Cholera- and anthrax-like toxins are among several new ADP-ribosyltransferases

Chelt, a cholera-like toxin from Vibrio cholerae, and Certhrax, an anthrax-like toxin from Bacillus cereus, are among six new bacterial protein toxins we identified and characterized using in silico and cell-based techniques. We also uncovered medically relevant toxins from Mycobacterium avium and Enterococcus faecalis. We found agriculturally relevant toxins in Photorhabdus luminescens and Vibrio splendidus. These toxins belong to the ADP-ribosyltransferase family that has conserved structure despite low sequence identity. Therefore, our search for new toxins combined fold recognition with rules for filtering sequences--including a primary sequence pattern--to reduce reliance on sequence identity and identify toxins using structure. We used computers to build models and analyzed each new toxin to understand features including: structure, secretion, cell entry, activation, NAD+ substrate binding, intracellular target binding and the reaction mechanism. We confirmed activity using a yeast growth test. In this era where an expanding protein structure library complements abundant protein sequence data--and we need high-throughput validation--our approach provides insight into the newest toxin ADP-ribosyltransferases.     

In vivo covalent cross-linking of cellular actin by the Vibrio cholerae RTX toxin

Enteric pathogens often export toxins that elicit diarrhea as a part of the etiology of disease, including toxins that affect cytoskeletal structure. Recently, we discovered that the intestinal pathogen Vibrio cholerae elicits rounding of epithelial cells that is dependent upon a gene we designated rtxA. Here we investigate the association of rtxA with the cell-rounding effect. We find that V. cholerae exports a large toxin, RTX (repeats-in-toxin) toxin, to culture supernatant fluids and that this toxin is responsible for cell rounding. Furthermore, we find that cell rounding is not due to necrosis, suggesting that RTX toxin is not a typical member of the RTX family of pore-forming toxins. Rather, RTX toxin causes depolymerization of actin stress fibers and covalent cross-linking of cellular actin into dimers, trimers and higher multimers. This RTX toxin-specific cross-linking occurs in cells previously rounded with cytochalasin D, indicating that G-actin is the toxin target. Although several models explain our observations, our simultaneous detection of actin cross-linking and depolymerization points toward a novel mechanism of action for RTX toxin, distinguishing it from all other known toxins.     

Retrograde transport of protein toxins through the Golgi apparatus

A number of protein toxins from plants and bacteria take advantage of transport through the Golgi apparatus to gain entry into the cytosol where they exert their action. These toxins include the plant toxin ricin, the bacterial Shiga toxins, and cholera toxin. Such toxins bind to lipids or proteins at the cell surface, and they are endocytosed both by clathrin-dependent and clathrin-independent mechanisms. Sorting to the Golgi and retrograde transport to the endoplasmic reticulum (ER) are common to these toxins, but the exact mechanisms turn out to be toxin and cell-type dependent. In the ER, the enzymatically active part is released and then transported into the cytosol, exploiting components of the ER-associated degradation system. In this review, we will discuss transport of different protein toxins, but we will focus on factors involved in entry and sorting of ricin and Shiga toxin into and through the Golgi apparatus.     

Detection of RTX toxin genes in gram-negative bacteria with a set of specific probes.

The family of RTX (RTX representing repeats in the structural toxin) toxins is composed of several protein toxins with a characteristic nonapeptide glycine-rich repeat motif. Most of its members were shown to have cytolytic activity. By comparing the genetic relationships of the RTX toxin genes we established a set of 10 gene probes to be used for screening as-yet-unknown RTX toxin genes in bacterial species. The probes include parts of apxIA, apxIIA, and apxIIIA from Actinobacillus pleuropneumoniae, cyaA from Bordetella pertusis, frpA from Neisseria meningitidis, prtC from Erwinia chrysanthemi, hlyA and elyA from Escherichia coli, aaltA from Actinobacillus actinomycetemcomitans and lktA from Pasteurella haemolytica. A panel of pathogenic and nonpathogenic gram-negative bacteria were investigated for the presence of RTX toxin genes. The probes detected all known genes for RTX toxins. Moreover, we found potential RTX toxin genes in several pathogenic bacterial species for which no such toxins are known yet. This indicates that RTX or RTX-like toxins are widely distributed among pathogenic gram-negative bacteria. The probes generated by PCR and the hybridization method were optimized to allow broad-range screening for RTX toxin genes in one step. This included the binding of unlabelled probes to a nylon filter and subsequent hybridization of the filter with labelled genomic DNA of the strain to be tested. The method constitutes a powerful tool for the assessment of the potential pathogenicity of poorly characterized strains intended to be used in biotechnological applications. Moreover, it is useful for the detection of already-known or new RTX toxin genes in bacteria of medical importance.     

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.     

Protein toxins from plants and bacteria: Probes for intracellular transport and tools in medicine

A number of protein toxins produced by bacteria and plants enter eukaryotic cells and inhibit protein synthesis enzymatically. These toxins include the plant toxin ricin and the bacterial toxin Shiga toxin, which we will focus on in this article. Although a threat to human health, toxins are valuable tools to discover and characterize cellular processes such as endocytosis and intracellular transport. Bacterial infections associated with toxin production are a problem worldwide. Increased knowledge about toxins is important to prevent and treat these diseases in an optimal way. Interestingly, toxins can be used for diagnosis and treatment of cancer.     

Insulinotropic toxins as molecular probes for analysis of glucagon-likepeptide-1 receptor-mediated signal transduction in pancreatic beta-cells

Cholera toxin, pertussis toxin, mastoparan, maitotoxin, and alpha-latrotoxin are complex protein or polyether-based toxins of bacterial, insect, or phytoplankton origin that act with high potency at the endocrine pancreas to stimulate secretion of insulin from beta-cells located in the islets of Langerhans. The remarkable insulinotropic properties of these toxins have attracted considerable attention by virtue of their use as selective molecular probes for analyses of beta-cell stimulus-secretion coupling. Targets of the toxins include heptahelical cell surface receptors, GTP-binding proteins, ion channels, Ca(2+) stores, and the exocytotic secretory apparatus. Here we review the value of insulinotropic toxins from the perspective of their established use in the study of signal transduction pathways activated by the blood glucose-lowering hormone glucagon-like peptide-1 (GLP-1). Our analysis of one insulinotropic toxin (alpha-latrotoxin) leads us to conclude that there exists a process of molecular mimicry whereby the 'lock and key'analogy inherent to hormone-receptor interactions is reproduced by a toxin related in structure to GLP-1.     

A system for the directed evolution of the insecticidal protein from Bacillus thuringiensis

Theoretically, the activity of AB-type toxin molecules such as the insecticidal toxin (Cry toxin) from B. thuringiensis, which have one active site and two binding site, is improved in parallel with the binding affinity to its receptor. In this experiment, we tried to devise a method for the directed evolution of Cry toxins to increase the binding affinity to the insect receptor. Using a commercial T7 phage-display system, we expressed Cry1Aa toxin on the phage surface as fusions with the capsid protein 10B. These recombinant phages bound to a cadherin-like protein that is one of the Cry1Aa toxin receptors in the model target insect Bombyx mori. The apparent affinity of Cry1Aa-expressing phage for the receptor was higher than that of Cry1Ab-expressing phage. Phages expressing Cry1Aa were isolated from a mixed suspension of phages expressing Cry1Ab and concentrated by up to 130,000-fold. Finally, random mutations were made in amino acid residues 369–375 in domain 2 of Cry1Aa toxin, the mutant toxins were expressed on phages, and the resulting phage library was screened with cadherin-like protein-coated beads. As a result, phages expressing abnormal or low-affinity mutant toxins were excluded, and phages with high-affinity mutant toxins were selected. These results indicate that a method combining T7 phage display with selection using cadherin-like protein-coated magnetic beads can be used to increase the activity of easily obtained, low-activity Cry toxins from bacteria.     

Localization of the voltage-sensor toxin receptor on KvAP

A variety of venomous animals produce small protein toxins that impair the function of voltage-dependent cation channels by affecting the motions of the voltage-sensor domains and altering the energetics of the opening of the channel. In this study, we investigate the location of the receptor for tarantula venom voltage-sensor toxins on the voltage-dependent K+ channel from Aeropyrum pernix (KvAP), an archeabacterial channel that is functionally inhibited by members of this toxin family. We show that it is possible to purify the same set of toxins from venom of the tarantula Grammostola spatulata using either the purified KvAP voltage-sensor domain or the full-length KvAP channel. The equivalence of toxin retention profiles for the two channel proteins implies that the tarantula voltage-sensor toxin receptor resides exclusively on the voltage-sensor domain and that the pore is not required for the toxin-channel interaction. We have identified and characterized the functional properties of a subset of the tarantula toxins that bind to the KvAP voltage-sensor domain. Some of these toxins, VSTX1 and GSMTX4, have been previously isolated, while others, VSTX2 and VSTX3, are new members of the tarantula voltage-sensor toxin family. Some but not all toxins that bind to the voltage-sensor domain affect voltage-dependent gating of KvAP channels in lipid membranes.     

Role of receptor interaction in the mode of action of insecticidal Cry and Cyt toxins produced by Bacillus thuringiensis

Cry toxins from Bacillus thuringiensis are used for insect control. Their primary action is to lyse midgut epithelial cells. In this review we will summarize recent findings on the Cry toxin-receptor interaction and the role of receptor recognition in their mode of action. Cry toxins interact sequentially with multiple receptors. In lepidopteran insects, Cry1A monomeric toxins interact with the first receptor and this interaction triggers oligomerization of the toxins. The oligomer then interacts with second receptor inducing insertion into membrane microdomains and larval death. In the case of mosquitocidal toxins, Cry and Cyt toxins play a part. These toxins have a synergistic effect and Cyt1Aa overcomes Cry toxin resistance. Recently, it was proposed that Cyt1Aa synergizes or suppresses resistance to Cry toxins by functioning as a membrane-bound receptor for Cry toxin.     

Scorpion toxins from Centruroides noxius and Tityus serrulatus. Primary structures and sequence comparison by metric analysis.

The complete primary structures of toxin II-14 from the Mexican scorpion Centruroides noxius Hoffmann and toxin gamma from the Brazilian scorpion Tityus serrulatus Lutz and Mello have been determined. Cleavage of toxin gamma after Met-6 with CNBr produced the 55-residue peptide 7-61, which maintained the four disulphide bonds but was not toxic to mice at a dose 3 times the lethal dose of native toxin gamma. Pairwise comparison by metric analysis of segment 1-50 of toxin gamma and the corresponding segments from two other South American scorpion toxins, five North American scorpion toxins, nine North African scorpion toxins and one Central Asian scorpion toxin showed that the three Brazilian toxins are intermediate between the North American and North African toxins. This result is consistent with the hypothesis that the South American and African continents were joined by a land connection in the distant past.     

Lipid requirements for entry of protein toxins into cells

The plant toxin ricin and the bacterial toxin Shiga toxin both belong to a group of protein toxins having one moiety that binds to the cell surface, and another, enzymatically active moiety, that enters the cytosol and inhibits protein synthesis by inactivating ribosomes. Both toxins travel all the way from the cell surface to endosomes, the Golgi apparatus and the ER before the ribosome-inactivating moiety enters the cytosol. Shiga toxin binds to the neutral glycosphingolipid Gb3 at the cell surface and is therefore dependent on this lipid for transport into the cells, whereas ricin binds both glycoproteins and glycolipids with terminal galactose. The different steps of transport used by these toxins have specific requirements for lipid species, and with the recent developments in mass spectrometry analysis of lipids and microscopical and biochemical dissection of transport in cells, we are starting to see the complexity of endocytosis and intracellular transport. In this article we describe lipid requirements and the consequences of lipid changes for the entry and intoxication with ricin and Shiga toxin. These toxins can be a threat to human health, but can also be exploited for diagnosis and therapy, and have proven valuable as tools to study intracellular transport.     

p97 Is in a complex with cholera toxin and influences the transport of cholera toxin and related toxins to the cytoplasm

Certain protein toxins, including cholera toxin, ricin, and Pseudomonas aeruginosa exotoxin A, are transported to the lumen of the endoplasmic reticulum where they retro-translocate across the endoplasmic reticulum membrane to enter the cytoplasm. The mechanism of retrotranslocation is poorly understood but may involve the endoplasmic reticulum-associated degradation pathway. The AAA ATPase p97 (also called valosin-containing protein) participates in the retro-translocation of cellular endoplasmic reticulum-associated degradation substrates and is therefore a candidate to participate in the retrotranslocation of protein toxins. To investigate whether p97 functions in toxin delivery to the cytoplasm, we measured the sensitivity to toxins of cells expressing either wild-type p97 or a dominant ATPase-defective p97 mutant under control of a tetracycline-inducible promoter. The rate at which cholera toxin and related toxins entered the cytoplasm was reduced in cells expressing the ATPase-defective p97, suggesting that the toxins might interact with p97. To detect interaction, the cholera toxin A chain was immunoprecipitated from cholera toxin-treated Vero cells, and co-immunoprecipitation of p97 was assessed by immunoblotting. The immunoprecipitates contained both cholera toxin A chain and p97, evidence that the two proteins are in a complex. Altogether, these results provide functional and structural evidence that p97 participates in the transport of cholera toxin to the cytoplasm.     

Exchange of a single amino acid switches the substrate properties of RhoA and RhoD toward glucosylating and transglutaminating toxins

Rho GTPases are the preferred targets of various bacterial cytotoxins, including Clostridium difficile toxins A and B, Clostridium sordellii lethal toxin, the cytotoxic necrotizing factors (CNF1) from Escherichia coli, and the dermonecrotizing toxin (DNT) from Bordetella species. The toxins inactivate or activate specific sets of Rho GTPases by mono-O-glucosylation and deamidation/transglutamination, respectively. Here we studied the structural basis of the recognition of RhoA, which is modified by toxin B, CNF1, and DNT, in comparison with RhoD, which is solely a substrate for lethal toxin. We found that a single amino acid residue in RhoA and RhoD defines the substrate specificity for toxin B and lethal toxin. Change of serine 73 to phenylalanine in RhoA turned RhoA into a substrate for lethal toxin. Accordingly, change of the equivalently positioned phenylalanine 85 in RhoD with serine allowed glucosylation by toxin B. Comparable results were achieved with the Rho-activating and transglutaminating enzymes CNF1 and DNT. Here, amino acid glutamate 64 of RhoA and the equivalent aspartate 76 of RhoD define substrate specificity for CNF1 and DNT, respectively. These data indicate that single amino acid residues located in the switch II region of Rho proteins determine enzyme specificity for diverse bacterial toxins.     

Production and excretion of okadaic acid,pectenotoxin-2 and a novel dinophysistoxin from the DSP-causing marine dinoflagellate Dinophysis acuta – Effects of light,food availability and growth phase

Diarrhetic shellfish poisoning (DSP) toxins constitute a severe economic threat to shellfish industries and a major food safety issue for shellfish consumers. The prime producers of the DSP toxins that end up in filter feeding shellfish are species of the marine mixotrophic dinoflagellate genus Dinophysis. Intraspecific toxin contents of Dinophysis spp. vary a lot, but the regulating factors of toxin content are still poorly understood. Dinophysis spp. have been shown to sequester and use chloroplasts from their ciliate prey, and with this rare mode of nutrition, irradiance and food availability could play a key role in the regulation of toxins contents and production. We investigated toxin contents, production and excretion of a Dinophysis acuta culture under different irradiances, food availabilities and growth phases. The newly isolated strain of D. acuta contained okadaic acid (OA), pectenotoxins-2 (PTX-2) and a novel dinophysistoxin (DTX) that we tentatively describe as DTX-1b isomer. We found that all three toxins were excreted to the surrounding seawater, and for OA and DTX-1b as much as 90% could be found in extracellular toxin pools. For PTX-2 somewhat less was excreted, but often >50% was found extracellularly. This was the case both in steady-state exponential growth and in food limited, stationary growth, and we emphasize the need to include extracellular toxins in future studies of DSP toxins. Cellular toxin contents were largely unaffected by irradiance, but toxins accumulated both intra- and extracellularly when starvation reduced growth rates of D. acuta. Toxin production rates were highest during exponential growth, but continued at decreased rates when cell division ceased, indicating that toxin production is not directly associated with ingestion of prey. Finally, we explore the potential of these new discoveries to shed light on the ecological role of DSP toxins.     

Action of different toxins from the scorpion Androctonus australis on a locust nerve-muscle preparation

The neuromuscular effects of four purified toxins and crude venom from the scorpion Androctonus australis were investigated in the extensor tibiae nerve-muscle preparation of the locust Locusta migratoria. Insect and crustacean toxin and the mammal toxins I and II which have previously been shown to act on fly larvae, isopods, and mice all paralyse locust larvae. The paralytic potencies decrease in the following order: insect toxin → mammal toxin I → crustacean toxin → mammal toxin II.The toxins and crude venom cause repetitive activity of the motor axons. This leads to long spontaneous trains of junction potentials in the case of crude venom and insect toxin. The other toxins chiefly cause short bursts of action and junction potentials following single stimuli.The ‘slow’ excitatory motor axon invariably is affected sooner than the inhibitory or the ‘fast’ excitatory one. The minimal doses of toxins required to affect the ‘slow’ motor axon decrease in an order somewhat different from that established for their paralytic potencies: insect toxin → crustacean toxin → mammal toxin I → mammal toxin II.Crude venom depolarises and destabilises the muscle membrane potential at low doses. At high doses it decreases the membrane resistance, whereas insect toxin leads to an increase.Crude venom and insect toxin enhance the frequency of mejps, whereas mammal toxin I leads to the occurrence of ‘giant’ mejps.The pattern of axonal activities indicates that the various peripheral branches of the motor nerve are the primary target of the toxins.The time course of nerve action potentials is affected by mammal toxin I and crustacean toxin which cause anomalous shapes and prolongations not caused by insect toxin.The results with other animals suggest that only the insect toxin is selective in its activity. The way it affects the axon might be quite different from that previously reported for scorpion venoms or toxins.     

Polymeric IgA is superior to monomeric IgA and IgG carrying the same variable domain in preventing Clostridium difficile toxin A damaging of T84 monolayers

The two exotoxins A and B produced by Clostridium difficile are responsible for antibiotic-associated enterocolitis in human and animals. When added apically to human colonic carcinoma-derived T84 cell monolayers, toxin A, but not toxin B, abolished the transepithelial electrical resistance and altered the morphological integrity. Apical addition of suboptimal concentration of toxin A made the cell monolayer sensitive to toxin B. Both toxins induced drastic and rapid epithelial alterations when applied basolaterally with a complete disorganization of tight junctions and vacuolization of the cells. Toxin A-specific IgG2a from hybridoma PCG-4 added apically with toxin A alone or in combination with toxin B abolished the toxin-induced epithelial alterations for up to 8 h. The Ab neutralized basolateral toxin A for 4 h, but not the mixture of the two toxins. Using an identical Ab:Ag ratio, we found that recombinant polymeric IgA (IgAd/p) with the same Fv fragments extended protection against toxin A for at least 24 h in both compartments. In contrast, the recombinant monomeric IgA counterpart behaved as the PCG-4 IgG2a Ab. The direct comparison between different Ig isotype and molecular forms, but of unique specificity, demonstrates that IgAd/p Ab is more efficient in neutralizing toxin A than monomeric IgG and IgA. We conclude that immune protection against C. difficile toxins requires toxin A-specific secretory Abs in the intestinal lumen and IgAd/p specific for both toxins in the lamina propria.     

Binding sites for Bacillus thuringiensis Cry2Ae toxin on heliothine brush border membrane vesicles are not shared with Cry1A, Cry1F, or Vip3A toxin

The use of combinations of Bacillus thuringiensis (Bt) toxins with diverse modes of action for insect pest control has been proposed as the most efficient strategy to increase target range and delay the onset of insect resistance. Considering that most cases of cross-resistance to Bt toxins in laboratory-selected insect colonies are due to alteration of common toxin binding sites, independent modes of action can be defined as toxins sharing limited or no binding sites in brush border membrane vesicles (BBMV) prepared from the target insect larvae. In this paper, we report on the specific binding of Cry2Ae toxin to binding sites on BBMV from larvae of the three most commercially relevant heliothine species, Heliothis virescens, Helicoverpa zea, and Helicoverpa armigera. Using chromatographic purification under reducing conditions before labeling, we detected specific binding of radiolabeled Cry2Ae, which allowed us to perform competition assays using Cry1Ab, Cry1Ac, Cry1Fa, Vip3A, Cry2Ae, and Cry2Ab toxins as competitors. In these assays, Cry2Ae binding sites were shared with Cry2Ab but not with the tested Cry1 or Vip3A toxins. Our data support the use of Cry2Ae toxin in combination with Cry1 or Vip3A toxins in strategies to increase target range and delay the onset of heliothine resistance.     

Understanding policing as a mechanism of cheater control in cooperating bacteria

Policing occurs in insect, animal and human societies, where it evolved as a mechanism maintaining cooperation. Recently, it has been suggested that policing might even be relevant in enforcing cooperation in much simpler organisms such as bacteria. Here, we used individual‐based modelling to develop an evolutionary concept for policing in bacteria and identify the conditions under which it can be adaptive. We modelled interactions between cooperators, producing a beneficial public good, cheaters, exploiting the public good without contributing to it, and public good‐producing policers that secrete a toxin to selectively target cheaters. We found that toxin‐mediated policing is favoured when (a) toxins are potent and durable, (b) toxins are cheap to produce, (c) cell and public good diffusion is intermediate, and (d) toxins diffuse farther than the public good. Although our simulations identify the parameter space where toxin‐mediated policing can evolve, we further found that policing decays when the genetic linkage between public good and toxin production breaks. This is because policing is itself a public good, offering protection to toxin‐resistant mutants that still produce public goods, yet no longer invest in toxins. Our work thus highlights that not only specific environmental conditions are required for toxin‐mediated policing to evolve, but also strong genetic linkage between the expression of public goods, toxins and toxin resistance is essential for this mechanism to remain evolutionarily stable in the long run.     

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.