proteins, such as thymosin ␤4 and profilin. This correlates with our recent findings that both proteins do not inhibit actin cross-linking by ACD (10). Both loops are mobile, unstructured, and protrude out of the body of the actin molecule, making them good substrates for enzymes.…”

Section: Acd-induced Cross-linking Precludes Actin Polymerization Intsupporting

confidence: 78%

“…To reveal the mechanism of actin cross-linking by ACD, we limited the extent of the cross-linking to yield mainly dimers (10). We then used limited proteolysis of purified ACD cross-linked actin dimers and uncross-linked monomers to identify the actin peptides that are covalently linked.…”

Section: Resultsmentioning

confidence: 99%

“…This toxin has been associated with V. cholerae virulence in both pulmonary and intestinal infection models and has been demonstrated to contribute to cholera pathogenesis by preventing clearance of the bacterium from the intestine at the earliest stages of infection (5)(6)(7). We have shown recently that the actin cross-linking domain (ACD) of MAR-TX Vc directly catalyzes a covalent cross-linking of monomeric G-actin in a Mg 2ϩ -ATP dependent manner, causing disassembly of the stress fibers and cell rounding (8)(9)(10)(11), but the nature of the cross-linked bond and the mechanism of the actin cytoskeleton disruption remained elusive.…”

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confidence: 99%

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Connecting actin monomers by iso-peptide bond is a toxicity mechanism of the Vibrio cholerae MARTX toxin

Kudryashov

Durer

Ytterberg

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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The Gram-negative bacterium Vibrio cholerae is the causative agent of a severe diarrheal disease that afflicts three to five million persons annually, causing up to 200,000 deaths. Nearly all V. cholerae strains produce a large multifunctional-autoprocessing RTX toxin (MARTXVc), which contributes significantly to the pathogenesis of cholera in model systems. The actin cross-linking domain (ACD) of MARTXVc directly catalyzes a covalent cross-linking of monomeric G-actin into oligomeric chains and causes cell rounding, but the nature of the cross-linked bond and the mechanism of the actin cytoskeleton disruption remained elusive. To elucidate the mechanism of ACD action and effect on actin, we identified the covalent cross-link bond between actin protomers using limited proteolysis, X-ray crystallography, and mass spectrometry. We report here that ACD catalyzes the formation of an intermolecular iso-peptide bond between residues E270 and K50 located in the hydrophobic and the DNaseI-binding loops of actin, respectively. Mutagenesis studies confirm that no other residues on actin can be cross-linked by ACD both in vitro and in vivo. This cross-linking locks actin protomers into an orientation different from that of F-actin, resulting in strong inhibition of actin polymerization. This report describes a microbial toxin mechanism acting via iso-peptide bond cross-linking between host proteins and is, to the best of our knowledge, the only known example of a peptide linkage between nonterminal glutamate and lysine side chains.cross-linking ͉ X-ray crystallography ͉ mass spectrometry A s a vital component of every eukaryotic cell, the actin cytoskeleton is a frequent target for microbial toxins. By impairing actin-orchestrated functions via production of specific toxins, pathogenic bacteria manage to escape immune cell surveillance and not only overcome cell barriers but also hijack the actin cytoskeleton for efficient invasion and dissemination. These toxins modulate activities of host proteins either via direct binding to or covalent modifications of their targets. Most of the toxins acting on the actin cytoskeleton shift the equilibrium between polymerized F-and monomeric G-actin by targeting actin adaptor and regulatory proteins such as the Arp2/3 complex and RhoGTPases (1). A lower number of protein toxins affect actin directly rather than through other components of the cytoskeleton. Two types of covalent modifications of actin by two distinct groups of bacterial toxins have been described so far. The first group consists of a family of interrelated toxins with ADP-ribosyltransferase activity (such as C2 toxin of Clostridium botulinum, Iota toxin of C. perfringens, and SpvB of Salmonella enterica), which block actin polymerization by adding an ADPribose moiety to Arg-177 of G-actin, thereby resulting in sterical clashes between actin protomers (2, 3).The second group is represented by MARTX Vc [multifunctional-autoprocessing repeats-in-toxins (RTX) toxin of V. cholerae], a toxin produced by nearly all environment...

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Section: Acd-induced Cross-linking Precludes Actin Polymerization Intsupporting

confidence: 78%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Connecting actin monomers by iso-peptide bond is a toxicity mechanism of the Vibrio cholerae MARTX toxin

Kudryashov

Durer

Ytterberg

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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“…In addition, purified MARTX effectors tend, in general, to behave poorly in solution, as they seem to be evolutionarily selected to be unstable, likely because of a need to unfold easily for type I secretion out of the bacterium and again for translocation into the eukaryotic cell cytosol. ACD and CPD are both known to require cofactors (50,51), and for the CPD, it has been demonstrated that the recombinant protein does not become stably folded in vitro until it associates with its activator (52). Thus, it is possible that VvExoY does require a cofactor that would increase both its in vitro stability and its activity.…”

Section: Discussionmentioning

confidence: 99%

Vibrio vulnificus Biotype 3 Multifunctional Autoprocessing RTX Toxin Is an Adenylate Cyclase Toxin Essential for Virulence in Mice

et al. 2014

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Vibrio vulnificus is an environmental organism that causes both food-borne and wound infections with high morbidity and mortality in humans. The annual incidence and global distribution of infections associated with this pathogen are increasing with climate change. In the late 1990s, an outbreak of tilapia-associated wound infections in Israel was linked to a previously unrecognized variant of V. vulnificus designated biotype 3. The sudden emergence and clonality of the outbreak suggest that this strain may be a true newly emergent pathogen with novel virulence properties compared to those of other V. vulnificus strains. In a subcutaneous infection model to mimic wound infection, the multifunctional autoprocessing RTX (MARTX) toxin of biotype 3 strains was shown to be an essential virulence factor contributing to highly inflammatory skin wounds with severe damage affecting every tissue layer. We conducted a sequencing-based analysis of the MARTX toxin and found that biotype 3 MARTX toxin has an effector domain structure distinct from that of either biotype 1 or biotype 2. Of the two new domains identified, a domain similar to Pseudomonas aeruginosa ExoY was shown to confer adenylate cyclase activity on the MARTX toxin. This is the first demonstration that the biotype 3 MARTX toxin is essential for virulence and that the ExoY-like MARTX effector domain is a catalytically active adenylate cyclase.

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“…The RhoGTPase-inactivation domain (RID) causes conversion of activated GTP-bound Rho, Rac, and CDC42 to the inactive GDP-bound forms resulting in depolymerization of actin (10). The adjacent actin cross-linking domain (ACD) catalyzes the covalent cross-linking of monomeric G-actin resulting in the irreversible destruction of the cytoskeleton (7,11,12). Together these domains rapidly round eukaryotic cells without causing cell lysis.…”

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confidence: 99%

Structure-Function Analysis of Inositol Hexakisphosphate-induced Autoprocessing of the Vibrio cholerae Multifunctional Autoprocessing RTX Toxin

Procházková

Satchell

2008

Journal of Biological Chemistry

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Vibrio cholerae secretes a large virulence-associated multifunctional autoprocessing RTX toxin (MARTX Vc ). Autoprocessing of this toxin by an embedded cysteine protease domain (CPD) is essential for this toxin to induce actin depolymerization in a broad range of cell types. A homologous CPD is also present in the large clostridial toxin TcdB and recent studies showed that inositol hexakisphosphate (Ins(1,2,3,4,5,6)P 6 or InsP 6 ) stimulated the autoprocessing of TcdB dependent upon the CPD (Egerer, M., Giesemann, T., Jank, T., Satchell, K. J., and Aktories, K. (2007) J. Biol. Chem. 282, 25314 -25321). In this work, the autoprocessing activity of the CPD within MARTX Vc is similarly found to be inducible by InsP 6 . The CPD is shown to bind InsP 6 (K d , 0.6 M), and InsP 6 is shown to stimulate intramolecular autoprocessing at both physiological concentrations and as low as 0.01 M. Processed CPD did not bind InsP 6 indicating that, subsequent to cleavage, the activated CPD may shift to an inactive conformation. To further pursue the mechanism of autoprocessing, conserved residues among 24 identified CPDs were mutagenized. In addition to cysteine and histidine residues that form the catalytic site, 2 lysine residues essential for InsP 6 binding and 5 lysine and arginine residues resulting in loss of activity at low InsP 6 concentrations were identified. Overall, our data support a model in which basic residues located across the CPD structure form an InsP 6 binding pocket and that the binding of InsP 6 stimulates processing by altering the CPD to an activated conformation. After processing, InsP 6 is shown to be recycled, while the cleaved CPD becomes incapable of further binding of InsP 6 .Vibrio cholerae is the etiologic agent of the acute intestinal infection cholera, that remains a world-wide problem with over 200,000 reported and an estimated 1 million actual cases each year (1, 2). To cause illness, V. cholerae colonizes the small intestine, where it secretes its major virulence factor, the ADPribosylating cholera toxin, which elicits massive fluid secretion resulting in the profuse diarrhea that is the hallmark of cholera infection. Nearly all O1, O139, and non-O1/non-O139 clinical isolates of V. cholerae produce another secreted toxin that is the founding member of a new family of bacterial protein toxins called the multifunctional autoprocessing repeats-in-toxins (MARTX) 2 toxins (3-7). In V. cholerae, this toxin has recently been shown to contribute to virulence in mice and is among three secreted factors associated with the ability of V. cholerae to establish an intestinal infection that persists beyond 24 h (8, 9). Hence, MARTX Vc is proposed to function during the earliest stages of human exposure to V. cholerae either to modify the intestinal tract allowing colonization to occur or to reduce the functionality of innate immune cells preventing clearance. The broad distribution of MARTX Vc among environmental isolates further suggests this toxin may have a role in extraintestinal survival (3, 5-7).MAR...

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Characterization of the Enzymatic Activity of the Actin Cross-linking Domain from the Vibrio cholerae MARTX Toxin

Cited by 41 publications

References 48 publications

Connecting actin monomers by iso-peptide bond is a toxicity mechanism of the Vibrio cholerae MARTX toxin

Connecting actin monomers by iso-peptide bond is a toxicity mechanism of the Vibrio cholerae MARTX toxin

Vibrio vulnificus Biotype 3 Multifunctional Autoprocessing RTX Toxin Is an Adenylate Cyclase Toxin Essential for Virulence in Mice

Structure-Function Analysis of Inositol Hexakisphosphate-induced Autoprocessing of the Vibrio cholerae Multifunctional Autoprocessing RTX Toxin

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