Structure of the claudin-binding domain of Clostridium perfringens enterotoxin

Clostridium perfringens enterotoxin is a common cause of food-borne and antibiotic-associated diarrhea. The toxin's receptors on intestinal epithelial cells include claudin-3 and -4, members of a large family of tight junction proteins. Toxin-induced cytolytic pore formation requires residues in the NH(2)-terminal half, whereas residues near the COOH terminus are required for binding to claudins. The claudin-binding COOH-terminal domain is not toxic and is currently under investigation as a potential drug absorption enhancer. Because claudin-4 is overexpressed on some human cancers, the toxin is also being investigated for targeting chemotherapy. Our aim was to solve the structure of the claudin-binding domain to advance its therapeutic applications. The structure of a 14-kDa fragment containing residues 194 to the native COOH terminus at position 319 was solved by x-ray diffraction to a resolution of 1.75A. The structure is a nine-strand beta sandwich with previously unappreciated similarity to the receptor-binding domains of several other toxins of spore-forming bacteria, including the collagen-binding domain of ColG from Clostridium histolyticum and the large Cry family of toxins (including Cry4Ba) of Bacillus thuringiensis. Correlations with previous studies suggest that the claudin-4 binding site is on a large surface loop between strands beta8 and beta9 or includes these strands. The sequence that was crystallized (residues 194-319) binds to purified human claudin-4 with a 1:1 stoichiometry and affinity in the submicromolar range similar to that observed for binding of native toxin to cells. Our results provide a structural framework to advance therapeutic applications of the toxin and suggest a common ancestor for several receptor-binding domains of bacterial toxins.     

Efficient extracellular production of hybrid E. coli heat-labile enterotoxin B subunits in a marine vibrio

Abstract Escherichia coli heat-labile enterotoxin B subunit (EtxB) has been proposed as a potential protein carrier for the delivery of heterologous peptides to target cells, particularly for the oral delivery of epitopes to the mucosal immune system. In this study, two extensions to the C-terminus of EtxB were genetically engineered that correspond to a well-characterized neutralising epitope of glycoprotein D from herpes simplex virus (EtxB-gD) and to the C-terminal nine amino acids from the 38 kDa subunit of HSV-encoded ribonucleotide reductase (EtxB-R2). Here we describe the extracellular secretion of the two hybrid EtxBs from a marine Vibrio harbouring a broad-host range inducible expression vector containing the hybrid genes. Large amounts of intact fusion proteins (15–20 mg per liter of culture) were secreted into the medium upon induction. These hybrid proteins maintained the receptor-binding activity of the native toxin as well as being cross-reactive with anti-EtxB and anti-heterologous peptide monoclonal antibodies.     

Structure of Cry2Aa suggests an unexpected receptor binding epitope

BACKGROUND: Genetically modified (GM) crops that express insecticidal protein toxins are an integral part of modern agriculture. Proteins produced by Bacillus thuringiensis (Bt) during sporulation mediate the pathogenicity of Bt toward a spectrum of insect larvae whose breadth depends upon the Bt strain. These transmembrane channel-forming toxins are stored in Bt as crystalline inclusions called Cry proteins. These proteins are the active agents used in the majority of biorational pesticides and insect-resistant transgenic crops. Though Bt toxins are promising as a crop protection alternative and are ecologically friendlier than synthetic organic pesticides, resistance to Bt toxins by insects is recognized as a potential limitation to their application. RESULTS: We have determined the 2.2 A crystal structure of the Cry2Aa protoxin by multiple isomorphous replacement. This is the first crystal structure of a Cry toxin specific to Diptera (mosquitoes and flies) and the first structure of a Cry toxin with high activity against larvae from two insect orders, Lepidoptera (moths and butterflies) and Diptera. Cry2Aa also provides the first structure of the proregion of a Cry toxin that is cleaved to generate the membrane-active toxin in the larval gut. CONCLUSIONS: The crystal structure of Cry2Aa reported here, together with chimeric-scanning and domain-swapping mutagenesis, defines the putative receptor binding epitope on the toxin and so may allow for alteration of specificity to combat resistance or to minimize collateral effects on nontarget species. The putative receptor binding epitope of Cry2Aa identified in this study differs from that inferred from previous structural studies of other Cry toxins.     

Bacillus thuringiensis (Bt) toxin susceptibility and isolation of resistance mutants in the nematode Caenorhabditis elegans

The protein toxins produced by Bacillus thuringiensis (Bt) are the most widely used natural insecticides in agriculture. Despite successful and extensive use of these toxins in transgenic crops, little is known about toxicity and resistance pathways in target insects since these organisms are not ideal for molecular genetic studies. To address this limitation and to investigate the potential use of these toxins to control parasitic nematodes, we are studying Bt toxin action and resistance in Caenorhabditis elegans. We demonstrate for the first time that a single Bt toxin can target a nematode. When fed Bt toxin, C. elegans hermaphrodites undergo extensive damage to the gut, a decrease in fertility, and death, consistent with toxin effects in insects. We have screened for and isolated 10 recessive mutants that resist the toxin's effects on the intestine, on fertility, and on viability. These mutants define five genes, indicating that more components are required for Bt toxicity than previously known. We find that a second, unrelated nematicidal Bt toxin may utilize a different toxicity pathway. Our data indicate that C. elegans can be used to undertake detailed molecular genetic analysis of Bt toxin pathways and that Bt toxins hold promise as nematicides.     

Opportunities for structure-based design of protease-directed drugs

As a result of the recent enormous technological progress, experimental structure determination has become an integral part of the development of drugs against disease-related target proteins. The post-translational modification of proteins is an important regulatory process in living organisms; one such example is lytic processing by peptidases. Many different peptidases represent disease targets and are being used in structure-based drug design approaches. The development of drugs such as aliskiren and tipranavir, which inhibit renin and HIV protease, respectively, testifies to the success of this approach.     

Expression and purification of functional Clostridium perfringens alpha and epsilon toxins in Escherichia coli

The alpha and epsilon toxins are 2 of the 4 major lethal toxins of the pathogen Clostridium perfringens. In this study, the expression of the epsilon toxin (etx) gene of C. perfringens was optimized by replacing rare codons with high-frequency codons, and the optimized gene was synthesized using overlapping PCR. Then, the etx gene or the alpha-toxin gene (cpa) was individually inserted into the pTIG-Trx expression vector with a hexahistidine tag and a thioredoxin (Trx) to facilitate their purification and induce the expression of soluble proteins. The recombinant alpha toxin (rCPA) and epsilon toxin (rETX) were highly expressed as soluble forms in the recipient Escherichia coli BL21 strain, respectively. The rCPA and rETX were purified using Ni(2+)-chelating chromatography and size-exclusion chromatography. And the entire purification process recovered about 40% of each target protein from the starting materials. The purified target toxins formed single band at about 42kDa (rCPA) or 31kDa (rETX) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their functional activity was confirmed by bioactivity assays. We have shown that the production of large amounts of soluble and functional proteins by using the pTIG-Trx vector in E. coli is a good alternative for the production of native alpha and epsilon toxins and could also be useful for the production of other toxic proteins with soluble forms.     

Analysis of the Actinobacillus actinomycetemcomitans leukotoxin gene. Delineation of unique features and comparison to homologous toxins

Actinobacillus actinomycetemcomitans leukotoxin has been implicated as a virulence factor in human infections. To initiate delineation of leukotoxin structure/function relationships, molecular cloning of the leukotoxin gene was carried out. When an A. actinomycetemcomitans genomic DNA library in lambda EMBL3 was screened using a 1.3-kilobase pair restriction fragment containing a portion of the leukotoxin gene, 13 positive recombinants were identified. One recombinant, designated lambda OP8, containing a 16-kilobase pair insert was selected for detailed study. Lysates from lambda OP8, but not control lysates, exhibited leukotoxic activity with target cell specificity identical to the native toxin. Western blots identified the recombinant-produced toxin as a 125-kDa protein doublet identical in mobility to the native toxin. Restriction enzyme and extensive DNA analyses demonstrated that the leukotoxin gene showed strong homology to two other toxins produced by Escherichia coli and Pasteurella haemolytica. As in the other two species, the A. actinomycetemcomitans toxin is contained in a cluster of four genes in which the A gene encodes the toxin and the products of the B, C, and D genes are involved in posttranslational modification of the toxin and its membrane insertion and secretion. The target cell specificity of the A. actinomycetemcomitans toxin differs from the other two toxins and is restricted to human and some non-human primate cells of the monomyelocytic lineage. The A. actinomycetemcomitans leukotoxin is not secreted but remains associated with the bacterial membrane, possibly through a hydrophobic domain at the carboxyl terminus which distinguishes it from the E. coli and P. haemolytica toxins.     

Structure and Orientation of a Voltage-Sensor Toxin in Lipid Membranes

Amphipathic protein toxins from tarantula venom inhibit voltage-activated potassium (Kv) channels by binding to a critical helix-turn-helix motif termed the voltage sensor paddle. Although these toxins partition into membranes to bind the paddle motif, their structure and orientation within the membrane are unknown. We investigated the interaction of a tarantula toxin named SGTx with membranes using both fluorescence and NMR spectroscopy. Depth-dependent fluorescence-quenching experiments with brominated lipids suggest that Trp³⁰ in SGTx is positioned ∼9 Å from the center of the bilayer. NMR spectra reveal that the inhibitor cystine knot structure of the toxin does not radically change upon membrane partitioning. Transferred cross-saturation NMR experiments indicate that the toxin's hydrophobic protrusion contacts the hydrophobic core of the membrane, whereas most surrounding polar residues remain at interfacial regions of the bilayer. The inferred orientation of the toxin reveals a twofold symmetry in the arrangement of basic and hydrophobic residues, a feature that is conserved among tarantula toxins. These results have important implications for regions of the toxin involved in recognizing membranes and voltage-sensor paddles, and for the mechanisms by which tarantula toxins alter the activity of different types of ion channels.     

Interaction of a toxin from the scorpion Tityus serrulatus with a cloned K+ channel from squid (sqKv1A)

A toxin from the scorpion Tityus serrulatus (TsTX-Kalpha) blocks native squid K(+) channels and their cloned counterpart, sqKv1A, at pH 8 ((native)K(d) approximately 20 nM; (sqKv1A)K(d) approximately 10 nM). In both cases, decreasing the pH below 7.0 significantly diminishes the TsTX-Kalpha effect (pK = 6.6). In the cloned squid channel, the pH dependence of the block is abolished by a single point mutation (H351G), and no change in toxin affinity was observed at higher pH values (pH > or =8.0). To further investigate the TsTX-Kalpha-sqKv1A interaction, the three-dimensional structure of TsTX-Kalpha was determined in solution by NMR spectroscopy, and a model of the TsTX-Kalpha-sqKv1A complex was generated. As found for other alpha-K toxins such as charybdotoxin (CTX), site-directed mutagenesis at toxin residue K27 (K27A, K27R, and K27E) significantly reduced the toxin's affinity for sqKv1A channels. This is consistent with the TsTX-Kalpha-sqKv1A model reported here, which has K27 of the toxin inserted into the ion conduction pathway of the K(+) channel. This toxin-channel model also illustrates a possible mechanism for the pH-dependent block whereby lysine residues from TsTX-Kalpha (K6 and K23) are repelled by protonated H351 on sqKv1A at low pH.     

Crystal structure of a non-toxic mutant of heat-labile enterotoxin, which is a potent mucosal adjuvant.

Two closely related bacterial toxins, heat-labile enterotoxin (LT-I) and cholera toxin (CT), not only invoke a toxic activity that affects many victims worldwide but also contain a beneficial mucosal adjuvant activity that significantly enhances the potency of vaccines in general. For the purpose of vaccine design it is most interesting that the undesirable toxic activity of these toxins can be eliminated by the single-site mutation Ser63Lys in the A subunit while the mucosal adjuvant activity is still present. The crystal structure of the Ser63Lys mutant of LT-I is determined at 2.0 A resolution. Its structure appears to be essentially the same as the wild-type LT-I structure. The substitution Ser63Lys was designed, based on the wild-type LT-I crystal structure, to decrease toxicity by interfering with NAD binding and/or catalysis. In the mutant crystal structure, the newly introduced lysine side chain is indeed positioned such that it could potentially obstruct the productive binding mode of the substrate NAD while at the same time its positive charge could possibly interfere with the critical function of nearby charged groups in the active site of LT-I. The fact that the Ser63Lys mutant of LT-I does not disrupt the wild-type LT-I structure makes the non-toxic mutant potentially suitable, from a structural point of view, to be used as a vaccine to prevent enterotoxigenic E. coli infections. The structural similarity of mutant and wild-type toxin might also be the reason why the inactive Ser63Lys variant retains its adjuvant activity.     

Prediction of a conserved, neutralizing epitope in ribosome-inactivating proteins

The secondary structures, side-chain solvent accessibilities, and superpositioned crystal structures of the A-chain of ricin and four other plant rRNA N-glycosidases (ribosome-inactivating proteins, RIPs) were examined. Previously, a 26-residue fragment from the A-chain of ricin was determined to bind to a neutralizing monoclonal antibody. The region in the native ricin A-chain, to which this peptide corresponds, is solvent-exposed and contains a negatively charged residue that has been hypothesized to participate in the toxin's function, namely, rRNA binding and/or enzymatic activity. This region appears to be conserved in all of the structurally defined plant RIPs examined. Moreover, other plant RIPs, whose tertiary structures are, as yet, unknown, were predicted to have an analogous, solvent-exposed region containing a conserved, negatively charged residue. By analogy, these conserved structural and functional features lead to the suggestion that this exposed region represents a logical starting point for experiments designed to locate neutralizing epitopes in these RIPs. In contrast, the tertiary structure of the analogous region in a bacteria-derived RIP (Shiga toxin) is a less solvent-exposed, truncated loop and is a structure that is not as likely to be a neutralizing epitope. Because most of the amino acid residues are not conserved within this exposed region, these RIPs are predicted to be antigenically distinct.     

Beta-barrel membrane protein folding and structure viewed through the lens of alpha-hemolysin

The beta-barrel is a transmembrane structural motif commonly encountered in bacterial outer membrane proteins and pore-forming toxins (PFTs). Alpha-hemolysin (alphaHL) is a cytotoxin secreted by Staphylococcus aureus that assembles from a water-soluble monomer to form a membrane-bound heptameric beta-barrel on the surface of susceptible cells, perforating the cell membranes, leading to cell death and lysis. The mechanism of heptamer assembly, which has been studied extensively, occurs in a stepwise manner, and the structures of the initial, monomeric form and final, membrane-embedded pore are known. The toxin's ability to assemble from an aqueous, hydrophilic species to a membrane-inserted oligomer is of interest in understanding the assembly of PFTs in particular and the folding and structure of beta-barrel membrane proteins in general. Here we review the structures of the monomeric and heptamer states of LukF and alphaHL, respectively, the mechanism of toxin assembly, and the relationships between alphaHL and nontoxin beta-barrel membrane proteins.     

Molecular cloning, overexpression in Escherichia coli, and purification of 6x his-tagged C-terminal domain of Clostridium difficile toxins A and B

Genomic DNA from ribotype-01 and -17 Clostridium difficile strains was used for amplification of the sequences encoding the carboxy-terminal domain of toxins A (TcdA) and B (TcdB). The deduced C-terminal TcdB ribotype-01 and -17 domains share 99.5% amino acid sequence identity while TcdA ribotype-17 comprises a 607 amino acid deletion compared to TcdA-01. When compared to previously sequenced C. difficile toxins, 99.3% amino acid identity was found between TcdA-01 and TcdA from strain VPI10643 and 98.8% identity between TcdA-17 and TcdA from strain F-1470. The obtained sequences were fused in 3' to a sequence encoding a hexahistidine tag and cloned into an Escherichia coli expression vector. The recombinant proteins were expressed in E. coli and purified using single-step metal-chelate chromatography. The recombinant carboxy-terminal domain of TcdA-01 was purified from the soluble E. coli lysate fraction whereas TcdA-17 and TcdB-17 carboxy-terminal domains were purified from inclusion bodies. At least 40 mg of each protein was purified per liter of bacterial culture. The recombinant toxin domains were detected specifically by Western blot and ELISA with antibodies against native C. difficile toxins. This study demonstrated that the carboxy-terminal domains of TcdA and TcdB can be produced using an E. coli expression system and easily purified. These recombinant, stable, and non-toxic proteins provide a convenient source for use in the diagnosis of C. difficile infections, instead of native toxins, as controls and calibrators in immunoassay kits and to obtain specific monoclonal antibodies.     

Crystal structure of heat-labile enterotoxin from Escherichia coli with increased thermostability introduced by an engineered disulfide bond in the A subunit.

Cholera toxin (CT) produced by Vibrio cholerae and heat-labile enterotoxin (LT-I), produced by enterotoxigenic Escherichia coli, are AB5 heterohexamers with an ADP-ribosylating A subunit and a GM1 receptor binding B pentamer. These toxins are among the most potent mucosal adjuvants known and, hence, are of interest both for the development of anti-diarrheal vaccines against cholera or enterotoxigenic Escherichia coli diarrhea and also for vaccines in general. However, the A subunits of CT and LT-I are known to be relatively temperature sensitive. To improve the thermostability of LT-I an additional disulfide bond was introduced in the A1 subunit by means of the double mutation N40C and G166C. The crystal structure of this double mutant of LT-I has been determined to 2.0 A resolution. The protein structure of the N40C/G166C double mutant is very similar to the native structure except for a few local shifts near the new disulfide bond. The introduction of this additional disulfide bond increases the thermal stability of the A subunit of LT-I by 6 degrees C. The enhancement in thermostability could make this disulfide bond variant of LT-I of considerable interest for the design of enterotoxin-based vaccines.     

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.     

Hemorrhagic toxins from Western diamondback rattlesnake (Crotalus atrox) venom: isolation and characterization of five toxins and the role of zinc in hemorrhagic toxin e.

Five previously unknown hemorrhagic proteins, designated hemorrhagic toxins a,b,c,d, and e, were isolated from the venom of the western diamondback rattlesnake (Crotalus atrox). Molecular weights of hemorrhagic toxins a-e were determined to be 68 000, 24 000, 24 000, 24 000, and 25 700, respectively, by sodium dodecyl sulfate-phosphate gel electrophoresis using various polyacrylamide gel concentrations. Amino acid composition showed a total of 636, 200, 213, 214, and 219 amino acids for hemorrhagic toxins a-e, respectively. All the hemorrhagic toxins were found to lose their hemorrhagic activities with the metal chelators ethylenediaminetetraacetic acid and 1, 10-phenanthroline. All the hemorrhagic toxins were found to contain approximately 1 mol of zinc/mol of toxin, and they were all demonstrated to be proteolytic when dimethylcasein and dimethylhemoglobin were used as substrates. When zinc was removed from hemorrhagic toxin e with 1,10-phenanthroline, both both the proteolytic and hemorrhagic activities were equally inhibited. When the apohemorrhagic toxin e thus produced was incubated with zinc, the hemorrhagic and proteolytic activities were regenerated to the same extent. CD, UV, and Raman spectroscopy were used to study the structure of native hemorrhagin toxin e as well as the structural changes caused by zinc removal. From CD spectroscopy the native toxin was estimated to consist of 23% alpha helix, 6% beta structure, and 71% random-coil conformation. When over 90% of the zinc was removed, the alpha-helix content dropped from 23 to 7%.     

Roles of Glu 349 and Asp 352 in membrane insertion and translocation by diphtheria toxin.

Acidic conditions within the endosomal lumen induce the T domain of receptor-bound diphtheria toxin (DT) to insert into the endosomal membrane and mediate translocation of the toxin's catalytic domain to the cytosol. A conformational rearrangement in the toxin occurring near pH5 allows a buried apolar helical hairpin of the native T domain (helices TH8 and TH9) to undergo membrane insertion. If the inserted hairpin spans the bilayer, as hypothesized, then the two acidic residues within the TL5 interhelical loop, Glu 349 and Asp 352, should become exposed at the neutral cytosolic face of the membrane and reionize. To investigate the roles of these residues in toxin action, we characterized mutant toxins in which one or both acidic residues had been replaced with nonionizable ones. Each of two double mutants examined showed a several-fold reduction in cytotoxicity in 24-h Vero cell assays (sixfold for E349A + D352A and fourfold for E349Q + D352N), whereas the individual E349Q and D352N mutations caused smaller reductions in toxicity. The single and double mutations also attenuated the toxin's ability to permeabilize Vero cells to Rb+ at low pH and decreased channel formation by the toxin in artificial planar bilayers. Neither of the double mutations affected the pH-dependence profile of the toxin's conformational rearrangement in solution, as measured by binding of the hydrophobic fluorophore, 2-p-toluidinyl-naphthalene 6-sulfonate. The results demonstrate that, although there is no absolute requirement for an acidic residue within the TL5 loop for toxicity, Glu 349 and Asp 352 do significantly enhance the biological activity of the protein. The data are consistent with a model in which ionization of these residues at the cytosolic face of the endosomal membrane stabilizes the TH8/TH9 hairpin in a transmembrane configuration, thereby facilitating channel formation and translocation of the toxin's catalytic chain.     

Common features of the NAD-binding and catalytic site of ADP-ribosylating toxins

Computer analysis of the three-dimensional structure of ADP-ribosylating toxins showed that in all toxins the NAD-binding site is located in a cavity. This cavity consists of 16 contiguous amino acids that form an a-helix bent over β-strand. The tertiary folding of this structure is strictly conserved despite the differences in the amino acid sequence. Catalysis is supported by two spatially conserved amino acids, each flanking the NAD-binding site. These are: a glutamic acid that is conserved in all toxins, and a nucleophillc residue, which is a histidine in the diphtheria toxin and Pseudomonas exotoxin A, and an arginine in the cholera toxin, the Escherichia coli heat-labile enterotoxins, the pertussis toxin and the mosquitocidal toxin of Bacillus sphaericus. The latter group of toxins presents an additional histidine that appears important for catalysis. This structure suggests a general mechanism of ADP-ribosylation evolved to work on different target proteins.     

AB(5) toxins: structures and inhibitor design

High-resolution crystal structures of AB(5) toxins in their native form or in complex with a variety of ligands have led to the structure-based design and discovery of inhibitors targeting different areas of the toxins. The most significant progress is the development of highly potent multivalent ligands that block binding of the toxins to their receptors.     

A plasmid encoding a combination of mosquito-larvicidal genes from Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus confers toxicity against a broad range of mosquito larvae when expressed in Gram-negative bacteria

A recombinant plasmid harboring cry4A, cry4B and cry11A from Bacillus thuringiensis subsp. israelensis and binary toxin genes from Bacillus sphaericus has been constructed. The three cry genes were placed under the control of the cry4B promoter whereas the binary toxin gene was controlled by its native promoter. The expression of toxins in Escherichia coli harboring the resulting plasmid, p4BDA-5142, was investigated. Cry4B expression was highest compared to other toxins. Although the level of toxin expression was low compared with E. coli expressing single toxins, the recombinant E. coli strain harboring p4BDA-5142 exhibited broad range mosquito-larvicidal activity against all Aedes, Culex and Anopheles larvae. This work has shown that the development of the recombinant plasmid can be used to broaden the host range spectrum of the appropriate bacterial host for mosquito control.