Production, purification and characterization of Clostridium difficile toxic proteins different from toxin A and from toxin B

The purification and characterization of three new proteins called C1, C2, and C3 from Clostridium difficile are described. Their estimated molecular mass were about 350 (C1), 270 (C2) and 140 (C3) kDa, consisting of subunits of 39 (C1), 43 (C2) and 41 (C3) kDa, respectively. Immunodiffusion revealed that the three proteins contained similar but not identical antigenic determinants to toxin A. Each protein induced a cytotonic effect on hamster ovaric cells; the combined proteins, had a specific activity on cells 5-times higher than that of toxin A. In rat intestinal loops, they induced a clear fluid secretion, while toxin A elicited a haemorrhagic fluid response. The cytotonic activities of all three proteins were abolished by antiserum against toxin A, while antiserum against toxin B inhibited only the activity of the 270 kDa protein. In contrast to toxin A, the cytotoxicity of the three proteins was inactivated by trypsin. Thus, the chemical, antigenic and biological properties of these proteins differed from those of toxin A and toxin B.     

Genetic exchange of the S2 and S3 subunits in pertussis toxin

Bordetella pertussis, the causative agent of whooping cough, produces a complex hetero-oligomeric exotoxin, named pertussis toxin (PTX), which is responsible for several of the clinical manifestations associated with whooping cough. The toxin is composed of five dissimilar subunits, named S1 through S5 and arranged in a hexameric structure with a 1S1:1S2:1S3:2S4:1S5 stoichiometry. Although S2 and S3 share 70% amino acid identity, these two subunits were previously thought not to be able to substitute for each other in toxin assembly/secretion and the biological activities of PTX. Here, we show that toxin analogues containing two S3 subunits and lacking S2 (PTXdeltaS2), or containing two S2 subunits and lacking S3 (PTXdeltaS3), can be produced, assembled and secreted by B. pertussis strains, in which the S2-encoding cistron or the S3-coding cistrons have been inactivated by internal in-frame deletions that avoid downstream effects. In fact, PTXdeltaS3 was produced in higher amounts in the bacterial culture supernatants than natural PTX, whereas PTXdeltaS2 was produced in lower amounts than PTX. The action of the toxin analogues on the clustering of Chinese Hamster Ovary cells was also affected differentially by the S2-S3 substitution. These toxin analogues constitute thus interesting probes for the study of cellular functions, in particular immune cell functions, for which natural PTX has already shown its usefulness.     

Hemorrhagic toxin from the venom of Agkistrodon bilineatus (common cantil)

1. Hemorrhagic toxin was isolated from Agkistrodon bilineatus (Common cantil) venom using a three-step purification procedure to obtain 32.8 mg of purified hemorrhagic toxin from 700 mg of crude venom. 2. The purified toxin was homogeneous by disc polyacrylamide gel electrophoresis at pH 8.3, and by isoelectric focusing. 3. Hemorrhagic toxin possessed lethal, hemorrhagic and proteolytic activities. These activities of this toxin were inhibited by ethylenediaminetetraacetic acid (EDTA) and ethyleneglycol-bis-(beta-aminoethylether)N,N'-tetraacetic acid (EGTA), but not by cysteine or soybean trypsin inhibitor (SBTI). 4. Its molecular weight was approximately 48 kDa and the isoelectric point was 4.2. 5. Purified preparation hydrolyzed the Asn(3)--Gln(4), His(10)--Leu(11), Ala(14)--Leu(15), Tyr(16)--Leu(17), Arg(22)--Gly(23) and Phe(24)--Phe(25) bonds of oxidized insulin B. chain. 6. The A alpha chain of fibrinogen was first split and B beta chain was cleaved later by this toxin. 7. Hemorrhagic toxin contains 1 mol of zinc and 2 mol of calcium per mol of protein.     

Effect of oxidizing agents and sulfhydryl group reagents on beta toxin from Clostridium perfringens type C

Purified beta toxin from Clostridium perfringens type C was inactivated by the oxidizing agents o-iodosobenzoate (OIBA), oxidized glutathione, and ferricyanide, and by the sulfhydryl group regents 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide, iodoacetamide, and iodoacetic acid, causing loss of activity in various degrees depending on the concentration used. The activity of the toxin was not influenced by exposure to 1.0 mM of p-chloromercuribenzoate. The toxin treated by OIBA or DTNB was reactivated by incubation with 2-mercaptoethanol and dithiothreitol. The data suggest that beta toxin contains thiol groups which are essential for the activity.     

Recombinant expression of in silico identified Bcell epitope of epsilon toxin of Clostridium perfringens in translational fusion with a carrier protein

Epsilon toxin secreted by Clostridium perfringens types B and D has been directly implicated as the causative agent of fatal enterotoxemia in domestic animals. The aim of the present study is to use in silico approach for identification of B-cell epitope(s) of epsilon toxin, and its expression in fusion with a carrier protein to analyze its potential as vaccine candidate(s). Using different computational analyses and bioinformatics tools, a number of antigenic determinant regions of epsilon toxin were identified. One of the B cell epitopes of epsilon toxin comprising the region (amino acids 40-62) was identified as a promising antigenic determinant. This Etx epitope (Etx_40-62) was cloned and expressed as a translational fusion with B-subunit of heat labile enterotoxin (LTB) of E. coli in a secretory expression system. Similar to the native LTB, the recombinant fusion protein retained the ability to pentamerize and bind to GM₁ ganglioside receptor of LTB. The rLTB.Etx_40-62 could be detected both with anti-Etx and anti-LTB antisera. The rLTB.Etx_40-62 fusion protein thus can be evaluated as a potential vaccine candidate against C. perfringens.

Abbreviations

aa - amino acid(s), Etx - epsilon toxin of Clostridium perfringens, LTB - B-subunit of heat labile enterotoxin of E. coli.     

构象稳定的破伤风毒素Hc 片段突变体 (HcM) 的制备及其免疫原性分析

破伤风是由破伤风杆菌侵入人体伤口、生长繁殖、产生毒素而引起的一种急性特异性感染,其死亡率高,严重危害人民生命健康。研究证实破伤风毒素重链C端 (Hc) 具有与毒素受体结合的活性,完全保留了全分子的免疫原性,有望开发成为新的基因工程破伤风亚单位疫苗以替换传统的甲醛灭活类毒素疫苗。由于野生型Hc蛋白 (HcW) 易形成分子间及分子内二硫键,且各构象分子之间易发生不稳定的转换,为疫苗的生产工艺带来困难,因此,通过将破伤风HcW蛋白的869位半胱氨酸突变为丙氨酸,构建构象稳定的破伤风亚单位疫苗突变体HcM,对Hc     

Characterization of a Cl. Perfringens type D strain, isolated in the field and optimization of epsilon toxin biosynthesis in a cell culture

A field strain of cl. perfringens, named Dt001, was isolated from kidney of ovine enterotoemia case. The isolate characterized as Cl. perfringens, type D was based on its cultural and biochemical characters and its factors of virulence. The strain was very toxinogenic and well adapted to culture conditions of biofermentation when the parameters related to ptt, incubation time, substrat ... were optimized. Thus, the use of carbon source as polymer (destrine), the continuous control of pH allowed improvement of the rate of biosynthesis of Epsilon toxine by 10 times. The study of the immunogenicity of the isolate showed that preparations of anacultures were more immunogenic then those of anatoxine type. The fact that the two forms of epsilon antigens (protoxin and active toxin) show similar immune response in rabbits, indicates that the proteolytic action of trypsin is limited only to the toxic sites and does not affect the immunogenic epsitopes of the toxin. It also suggests a molecular organization of epsilon toxin in which the immunogenic epsitopes and the toxin sites are apart. The biotechnological performances and the immunogenicity and toxinogenical of the Dt001 isolate are in favor of its possible use as a component of an inactivated vaccine against enterotoxenia.     

Contraction induced by Clostridium perfringens epsilon toxin in the isolated rat ileum

Clostridium perfringens epsilon toxin caused contraction of the isolated ileum of the rat in a dose-dependent manner. The contraction caused by the toxin was inhibited by a low Na medium, tetrodotoxin (TTX), atropine, mecamylamine or tetraethylammonium (TEA). Furthermore, the contractile response induced by the toxin was abolished by incubation in Ca-free medium, and completely restored by and addition of Ca2+. In addition, verapamil inhibited contraction induced by the toxin in a dose-dependent manner. These data suggest that epsilon toxin induces contraction of the isolated ileum and that the toxin-elicited contraction is the result of an indirect action mediated through the nervous systems.     

Tyrosine alpha 244 is derivatized when the bovine heart mitochondrial F1-ATPase is inactivated with 5'-p-fluorosulfonylbenzoylethenoadenosine

The bovine heart mitochondrial F1-ATPase (MF1) is inactivated by 5'-p-fluorosulfonylbenzoylethenoadenosine (FSB epsilon A) with pseudo-first order kinetics. The dependence of the rate of inactivation on the concentration of FSB epsilon A revealed an apparent Kd of 0.25 mM. ATP and ADP, and to a lesser extent, ITP and IDP provide partial protection against inactivation by the reagent. Isolation and sequence analysis of major radioactive fragments in peptic or cyanogen bromide digests of MF1 inactivated with [3H]FSB epsilon A indicate that modification of Tyr-alpha 244 is associated with the loss of activity observed. Assessment of the amount of Tyr-alpha 244 derivatized with [3H]FSB epsilon A at specific points during inactivation of the ATPase indicates that maximal inactivation is achieved on modification of this residue in slightly greater than one copy of the alpha subunit. The following characteristics of inactivation of MF1 by FSB epsilon A have also been determined. (a) The rate of inactivation of ITPase activity by FSB epsilon A is 1.4 times greater than that observed for inactivation of ATPase activity under identical conditions. (b) After maximally inactivating the capacity of MF1 to hydrolyze saturating ATP with FSB epsilon A, the modified enzyme retained its capacity to hydrolyze substoichiometric ATP. (c) Inactivation of the ATPase by FSB epsilon A is accelerated by Pi. In each of the above characteristics, MF1 modified by FSB epsilon A resembles enzyme inactivated with 5'-p-fluorosulfonylbenzoyladenosine (FSBA) more than it does enzyme inactivated with 5'-p-fluorosulfonylbenzoylinosine (FSBI). Furthermore, prior inactivation of MF1 with FSBA completely prevents labeling of Tyr-alpha 244 with [3H]FSB epsilon A, whereas prior inactivation of the enzyme with FSBI does not. Since a single catalytic site is modified when FSBI inactivates MF1 whereas three noncatalytic sites are modified when it is maximally inactivated with FSBA, it is concluded that FSB epsilon A also modifies noncatalytic sites.     

1-N6-Etheno-ADP-ribosylation of elongation factor-2 by diphtheria toxin

Diphtheria toxin fragment A is able to inhibit protein synthesis in the eukaryotic cell by ADP-ribosylating the diphthamide residue of elongation factor-2 (EF-2) [(1980) J. Biol. Chem. 255, 10710-10720]. The reaction requires NAD as ADP-ribose donor. This work reports on the capacity of an NAD analog, the nicotinamide 1-N6-ethenoadenine dinucleotide (epsilon NAD), to be a substrate of diphtheria toxin fragment A in the transferring reaction of the fluorescent moiety, the epsilon ADP-ribose, to the EF-2. As a consequence of the transfer of the epsilon ADP-ribosyl moiety to the EF-2, there is an increase in the emission intensity of the fluorophore and a blue shift in its emission maximum. The epsilon ADP-ribosylated EF-2, like ADP-ribosylated EF-2, retains the capacity to bind GTP and ribosome. The utility of introducing a fluorescent probe in a well defined point of the EF-2 molecule for conformational or binding studies is discussed.     

Clostridium perfringens epsilon toxin induces a rapid change of cell membrane permeability to ions and forms channels in artificial lipid bilayers

Epsilon toxin is a potent toxin produced by Clostridium perfringens types B and D, which are responsible for a rapidly fatal enterotoxemia in animals. One of the main properties of epsilon toxin is the production of edema. We have previously found that epsilon toxin causes a rapid swelling of Madin-Darby canine kidney cells and that the toxin does not enter the cytosol and remains associated with the cell membrane by forming a large complex (Petit, L., Gibert, M., Gillet, D., Laurent-Winter, C., Boquet, P., and Popoff, M. R. (1997) J. Bacteriol. 179, 6480-6487). Here, we report that epsilon toxin induced in Madin-Darby canine kidney cells a rapid decrease of intracellular K(+), and an increase of Cl(-) and Na(+), whereas the increase of Ca(2+) occurred later. The entry of propidium iodide that was correlated with the loss of cell viability monitored by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test indicates that epsilon toxin formed large pores. In artificial lipid bilayers, epsilon toxin caused current steps with a single-channel conductance of 60 pS in 100 mm KCl, which represented general diffusion pores. The channels were slightly selective for anions, but cations could also penetrate. Epsilon toxin formed wide and water-filled channels permeable to hydrophilic solutes up to a molecular mass of at least 1 kDa, which probably represents the basic mechanism of toxin action on target cells.     

Evaluation of a new cytotoxicity assay for Clostridium perfringens type D epsilon toxin

Abstract A new cytotoxicity assay for determining the activity of epsilon toxin produced by Clostridium perfringens type D has been developed. Viability of cultured cells was determined by the ability of only live cells to convert 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3-(4-sulfophenyl)tetrazolium to the coloured product formazan in the presence of phenazine methosulfate. Of the 12 cell lines tested, only the MDCK cell line was susceptible to epsilon toxin. Specificity was confirmed by the ability of only specific monoclonal antibodies to inhibit cytotoxicity. Good correlation was obtained with the mouse lethality assay ( r = 0.991) and over a wide range of viability (15–75%) as determined by ethidium bromide/acridine orange staining ( r = 0.995).     

In vitro effects of Clostridium perfringens type D epsilon toxin on water and ion transport in ovine and caprine intestine

Clostridium perfringens type D produces enterotoxaemia in sheep, goats and other animals. The disease is caused by C. perfringens epsilon toxin, and while enterotoxaemia in goats is usually characterized by enterocolitis, the disease in sheep is characterized by systemic lesions (such as lung and brain oedema) with minor and inconsistent changes observed in the intestine. A possible explanation for these differences is that epsilon toxin is more promptly absorbed by sheep than goat intestine. In an attempt to clarify this, we examined the in vitro effects of epsilon toxin on sheep and goat intestine. Pieces of intestinal mucosa from recently slaughtered animals were mounted in a modified Ussing-type chamber where net water flux (J(w)), short-circuit current (I(sc)) and tissue conductance (G(t)) were simultaneously recorded. After 70 min of incubation with epsilon toxin a reduction in absorptive J(w) and an increase in I(sc) and G(t) were observed in colonic tissues of both sheep and goats, but no alterations were registered in the ileum of either species. These in vitro results show that epsilon toxin affects the transport function of the colonic mucosa but it does not seem to produce any transport alteration in the ileum mucosa.     

Synthesis of potent inhibitors of anthrax toxin based on poly-L-glutamic acid

We report the synthesis of biodegradable polyvalent inhibitors of anthrax toxin based on poly-L-glutamic acid (PLGA). These biocompatible polyvalent inhibitors are at least 4 orders of magnitude more potent than the corresponding monovalent peptides in vitro and are comparable in potency to polyacrylamide-based inhibitors of anthrax toxin assembly. We have elucidated the influence of peptide density on inhibitory potency and demonstrated that these inhibitory potencies are limited by kinetics, with even higher activities seen when the inhibitors are preincubated with the heptameric receptor-binding subunit of anthrax toxin prior to exposure to cells. These polyvalent inhibitors are also effective at neutralizing anthrax toxin in vivo and represent attractive leads for designing biocompatible anthrax therapeutics.     

Evaluation of different fluids for detection of Clostridium perfringens type D epsilon toxin in sheep with experimental enterotoxemia

Enterotoxemia caused by Clostridium perfringens type D is a highly lethal disease of sheep, goats and other ruminants. The diagnosis of this condition is usually confirmed by detection of epsilon toxin, a major exotoxin produced by C. perfringens types B and D, in the intestinal content of affected animals. It has been suggested that other body fluids can also be used for detection of epsilon toxin. This study was performed to evaluate the usefulness of intestinal content versus other body fluids in detecting epsilon toxin in cases of sheep enterotoxemia. Samples of duodenal, ileal and colon contents, pericardial and abdominal fluids, aqueous humor and urine from 15 sheep with experimentally induced enterotoxemia, were analysed for epsilon toxin using a capture ELISA. Epsilon toxin was detected in 92% of the samples of ileal content, 64% of the samples of duodenal content, 57% of the samples of colon content and in 7% of the samples of pericardial fluid and aqueous humor. No epsilon toxin was found in samples of abdominal fluid or urine from the animals with enterotoxemia or in any samples from six clinically healthy sheep used as negative controls. The results of this study indicate that with the diagnostic capture ELISA used, intestinal content (preferably ileum) should be used for C. perfringens type D epsilon toxin detection in suspected cases of sheep enterotoxemia.     

Anthrax toxin lethal factor contains a zinc metalloprotease consensus sequence which is required for lethal toxin activity

Comparison of the anthrax toxin lethal factor (LF) amino acid sequence with sequences in the Swiss protein database revealed short regions of similarity with the consensus zinc-binding site, HEXXH, that is characteristic of metalloproteases. Several protease inhibitors, including bestatin and captopril, prevented intoxication of macrophages by lethal toxin. LF was fully inactivated by site-directed mutagenesis that substituted Ala for either of the residues (H-686 and H-690) implicated in zinc binding. Similarly, LF was inactivated by substitution of Cys for E-687, which is thought to be an essential part of the catalytic site. In contrast, replacement of E-720 and E-721 with Ala had no effect on LF activity. LF bound ⁶⁵Zn both in solution and on protein blots. The ⁶⁵Zn binding was reduced for several of the LF mutants. These data suggest that anthrax toxin LF is a zinc metallopeptidase, the catalytic function of which is responsible for the lethal activity observed in cultured cells and in animals.     

水稻基腐细菌毒素的分离纯化、性质和生物学作用

[目的]水稻基腐细菌毒素迄今未见报道.毒素是病原微生物重要的致病因子之一,毒素的分离纯化是研究病菌毒素功能和作用的前提和基础.[方法]通过几种层析柱的多次层析分离及对水稻 ,幼苗的生物活性跟踪测定,分离纯化水稻基腐细菌毒素;采用化学及生物化学方法,研究毒素的性质及生物学作用.[结果]获得了水稻基腐细菌毒素的一个成分T<,3>,该成分为黄色固体,溶于甲醇、正丁醇、水和甲酸;不溶于三氯甲烷、乙酸乙酯;微溶于丙酮,是非糖类和非蛋白质类物质,对紫外线敏感.毒素具有抑制水稻生根、使水稻秧苗萎蔫和对烟草细胞坏死的作用.高浓度毒素抑制水稻、玉米、番茄和烟草种子萌发,低浓度毒素则具有促进根、芽生长的作用.毒素对来自5个属的10种植物病原细菌具有抑菌活性,同时具有诱导水稻PAL和POD活性,且对抗病品种128的POD和PAL诱导活性均高于感病品种特籼13.[结论]首次建立了水稻基腐细菌毒素的分离纯化方法.该毒素具有抑制水稻幼根生长、导致秧苗萎蔫、引起烟草细胞坏死、抑制植物病原细菌和诱导水稻防卫酶活性等生物学作用.     

Clostridium perfringens toxin types from wild-caught Atlantic cod (Gadus morhua L.), determined by PCR and ELISA

Ninety-five fecal samples from Atlantic cod (Gadus morhua L.), caught along the northern Norwegian coast, were examined bacteriologically for occurrence of C. perfringens. Isolates were examined by polymerase chain reaction (PCR) for genes encoding the four lethal toxins (alpha, beta, epsilon, and iota) for classification into toxin types and for genes encoding enterotoxin and the novel beta2 toxin for further subclassification. In addition, a commercial enzyme-linked immunosorbent assay (ELISA) kit for detection of C. perfringens alpha, beta, and epsilon toxin was used. Clostridium perfringens could be isolated in 37 fecal samples (38.9%) from cod. All isolates were C. perfringens toxin type A (alpha toxin positive) as determined by PCR and also ELISA. In addition, in isolates from two cod (2.1%) the gene encoding for beta2 toxin was found (A, beta2) by PCR. Genes encoding for beta, epsilon, and iota toxins and enterotoxin were not found. This is the first detection of C. perfringens alpha and beta2 toxin in cod and of beta2 toxin in fish in general. The origin of this bacterium in cod is discussed.     

Clostridium perfringens epsilon toxin rapidly decreases membrane barrier permeability of polarized MDCK cells

Epsilon toxin is produced by Clostridium perfringens types B and D which are responsible for fatal intestinal diseases in animals. The main biological activity of epsilon toxin is the production of oedema in various organs. We have previously found that epsilon toxin forms a large membrane complex in MDCK cells which is not internalized into cell, and induces cell volume enlargement and loss of cell viability (Petit, L., Gibert, M., Gillet, D., Laurent-Winter, C., Boquet, P., Popoff, M. R. (1997) J Bacteriol 179, 6480-6487). Here, we show that epsilon toxin is very potent to decrease the trans-epithelial electrical resistance of polarized MDCK cells grown on filters without altering the organization of the junctional complexes. The dose-dependent decrease in trans-epithelial electrical resistance, more marked when the toxin was applied to the apical side than to the basal side of MDCK cells, was associated with a moderate increase of the paracellular permeability to low-molecular-weight compounds but not to macromolecules. Epsilon toxin probably acts by forming large membrane pores which permit the flux of ions and other molecules such as the entry of propidium iodide and finally to the loss of cell viability.     

Effect of Clostridium perfringens epsilon toxin on MDCK cells

Epsilon toxin is one of the major lethal toxins produced by Clostridium perfringens type D and B. It is responsible for a rapidly fatal disease in sheep and other farm animals. Many facts have been published about the physical properties and the biological activities of the toxin, but the molecular mechanism of the action inside the cells remains unclear. We have found that the C. perfringens epsilon toxin caused a significant decrease of the cell numbers and a significant enlargement of the mean cell volume of MDCK cells. The flow cytometric analysis of DNA content revealed the elongation of the S phase and to a smaller extent of the G2+M phase of toxin-treated MDCK cells in comparison to untreated MDCK cells. The results of ultrastructural studies showed that the mitosis is disturbed and blocked at a very early stage, and confirmed the toxin influence on the cell cycle of MDCK cells.