Inactivation of small Rho GTPases by the multifunctional RTX toxin from Vibrio cholerae

Many bacterial toxins target small Rho GTPases in order to manipulate the actin cytoskeleton. The depolymerization of the actin cytoskeleton by the Vibrio cholerae RTX toxin was previously identified to be due to the unique mechanism of covalent actin cross-linking. However, identification and subsequent deletion of the actin cross-linking domain within the RTX toxin revealed that this toxin has an additional cell rounding activity. In this study, we identified that the multifunctional RTX toxin also disrupts the actin cytoskeleton by causing the inactivation of small Rho GTPases, Rho, Rac and Cdc42. Inactivation of Rho by RTX was reversible in the presence of cycloheximide and by treatment of cells with CNF1 to constitutively activate Rho. These data suggest that RTX targets Rho GTPase regulation rather than affecting Rho GTPase directly. A novel 548-amino-acid region of RTX was identified to be responsible for the toxin-induced inactivation of the Rho GTPases. This domain did not carry GAP or phosphatase activities. Overall, these data show that the RTX toxin reversibly inactivates Rho GTPases by a mechanism distinct from other Rho-modifying bacterial toxins.     

The Actin cross-linking domain of the Vibrio cholerae RTX toxin directly catalyzes the covalent cross-linking of actin

Vibrio cholerae is a Gram-negative bacterial pathogen that exports enterotoxins to alter host cells and to elicit diarrheal disease. Among the secreted toxins is the multifunctional RTX toxin, which causes cell rounding and actin depolymerization by covalently cross-linking actin monomers into dimers, trimers, and higher multimers. The region of the toxin responsible for cross-linking activity is the actin cross-linking domain (ACD). In this study, we further investigated the role of the ACD in the actin cross-linking reaction. We show that the RTX toxin cross-links actin independently of tissue transglutaminase, thus eliminating an indirect model of ACD activity. We demonstrate that a fusion protein of the ACD and the N-terminal portion of lethal factor from Bacillus anthracis (LF(N)ACD) has cross-linking activity in vivo and in crude cell extracts. Furthermore, we determined that LF(N)ACD directly catalyzes the formation of covalent linkages between actin molecules in vitro and that Mg(2+) and ATP are essential cofactors for the cross-linking reaction. In addition, G-actin is proposed as a cytoskeletal substrate of the RTX toxin in vivo. Future studies of the in vitro cross-linking reaction will facilitate characterization of the enzymatic properties of the ACD and contribute to our knowledge of the novel mechanism of covalent actin cross-linking.     

Characterization of the enzymatic activity of the actin cross-linking domain from the Vibrio cholerae MARTX Vc toxin

Vibrio cholerae is a Gram-negative bacterial pathogen that exports enterotoxins, which alter host cells through a number of mechanisms resulting in diarrheal disease. Among the secreted toxins is the multifunctional, autoprocessing RTX toxin (MARTX(Vc)), which disrupts actin cytoskeleton by covalently cross-linking actin monomers into oligomers. The region of the toxin responsible for cross-linking activity is the actin cross-linking domain (ACD). In this study, we demonstrate unambiguously that ACD utilizes G- and not F-actin as a substrate for the cross-linking reaction and hydrolyzes one molecule of ATP per cross-linking event. Furthermore, major actin-binding proteins that regulate actin cytoskeleton in vivo do not block the cross-linking reaction in vitro. Cofilin inhibits the cross-linking of G- and F-actin, at a high mole ratio to actin but accelerates F-actin cross-linking at low mole ratios. DNase I completely blocks the cross-linking of actin, likely due to steric hindrance with one of the cross-linking sites on actin. In the context of the holotoxin, the inhibition of Rho by the Rho-inactivating domain of MARTX(Vc) (Sheahan, K. L., and Satchell, K. J. F. (2007) Cell. Microbiol. 9, 1324-1335) would accelerate F-actin depolymerization and provide G-actin, alone or in complex with actin-binding proteins, for cross-linking by ACD, ultimately leading to the observed rapid cell rounding.     

Autoprocessing of the Vibrio cholerae RTX toxin by the cysteine protease domain

Vibrio cholerae RTX is a large multifunctional bacterial toxin that causes actin crosslinking. Due to its size, it was predicted to undergo proteolytic cleavage during translocation into host cells to deliver activity domains to the cytosol. In this study, we identified a domain within the RTX toxin that is conserved in large clostridial glucosylating toxins TcdB, TcdA, TcnA, and TcsL; putative toxins from V. vulnificus, Yersinia sp., Photorhabdus sp., and Xenorhabdus sp.; and a filamentous/hemagglutinin-like protein FhaL from Bordetella sp. In vivo transfection studies and in vitro characterization of purified recombinant protein revealed that this domain from the V. cholerae RTX toxin is an autoprocessing cysteine protease whose activity is stimulated by the intracellular environment. A cysteine point mutation within the RTX holotoxin attenuated actin crosslinking activity suggesting that processing of the toxin is an important step in toxin translocation. Overall, we have uncovered a new mechanism by which large bacterial toxins and proteins deliver catalytic activities to the eukaryotic cell cytosol by autoprocessing after translocation.     

Actin cross-linking domain of Aeromonas hydrophila repeat in toxin A (RtxA) induces host cell rounding and apoptosis

The repeat in toxin (Rtx) of an environmental isolate ATCC 7966 of Aeromonas hydrophila consists of six genes (rtxACHBDE) organized in an operon similar to the gene organization found for the Rtx of the Vibrio species. The first gene in this operon (rtxA) encodes an exotoxin in vibrios, while other genes code for proteins needed for proper activation of RtxA and in secretion of this toxin from Vibrio cholerae. However, the RtxA of ATCC 7966, as well as from the clinical isolate SSU of A. hydrophila, was exclusively expressed and produced during co-infection of this pathogen with the host, e.g., HeLa cells, indicating that rtxA gene expression required host cell contact. Within the RtxA, an actin cross-linking domain (ACD) exists and to investigate the functionality of this domain, several truncated versions of ACD were generated to discern its minimal biological active region. Such genetically modified genes encoding ACD, which were truncated on either the NH(2) or the COOH terminal, as well as on both ends, were expressed from a bidirectional promoter of the pBI-enhanced green fluorescent protein (EGFP) vector in a HeLa-Tet-Off cell system. We demonstrated that only the full-length ACD of RtxA from A. hydrophila catalyzed the covalent cross-linking of the host cellular actin, whereas the ACD truncated on the NH(2), COOH or both ends did not exhibit such actin cross-linking characteristics. Further, we showed that the full-length ACD of A. hydrophila RtxA disrupted the actin cytoskeleton of HeLa cells, resulting in their rounding phenotype. Finally, our data provided evidence that the full-length ACD of RtxA induced host cell apoptosis. Our study is the first to report that A. hydrophila possesses a functional RtxA having an ACD that contributes to the host cell apoptosis, and hence could represent a potential virulence factor of this emerging human pathogen.     

Vibrio vulnificus RTX toxin kills host cells only after contact of the bacteria with host cells

Vibrio vulnificus causes acute cell death and a fatal septicaemia. In this study, we show that contact with host cells is a prerequisite to the acute cytotoxicity. We screened transposon mutants defective in the contact-dependent cytotoxicity . Two mutants had insertions within two open reading frames in a putative RTX toxin operon, the rtxA1 or rtxD encoding an RTX toxin (4701 amino acids) or an ABC type transporter (467 amino acids). An rtxA1 mutation resulted in a cytotoxicity defect, which was fully restored by in trans complementation. The expression of RtxA1 toxin increased after host cell contact in a time-dependent manner. The RtxA1 toxin induced cytoskeletal rearrangements and plasma membrane blebs, which culminated in a necrotic cell death. RtxA1 colocalized with actin and caused actin aggregation coinciding with a significant decrease in the F/G actin ratio. The RtxA1 toxin caused haemolysis through pore formation (radius 1.63 nm). The rtxA1 deletion mutant was defective in invading the blood stream from ligated ileal loops of CD1 mice. The rtxA1 null mutation resulted in over 100-fold increase in both intragastric and intraperitoneal LD₅₀s against mice. Overall, these results show that the RtxA1 toxin is a multifunctional cytotoxin and plays an essential role in the pathogenesis of V. vulnificus infections.     

Identification of a new RTX-like gene cluster in Vibrio cholerae

A gene cluster containing two genes in tandem has been identified in Vibrio cholerae ElTor N16961. Each has more than one cadherin domain and is homologous to the RTX toxin family and was common in various V. cholerae strains. Insertional mutagenesis demonstrated that each gene has a role in Hep-2 cell rounding, hemolytic activity towards human and sheep RBCs and biofilm formation. The mutants showed reduced adherence to intestinal epithelial cells as well as reduction of in vivo colonization in suckling mice. These two genes thus code for RTX-like toxins in V. cholerae and are associated with the pathogenecity of this organism.     

Genetic determination of essential residues of the Vibrio cholerae actin cross-linking domain reveals functional similarity with glutamine synthetases

Actin cross-linking domains (ACDs) are distinct domains found in several bacterial toxins, including the Vibrio cholerae MARTX toxin. The ACD of V. cholerae (ACD_Vc) catalyses the formation of an irreversible iso-peptide bond between lysine 50 and glutamic acid 270 on two actin molecules in an ATP- and Mg/Mn²⁺-dependent manner. In vivo , cross-linking depletes the cellular pool of G-actin leading to actin cytoskeleton depolymerization. While the actin cross-linking reaction performed by these effector domains has been significantly characterized, the ACD_Vc catalytic site has remained elusive due to lack of significant homology to known proteins. Using multiple genetic approaches, we have identified regions and amino acids of ACD_Vc required for full actin cross-linking activity. Then, using these functional data and structural homology predictions, it was determined that several residues demonstrated to be important for ACD_Vc activity are conserved with active-site residues of the glutamine synthetase family of enzymes. Thus, the ACDs are a family of bacterial toxin effectors that may be evolutionarily related to ligases involved in amino acid biosynthesis.     

New insights into the mode of action of the actin ADP-ribosylating virulence factors Salmonella enterica SpvB and Clostridium botulinum C2 toxin

The C2 toxin from Clostridium botulinum represents the prototype of the family of binary actin-ADP-ribosylating toxins. These toxins covalently transfer ADP-ribose from nicotinamide adenine dinucleotide (NAD(+)) onto arginine-177 of actin in the cytosol of eukaryotic cells resulting in depolymerization of actin filaments and cell rounding. The C2 toxin consists of two non-linked proteins, the enzyme component C2I and the binding and translocation component C2II, which delivers C2I into host cells. The ADP-ribosyltransferase SpvB from Salmonella enterica also modifies actin, but is delivered into the host cell cytosol from intracellular growing Salmonella, most likely via type-III-secretion. We characterized the mode of action of SpvB in comparison to C2 toxin in vitro and in intact cells. We identified arginine-177 as the target for SpvB-catalyzed mono-ADP-ribosylation of actin. To compare the cellular responses following modification of actin by SpvB or by the binary toxins without the influence of other Salmonella virulence factors, we constructed a cell-permeable fusion toxin to deliver the catalytic domain of SpvB (C/SpvB) into the cytosol of target cells. This review summarizes recent findings of research on the actin ADP-ribosylating toxins regarding their cellular uptake, molecular mode of action and the cellular consequences following ADP-ribosylation of actin.     

A cell-permeable fusion toxin as a tool to study the consequences of actin-ADP-ribosylation caused by the salmonella enterica virulence factor SpvB in intact cells

The virulence factor SpvB is a crucial component for the intracellular growth and infection process of Salmonella enterica. The SpvB protein mediates the ADP-ribosylation of actin in infected cells and is assumed to be delivered directly from the engulfed bacteria into the host cell cytosol. Here we used the binary Clostridium botulinum C2 toxin as a transport system for the catalytic domain of SpvB (C/SpvB) into the host cell cytosol. A recombinant fusion toxin composed of the enzymatically inactive N-terminal domain of C. botulinum C2 toxin (C2IN) and C/SpvB was cloned, expressed, and characterized in vitro and in intact cells. When added together with C2II, the C2IN-C/SpvB fusion toxin was efficiently delivered into the host cell cytosol and ADP-ribosylated actin in various cell lines. The cellular uptake of the fusion toxin requires translocation from acidic endosomes into the cytosol and is facilitated by Hsp90. The N- and C-terminal domains of SpvB are linked by 7 proline residues. To elucidate the function of this proline region, fusion toxins containing none, 5, 7, and 9 proline residues were constructed and analyzed. The existence of the proline residues was essential for the translocation of the fusion toxins into host cell cytosol and thereby determined their cytopathic efficiency. No differences concerning the mode of action of the C2IN-C/SpvB fusion toxin and the C2 toxin were obvious as both toxins induced depolymerization of actin filaments, resulting in cell rounding. The acute cellular responses following ADP-ribosylation of actin did not immediately induce cell death of J774.A1 macrophage-like cells.     

Actin as target for modification by bacterial protein toxins

Various bacterial protein toxins and effectors target the actin cytoskeleton. At least three groups of toxins/effectors can be identified, which directly modify actin molecules. One group of toxins/effectors causes ADP-ribosylation of actin at arginine-177, thereby inhibiting actin polymerization. Members of this group are numerous binary actin-ADP-ribosylating exotoxins (e.g. Clostridium botulinum C2 toxin) as well as several bacterial ADP-ribosyltransferases (e.g. Salmonella enterica SpvB) which are not binary in structure. The second group includes toxins that modify actin to promote actin polymerization and the formation of actin aggregates. To this group belongs a toxin from the Photorhabdus luminescens Tc toxin complex that ADP-ribosylates actin at threonine-148. A third group of bacterial toxins/effectors (e.g. Vibrio cholerae multifunctional, autoprocessing RTX toxin) catalyses a chemical crosslinking reaction of actin thereby forming oligomers, while blocking the polymerization of actin to functional filaments. Novel findings about members of these toxin groups are discussed in detail.     

Identification of a His-Asp-Cys Catalytic Triad Essential for Function of the Rho Inactivation Domain (RID) of Vibrio cholerae MARTX Toxin

Vibrio cholerae is the causative agent of the severe diarrheal disease cholera. For V. cholerae to colonize the intestinal epithelium, accessory toxins such as the multifunctional autoprocessing repeats-in-toxin (MARTX_Vc) toxin are required. MARTX toxins are composite toxins comprised of arrayed effector domains that carry out distinct functions inside the host cell. Among the three effector domains of MARTX_Vc is the Rho inactivation domain (RID_Vc) known to cause cell rounding through inactivation of small RhoGTPases. Using alanine scanning mutagenesis in the activity subdomain of RID_Vc, four residues, His-2782, Leu-2851, Asp-2854, and Cys-3022, were identified as impacting RID_Vc function in depolymerization of the actin cytoskeleton and inactivation of RhoA. Tyr-2807 and Tyr-3015 were identified as important potentially for forming the active structure for substrate contact but are not involved in catalysis or post translational modifications. Finally, V. cholerae strains modified to carry a catalytically inactive RID_Vc show that the rate and efficiency of MARTX_Vc actin cross-linking activity does not depend on a functional RID_Vc, demonstrating that these domains function independently in actin depolymerization. Overall, our results indicate a His-Asp-Cys catalytic triad is essential for function of the RID effector domain family shared by MARTX toxins produced by many Gram-negative bacteria.     

Domain organization and evolution of multifunctional autoprocessing repeats-in-toxin (MARTX) toxin in Vibrio vulnificus

The objective of this study was to analyze multifunctional autoprocessing repeats-in-toxin (MARTX) toxin domain organization within the aquatic species Vibrio vulnificus as well as to study the evolution of the rtxA1 gene. The species is subdivided into three biotypes that differ in host range and geographical distribution. We have found three different types (I, II, and III) of V. vulnificus MARTX (MARTX(Vv)) toxins with common domains (an autocatalytic cysteine protease domain [CPD], an α/β-hydrolase domain, and a domain resembling that of the LifA protein of Escherichia coli O127:H6 E2348/69 [Efa/LifA]) and specific domains (a Rho-GTPase inactivation domain [RID], a domain of unknown function [DUF], a domain resembling that of the rtxA protein of Photorhabdus asymbiotica [rtxA(PA)], and an actin cross-linking domain [ACD]). Biotype 1 isolates harbor MARTX(Vv) toxin types I and II, biotype 2 isolates carry MARTX(Vv) toxin type III, and biotype 3 isolates have MARTX(Vv) toxin type II. The analyzed biotype 2 isolates harbor two identical copies of rtxA1, one chromosomal and the other plasmidic. The evolutionary history of the gene demonstrates that MARTX(Vv) toxins are mosaics, comprising pieces with different evolutionary histories, some of which have been acquired by intra- or interspecific horizontal gene transfer. Finally, we have found evidence that the evolutionary history of the rtxA1 gene for biotype 2 differs totally from the gene history of biotypes 1 and 3.     

Crystal Structure of the VgrG1 Actin Cross-linking Domain of the Vibrio cholerae Type VI Secretion System

Vibrio cholerae is the cause of the diarrheal disease cholera. V. cholerae produces RtxA, a large toxin of the MARTX family, which is targeted to the host cell cytosol, where its actin cross-linking domain (ACD) cross-links G-actin, leading to F-actin depolymerization, cytoskeleton rearrangements, and cell rounding. These effects on the cytoskeleton prevent phagocytosis and bacterial engulfment by macrophages, thus preventing V. cholerae clearance from the gut. The V. cholerae Type VI secretion-associated VgrG1 protein also contains a C-terminal ACD, which shares 61% identity with MARTX ACD and has been shown to covalently cross-link G-actin. Here, we purified the VgrG1 C-terminal domain and determined its crystal structure. The VgrG1 ACD exhibits a V-shaped three-dimensional structure, formed of 12 β-strands and nine α-helices. Its active site comprises five residues that are conserved in MARTX ACD toxin, within a conserved area of ∼10 Å radius. We showed that less than 100 ACD molecules are sufficient to depolymerize the actin filaments of a fibroblast cell in vivo. Mutagenesis studies confirmed that Glu-16 is critical for the F-actin depolymerization function. Co-crystals with divalent cations and ATP reveal the molecular mechanism of the MARTX/VgrG toxins and offer perspectives for their possible inhibition.     

Aeromonas exoenzyme T of Aeromonas salmonicida is a bifunctional protein that targets the host cytoskeleton

Type III protein secretion has been shown recently to be important in the virulence of the fish pathogen Aeromonas salmonicida. The ADP-ribosylating toxin Aeromonas exoenzyme T (AexT) is one effector protein targeted for secretion via this system. In this study, we identified muscular and nonmuscular actin as substrates of the ADP-ribosylating activity of AexT. Furthermore, we show that AexT also functions as a GTPase-activating protein (GAP), displaying GAP activity against monomeric GTPases of the Rho family, specifically Rho, Rac, and Cdc42. Transfection of fish cells with wild type AexT resulted in depolymerization of the actin cytoskeleton and cell rounding. Point mutations within either the GAP or the ADP-ribosylating active sites of AexT (Arg-143 as well as Glu-398 and Glu-401, respectively) abolished enzymatic activity, yet did not prevent actin filament depolymerization. However, inactivation of the two catalytic sites simultaneously did. These results suggest that both the GAP and ADP-ribosylating domains of AexT contribute to its biological activity. This is the first bacterial virulence factor to be described that has a specific actin ADP-ribosylation activity and GAP activity toward Rho, Rac, and Cdc42, both enzymatic activities contributing to actin filament depolymerization.     

Protein kinase C signaling regulates ZO-1 translocation and increased paracellular flux of T84 colonocytes exposed to Clostridium difficile toxin A.

Clostridium difficile toxin A increases paracellular permeability in colonic epithelial T84 cells by mechanisms involving RhoA glucosylation and actin depolymerization. However, we previously observed that toxin A-mediated decline in transepithelial electrical resistance preceded changes in cell morphology and tight junction ultrastructure (Hecht, G., Pothoulakis, C., LaMont, J. T., and Madara, J. L. (1988) J. Clin. Invest. 82, 1516-1524). Recent studies also showed that C. difficile toxins induce early cellular responses, including activation of mitogen-activated protein kinases, generation of reactive oxygen metabolites, and calcium influx. The aim of this study was to investigate whether toxin A-induced early cellular responses contribute to the permeability changes. We found that toxin A stimulated the activities of membrane and cytosolic protein kinase Calpha (PKCalpha) and cytosolic PKCbeta. A specific PKCalpha/beta antagonist (myristoylated PKCalpha/beta peptide) blocked toxin A-mediated RhoA glucosylation. Furthermore, decreased transepithelial electrical resistance and increased translocation of ZO-1 from tight junction occurred within 2-3 h of toxin A exposure and were also inhibited by PKCalpha/beta antagonist. During this time period, toxin exposure did not induce translocation of ZO-2, dephosphorylation or translocation of occludin, or cell rounding. Our data indicate that PKC signaling regulates toxin A-mediated paracellular permeability changes and ZO-1 translocation.     

Uptake of binary actin ADP-ribosylating toxins

The focus of this article is on the cellular uptake mechanism of the family of binary actin ADP-ribosylating toxins from clostridia. These toxins are special-type AB toxins, because they are composed of two nonlinked proteins, which have to assemble on the surface of eukaryotic cells to act cytotoxically. The enzymatically active component (A), ADP-ribosylates G-actin in the cytosol of target cells. This leads to a complete depolymerization of the actin filaments and, thereby, to rounding up of cultured cells. The second component of these toxins, the binding/translocation component (B), mediates the transport of the enzyme component into the cytosol.     

Cross-reactivity of anthrax and C2 toxin: protective antigen promotes the uptake of botulinum C2I toxin into human endothelial cells

Binary toxins are among the most potent bacterial protein toxins performing a cooperative mode of translocation and exhibit fatal enzymatic activities in eukaryotic cells. Anthrax and C2 toxin are the most prominent examples for the AB(7/8) type of toxins. The B subunits bind both host cell receptors and the enzymatic A polypeptides to trigger their internalization and translocation into the host cell cytosol. C2 toxin is composed of an actin ADP-ribosyltransferase (C2I) and C2II binding subunits. Anthrax toxin is composed of adenylate cyclase (EF) and MAPKK protease (LF) enzymatic components associated to protective antigen (PA) binding subunit. The binding and translocation components anthrax protective antigen (PA(63)) and C2II of C2 toxin share a sequence homology of about 35%, suggesting that they might substitute for each other. Here we show by conducting in vitro measurements that PA(63) binds C2I and that C2II can bind both EF and LF. Anthrax edema factor (EF) and lethal factor (LF) have higher affinities to bind to channels formed by C2II than C2 toxin's C2I binds to anthrax protective antigen (PA(63)). Furthermore, we could demonstrate that PA in high concentration has the ability to transport the enzymatic moiety C2I into target cells, causing actin modification and cell rounding. In contrast, C2II does not show significant capacity to promote cell intoxication by EF and LF. Together, our data unveiled the remarkable flexibility of PA in promoting C2I heterologous polypeptide translocation into cells.     

The chaperone Hsp90 and PPIases of the cyclophilin and FKBP families facilitate membrane translocation of Photorhabdus luminescens ADP‐ribosyltransferases

TccC3 and TccC5 from Photorhabdus luminescens are ADP‐ribosyltransferases, which modify actin and Rho GTPases, respectively, thereby inducing polymerization and clustering of actin. The bacterial proteins are components of the Photorhabdus toxin complexes, consisting of the binding and translocation component TcdA1, a proposed linker component TcdB2 and the enzymatic component TccC3/5. While the action of the toxins on target proteins is clearly defined, uptake and translocation of the toxins into the cytosol of target cells are not well understood. Here we show by using pharmacological inhibitors that heat shock protein 90 (Hsp90) and peptidyl prolyl cis/trans isomerases (PPIases) including cyclophilins and FK506‐binding proteins (FKBPs) facilitate the uptake of the ADP‐ribosylating toxins into the host cell cytosol. Inhibition of Hsp90 and/or PPIases resulted in decreased intoxication of target cells by Photorhabdus toxin complexes determined by cell rounding and reduction of transepithelial electrical resistance of cell monolayers. ADP‐ribosyltransferase activity of toxins and toxin‐induced pore formation were notimpaired by the inhibitors of Hsp90 and PPIases. The Photorhabdus toxins interacted with Hsp90, FKBP51, Cyp40 and CypA, suggesting a role of these host cell factors in translocation and/or refolding of the ADP‐ribosyltransferases.