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

Abstract

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...

A Nucleotide State-sensing Region on Actin

“…Filament structures are stabilized through the binding of phalloidin at the interface of three subunits in F-actin, and thus, the assembly of polymerization-impaired actins can be frequently rescued by this reagent. Similarly, cofilin can also rescue the polymerization of some assembly impaired actin mutants and chemically or enzymatically modified actins by bridging adjacent subunits and remodeling the intersubunit interface in the filament (34,35). However, for the F169C actin mutant, only phalloidin (but not and on plates at 24 and 30°C.…”

Persistent nuclear actin filaments inhibit transcription by RNA polymerase II

“…First, we pre-incubated HeLa nuclear extracts with vehicle or the purified actin-crosslinking domain (ACD) of V. cholera MARTX. ACD toxin catalyzes inter-molecular amide bond formation between actin monomers, covalently crosslinking monomeric actin (Kudryashov et al, 2008). Western blots of ACD-treated nuclear extracts showed a ladder of actin crosslinked into oligomers of increasing molecular weights and a concomitant decrease in the amount of non-crosslinked actin (Fig.…”

“…Briefly, purified HeLa nuclear extract (30 µg, Promega) in transcription buffer (20 mM HEPES pH 7.9, 20% glycerol, 100 mM KCl, 0.2 mM EDTA, 0.5 mM PMSF, and 0.5 mM DTT) with 1 mM ATP and 4 mM MgCl 2 was pre-incubated with 0.5 µM purified ACD (Kudryashov et al, 2008) or buffer alone for 1 h at room temperature or with 10 µM phalloidin for 30 min at 4°C. After pre-incubation, RNAse T1 was added, along with 200 ng P41 DNA template containing the adenoviral major late promoter and G-less transcribing region and 0.4 mM of ATP, UTP and 0.02 mM CTP.…”

In vivo actin cross-linking induced by Vibrio cholerae type VI secretion system is associated with intestinal inflammation

“…Additionally, many VgrG proteins contain C-terminal extensions, some of which possess effector function, such as the actin cross-linking domain (ACD) of VgrG-1 in V. cholerae (11). This domain covalently cross-links actin monomers by catalyzing intermolecular isopeptide bonds (15). After uptake of bacteria into macrophage cell lines, VgrG-1 or its ACD domain was translocated into the host cell cytosol to cross-link cellular actin, thereby impairing subsequent phagocytic function (11,16,17).…”