Repeats-in-toxin (RTX) exoproteins of Gram-negative bacteria form a steadily growing family of proteins with diverse biological functions. Their common feature is the unique mode of export across the bacterial envelope via the type I secretion system and the characteristic, typically nonapeptide, glycine- and aspartate-rich repeats binding Ca2+ ions. In this review, we summarize the current state of knowledge on the organization of rtx loci and on the biological and biochemical activities of therein encoded proteins. Applying several types of bioinformatic screens on the steadily growing set of sequenced bacterial genomes, over 1000 RTX family members were detected, with the biological functions of most of them remaining to be characterized. Activities of the so far characterized RTX family members are then discussed and classified according to functional categories, ranging from the historically first characterized pore-forming RTX leukotoxins, through the large multifunctional enzymatic toxins, bacteriocins, nodulation proteins, surface layer proteins, up to secreted hydrolytic enzymes exhibiting metalloprotease or lipase activities of industrial interest.

The multifunctional autoprocessing repeats-in-toxin (MARTX) toxin of Vibrio cholerae causes destruction of the actin cytoskeleton by covalent cross-linking of actin and inactivation of Rho GTPases. The effector domains responsible for these activities are here shown to be independent proteins released from the large toxin by autoproteolysis catalyzed by an embedded cysteine protease domain (CPD). The CPD is activated upon binding inositol hexakisphosphate (InsP 6 ). In this study, we demonstrated that InsP 6 is not simply an allosteric cofactor, but rather binding of InsP 6 stabilized the CPD structure, facilitating formation of the enzyme-substrate complex. The 1.95-Å crystal structure of this InsP 6 -bound unprocessed form of CPD was determined and revealed the scissile bond Leu 3428 -Ala 3429 captured in the catalytic site. Upon processing at this site, CPD was converted to a form with 500-fold reduced affinity for InsP 6 , but was reactivated for high affinity binding of InsP 6 by cooperative binding of both a new substrate and InsP 6 . Reactivation of CPD allowed cleavage of the MARTX toxin at other sites, specifically at leucine residues between the effector domains. Processed CPD also cleaved other proteins in trans, including the leucine-rich protein YopM, demonstrating that it is a promiscuous leucine-specific protease.Multifunctional-autoprocessing repeats-in-toxin (MARTX) 3 toxins are a family of large bacterial protein toxins with conserved repeat regions at the N and C termini that are predicted to transfer effector domains located between the repeats across the eukaryotic cell plasma membrane (1). The best characterized MARTX is the Ͼ450-kDa secreted virulence-associated MARTX of Vibrio cholerae. This toxin causes disassembly of the actin cytoskeleton and enhances V. cholerae colonization of the small intestine, possibly by facilitating evasion of phagocytic cells (2, 3). The central region of the V. cholerae MARTX toxin contains four discrete domains: the actin cross-linking domain (ACD) that introduces lysine-glutamate cross-links between actin protomers (4, 5), the Rho-inactivating domain (RID) that disables small Rho GTPases (6), an ␣␤ hydrolase of unknown function (1), and an autoprocessing cysteine protease domain (CPD) (7,8).The CPD is a 25-kDa domain found in all MARTX toxins located just before the start of the C-terminal repeats (7,8). This domain is activated for autoproteolysis upon binding inositol hexakisphosphate (InsP 6 ) (7), a molecule ubiquitously present in eukaryotic cell cytosol (9 -11), but absent in extracellular spaces and bacteria. Thus, autocatalytic processing would not occur until after translocation of the CPD and effector domains is completed. In the context of the holotoxin, catalytic residue Cys 3568 was found to be essential for the toxin to induce efficient actin cross-linking by the ACD and Rho inactivation by the RID, demonstrating that autoprocessing is essential for MARTX to induce cell rounding (8).While it is clear that InsP 6 activates the CPD and that auto...

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...

Lens epithelium-derived growth factor (LEDGF/p75) is an epigenetic reader and attractive therapeutic target involved in HIV integration and the development of mixed lineage leukaemia (MLL1) fusion-driven leukaemia. Besides HIV integrase and the MLL1-menin complex, LEDGF/p75 interacts with various cellular proteins via its integrase binding domain (IBD). Here we present structural characterization of IBD interactions with transcriptional repressor JPO2 and domesticated transposase PogZ, and show that the PogZ interaction is nearly identical to the interaction of LEDGF/p75 with MLL1. The interaction with the IBD is maintained by an intrinsically disordered IBD-binding motif (IBM) common to all known cellular partners of LEDGF/p75. In addition, based on IBM conservation, we identify and validate IWS1 as a novel LEDGF/p75 interaction partner. Our results also reveal how HIV integrase efficiently displaces cellular binding partners from LEDGF/p75. Finally, the similar binding modes of LEDGF/p75 interaction partners represent a new challenge for the development of selective interaction inhibitors.

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...