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.     

艰难梭菌A毒素的细胞致死活性研究

本文报道艰难梭菌Ａ毒素对４种培养细胞的细胞致死活性的探讨。４种培养细胞为Ｖｅｒｏ（非洲绿猴肾细胞）细胞、ＴＰＣ─１（人甲状腺肿瘤细胞）细胞、ＮＩＨ３Ｔ３细胞（小鼠成纤维细胞）及将ｒａｓ癌基因转基因于ＮＩＨ３Ｔ３细胞的ＮＩＨ３Ｔ３ｒａｓ细胞。应用台酚蓝排除能试验、噻唑蓝（ＭＴＳ）比色、细胞膜损害测定试验、荧光显微术观察细胞核的形态变化等测定Ａ毒素细胞致死活性。实验表明：４种培养细胞系对Ａ毒素细胞圆缩化作用的敏感性依次为ＮＩＨ３Ｔ３ｒａｓ,ＴＰＣ─１,Ｖｅｒｏ,ＮＩＨ３Ｔ３细胞。而对Ａ毒素细胞致死活性的敏感性依次为ＴＰＣ─１,ＮＩＨ３Ｔ３,Ｖｅｒｏ,ＮＩＨ３Ｔ３ｒａｓ细胞。从而得知Ａ毒素的细胞致死活性不但依细胞种类不同而不同,而且也不一定与Ａ毒素的细胞圆缩化作用有关。肿瘤细胞ＴＰＣ─１细胞对Ａ毒素致死活性有特殊敏感性。以上结果对探索抗癌新药的研制具有重要意义。     

Cloning and characterization of overlapping DNA fragments of the toxin A gene of clostridium difficile

Clostridium difficile, a human pathogen, produces two very large protein toxins, A and B (250-600 kDa), which resist dissociation into subunits. To clone the toxin A gene, a genomic library of 3-8 kb chromosomal DNA fragments of C. difficile strain VPI 10463 established in pUC12 was screened with a rabbit polyclonal toxin A antiserum. Thirty-five clones were isolated which carried 2.5-7.0 kb inserts representing a 10 kb region of the C. difficile genome. All the inserts were oriented in the same direction, suggesting that toxin A gene expression was under control of the lac promoter of the pUC12 vector. Western blot experiments revealed the presence of low amounts of fusion proteins of variable size (30-170 kDa) in Escherichia coli strains harbouring recombinant plasmids. As deduced from subcloning experiments, the DNA sequences encoding toxin A comprised about 4 kb, corresponding to about 140 kDa of the 300-600 kDa protein. This was either due to incomplete cloning of the gene or it might indicate a subunit composition of toxin A. No additional gene(s) with homology to the cloned toxin A gene was detected.     

Detection of Shiga-like toxin on the outer membranes of Shigella species and Escherichia coli K-12

Abstract Outer membranes of Shigella species and E. coli K-12 carrying large invasive plasmids and isogenic non-invasive strains without plasmids were analyzed by SDS-PAGE. The immunoblotting analysis of the outer membrane proteins of these bacteria was performed with monoclonal antibody (mAb) made against A and B subunits of Shiga-like toxin (SLT). The SLT was detected in the outer membranes of S. dysenteriae 1 IDBM11, S. sonnei PNS20, S. flexneri M90T, S. dysenteriae 60R, and E. coli K-12 strain AB2463. The two other E. coli K-12 strains, C600 and 933J were included as controls for low and high toxin producers respectively. The outer membrane protein band of molecular weight 70 kDa was common to all bacterial strains studied. The most prominent band of 70 kDa protein was seen to be present in the high toxin producing plasmidless strain of S. dysenteriae 60R and the lysogenic strain of E. coli 933J. The invasive strains of S. dysenteriae 1 and S. flexneri M90T which carry the large invasive plasmids showed the least prominent band of 70 kDa protein.
The immunoblotting analysis of Shiga-toxin partially purified from the S. dysenteriae 60R strain revealed the absence of 70 kDa band on SDS-PAGE, instead the two dissociated subunits were seen. Furthermore, periplasmic Shiga-toxin proteins also showed the complete dissociation into A and B subunits. However, under the same denaturing conditions, the 70 kDa protein band cross-reacting with mAb against A and B subunits was still present in the outer membranes of all different strains.     

Purification Of Mouse and Human Interferons: Detection Of Subunit Structure

During the purification of mouse and human interferons, multiple active components have been detected. Mouse interferon was purified over 500-fold by differential precipitation, centrifugation, gel chromatography, and isoelectric focusing. On electrofocusing, two molecular forms (A and B) were noted. Form B (pI 7.35) had a molecular weight of about 38,000 and Form A (pI 7.15), which was equally active, a molecular weight of 19,000. Purified Form B was dissociable into Form A, but the reverse reaction occurred to a much lesser extent. Human interferon, purified about 1500-fold, is also composed of multiple molecular forms. Form B (pi 5.60) had a molecular weight of about 24,000 and Form A (pi 5.35), which may contain up to 85% of the total activity, a molecular weight of 12,000. Both forms appear to have equal specific activities. The dissociation of both human and mouse interferons into subunits appeared to take place during dialysis versus low salt (0.01 M tris pH 7.A). The data are consistent with the idea that the native molecule exists as a dimer of similar or identical subunits. Dimer formation, which probably occurs within the cells, does not seem to lead to a measurable cooperative effect between the subunits.     

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.     

R-Ras glucosylation and transient RhoA activation determine the cytopathic effect produced by toxin B variants from toxin A-negative strains of Clostridium difficile

Clostridium difficile induces antibiotic-associated diarrhea through the production of toxin A and toxin B; the former toxin has been assumed to be responsible for the symptoms of the disease. Several toxin A-negative strains from C. difficile have recently been isolated from clinical cases and have been reported to produce toxin B variants eliciting an atypical cytopathic effect. Ultrastructural analysis indicated these toxins induce a rounding cytopathic effect and filopodia-like structures. Toxin B variants glucosylated R-Ras, and transfection with a constitutively active mutant of this GTPase protected cells against their cytopathic effect. Treatment of cells with toxin B variants induced detachment from the extracellular matrix and blockade of the epidermal growth factor-mediated phosphorylation of extracellular-regulated protein kinases, demonstrating a deleterious effect on the R-Ras-controlled avidity of integrins. Treatment with toxin B variants also induced a transient activation of RhoA probably because of inactivation of Rac1. Altogether, these data indicate that the cytopathic effect induced by toxin B variants is because of cell rounding and detachment mediated by R-Ras glucosylation, and the induction of filopodia-like structures is mediated by RhoA activation. Implications for the pathophysiology of C. difficile-induced diarrhea are discussed.     

Ultrastructural effects of Clostridium difficile toxin B on smooth muscle cells and fibroblasts

The mechanism by which Clostridium difficile toxin B causes cells in culture to round was investigated. Cultured human lung fibroblasts and rabbit aortic smooth muscle cells were treated with partially purified or purified toxin B and monitored by light and transmission electron microscopy (TEM). Both preparations caused progressive cell rounding which correlated with disorganization of actin-containing myofilament bundles. Thin myofilaments became fragmented and finally disappeared (after 24 h) and dense bodies became more prominent, while all other organelles appeared unaffected.     

Reconstitution of urea-dissociated subunits of a pig heart phosphoprotein phosphatase

Pig heart phosphoprotein phosphatase [phosphoprotein phosphophydrolase, EC 3.1.3.16] of Mr 224,000 was dissociated by gel-filtration on Sephacryl S-300, into an active subunit (alpha subunit) of Mr 31,000 and inactive subunits of higher molecular weight in the presence of 6 M urea. After the removal of urea, these subunits reassociated, forming two enzyme forms of Mr 237,000 (Form 1) and Mr 123,000 (Form 2). Form 2 was produced by association of the alpha subunit with an inactive subunit (beta subunit) of Mr 80,000, while Form 1 was formed by combination of the alpha subunit with a complex of inactive subunits which was eluted from a Sephadex G-150 column in fractions of molecular weight range greater than 80,000. The dissociation and reassociation of the subunits of Form 1 by the same urea method produced not only Form 1, but also significant amounts of Form 2, indicating that the inactive subunits of Form 1 were a complex of the beta subunit with another inactive subunit(s). The molecular parameters and other properties of Form 1 were very close to those of the original enzyme. By the conversion of Form 1 to Form 2, the activities of Form 1 towards phosphorylase a and glycogen synthetase b were enhanced 2-3 fold with no significant change in activity towards P-H1 histone or in response to the stimulatory effect of Mg(CH3COO)2 on the dephosphorylation of P-H2B histone. However, removal of the beta subunit from From 2 resulted in strong suppression of activity towards P-H1 histone and response to the salt effect with lesser effects on the activities of Form 2 towards phosphorylase a and glycogen synthase b.     

Stimulation of proliferation of a human osteosarcoma cell line by exogenous acidic fibroblast growth factor requires both activation of receptor tyrosine kinase and growth factor internalization.

U2OS Dr1 cells, originating from a human osteosarcoma, are resistant to the intracellular action of diphtheria toxin but contain toxin receptors on their surfaces. These cells do not have detectable amounts of fibroblast growth factor receptors. When these cells were transfected with fibroblast growth factor receptor 4, the addition of acidic fibroblast growth factor to the medium induced tyrosine phosphorylation, DNA synthesis, and cell proliferation. A considerable fraction of the cell-associated growth factor was found in the nuclear fraction. When the growth factor was fused to the diphtheria toxin A fragment, it was still bound to the growth factor receptor and induced tyrosine phosphorylation but did not induce DNA synthesis or cell proliferation, nor was any fusion protein recovered in the nuclear fraction. On the other hand, when the fusion protein was associated with the diphtheria toxin B fragment to allow translocation to the cytosol by the toxin pathway, the fusion protein was targeted to the nucleus and stimulated both DNA synthesis and cell proliferation. In untransfected cells containing toxin receptors but not fibroblast growth factor receptors, the fusion protein was translocated to the cytosol and targeted to the nucleus, but in this case, it stimulated only DNA synthesis. These data indicate that the following two signals are required to stimulate cell proliferation in transfected U2OS Dr1 cells: the tyrosine kinase signal from the activated fibroblast growth factor receptor and translocation of the growth factor into the cell.     

K-ras transformation greatly increases the toxin-dependent ADP-ribosylation of GTP binding proteins in thyroid cells. Involvement of an inhibitor of the ADP-ribosylation reaction.

The GTP binding (G) proteins of normal (FRTL5) and ras-transformed thyroid cells (KiKi) were characterized by cholera and pertussis toxin-induced ADP-ribosylation and immunoblot analysis. Two pertussis toxin substrates with molecular masses of 40 and 41 kDa were identified in normal cells as the alpha i2 and alpha i3 subunits. The molecular masses of the cholera toxin substrates were 42 and 45 kDa. The same cholera and pertussis toxin substrates were present in the K-ras-transformed cell line. However, the toxin-dependent ADP-ribosylation was markedly higher in KiKi than in normal cell membranes (more than 50-fold). The reason for this difference was investigated; it could not be explained by the relative amounts of G proteins in the two cell systems, since the levels of alpha i2 subunit as measured by quantitative immunoblot in K-ras-transformed cells were only slightly (65%) higher than in normal cells. The difference in ADP-ribosylation was not due to poly-ADP-ribosylation nor to a different degree of subunit dissociation of G proteins in the two cell lines. Rather, the enhanced ADP-ribosylation in K-ras-transformed cells appears to be due to the loss of an inhibitory factor present in the normal cells. Partial characterization indicates that such a factor is a peripheral membrane protein of less than 25 kDa capable of directly interfering with the ADP-ribosylation reaction.     

Purification and identification of two pertussis-toxin-sensitive GTP-binding proteins of bovine spleen

Two alpha subunits of GTP-binding proteins were purified from bovine spleen membranes. Both proteins were ADP-ribosylated by pertussis toxin in the presence of beta gamma subunits. The major protein had a molecular mass of 40 kDa and its immunological reactivity and fragmentation pattern by limited proteolysis were identical with those of the alpha subunit of Gi2. The minor protein had a molecular mass of 41 kDa and its partial amino acid sequences completely matched with those predicted from human and rat Gi3 alpha cDNAs.     

Cloning and expression of the gene for diphtheria toxin and its subunits in Escherichia coli

The results of cloning Corynebacterium diphtheriae phi 984 tox gene and its A and B subunits in Escherichia coli are presented. Regulatory sequences of tox gene are capable to promote effective expression in E. coli cells. A set of recombinant plasmids has been obtained which can determine the synthesis of A and B individual subunits and are suitable for constructing immunotoxins by gene engineering. The diphtheria toxin of 62 kDa synthesized in E. coli has enzymatic activity and reacts with antitoxin sera. Some sites for E. coli proteases are present in tox-specific polypeptides.     

The subunit S1 is important for pertussis toxin secretion

Pertussis toxin is a protein containing five noncovalently linked subunits which are assembled into the monomer A (containing the subunit S1) and the oligomer B (containing subunits S2, S3, S4, and S5 in a 1:1:2:1 ratio). Each of the five subunits is synthesized as a precursor containing a secretory leader peptide and is secreted into the periplasm of Bordetella pertussis where the five subunits are assembled into the oligomeric structure and then released into the culture medium. In the absence of subunit S3 the remaining subunits are not secreted into the medium, thus suggesting that the assembled structure is necessary for the release of the toxin into the supernatant. In this study we describe four B. pertussis mutants which secrete into the medium low amounts of the B oligomer of pertussis toxin. These mutants have single or multiple changes in the gene encoding the S1 subunit and synthesize S1 proteins with altered conformation which are not assembled into the holotoxin and are apparently degraded in the periplasm. These data indicate that while the B oligomer alone has the structural information necessary for the extracellular export of pertussis toxin, the S1 subunit is required for its efficient release into the medium.     

Purification and properties of Clostridium difficile cytotoxin B

Toxin B, a potent cytotoxin produced by Clostridium difficile, was purified to homogeneity from 6-day broth cultures of a toxigenic isolate. Cytotoxin was purified approximately 4000-fold by sequential ammonium sulfate precipitation, DEAE-Sepharose chromatography, and high performance liquid chromatography on a Mono Q anion-exchange column. The molecular weight of reduced purified toxin was 50,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, compared to 150,000 for unreduced toxin. Dose-response studies indicated that subpicogram concentrations of purified toxin caused rounding of approximately 20,000 IMR-90 fibroblasts. The phenomenon of cell rounding caused by toxin B was correlated with the ratio of globular to filamentous actin in fibroblasts as measured by two techniques. The toxin caused a significant increase in the ratio of globular to filamentous actin which was nearly completed prior to the onset of rounding. We conclude that cell rounding of fibroblasts exposed to toxin B is related to an increase in the ratio of globular to filamentous actin which is produced by small numbers of toxin molecules/cell.     

Conformational studies of a synthetic peptide corresponding to the repeat motif of C hordein.

Cytosolic glutathione S-transferases were purified from the epithelial cells of human small and large intestine. These preparations were characterized with regard to specific activities, subunit and isoenzyme composition. Isoenzyme composition and specific activity showed little variation from proximal to distal small intestine. Specific activities of hepatic and intestinal enzymes from the same patient were comparable. Hepatic enzymes were mainly composed of 25 kDa subunits. Transferases from small intestine contained 24 and 25 kDa subunits, in variable amounts. Colon enzymes were composed of 24 kDa subunits. In most preparations, however, minor amounts of 27 and 27.5 kDa subunits were detectable. Separation into isoforms by isoelectric focusing revealed striking differences: glutathione S-transferases from liver were mainly basic or neutral, enzymes from small intestine were basic, neutral and acidic, whereas large intestine contained acidic isoforms only. The intestinal acidic transferase most probably was identical with glutathione S-transferase Pi, isolated from human placenta. In the hepatic preparation, this isoform was hardly detectable. The specific activity of glutathione S-transferase showed a sharp fall from small to large intestine. In proximal and distal colon, activity seemed to be about equal. In the ascending colon there might be a relationship between specific activity of glutathione S-transferases and age of the patient, activity decreasing with increasing age.     

Characterization of an Enterotoxin Produced by Vibrio cholerae O139

A cholera-like enterotoxin was purified from Vibrio cholerae O139 strain AI-1841 isolated from a diarrheal patient in Bangladesh. Its characteristics were compared with that of cholera toxins (CTs) of classical strain 569B and El Tor strain KT25. Al-1841 produced as much toxin as O1 strains. The toxins were indistinguishable in terms of their migration profiles in conventional polyacrylamide gel disc electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectrofocusing as well as their affinity for hydroxyapatite. The skin permeability factor activity and the fluid accumulation induced in rabbit ileal loops of the toxin of AI-1841 were identical to those of the CTs. Three toxins equally reacted against anti-569B CT antiserum in Western blotting, and their B subunits formed a precipitin line against any anti-B subunit antiserum by double gel immunodiffusion. Anti-569B CTB antibody neutralized the three toxins in their PF activities and enterotoxicities. The amino acid sequence of 1841 toxin B subunit was identical with that of KT25 CTB, corresponding to the DNA sequence of ctxB from El Tor strains of the seventh pandemic. We concluded 1841 toxin was identical to CT of the seventh pandemic El Tor vibrios.     

The arrangement of subunits in cholera toxin.

Cholera toxin consists of five similar B subunits of apparent molecular weight about 10 600 and one A subunit (29 000) consisting of two peptides (A1 23 000-24 000 and A2 about 5500) linked by a single disulfide bond. Each B subunit also contains one internal disulfide bond which is readily reduced but is protected from carboxymethylation unless the reduced subunits are heated in urea. Tyrosine residues in A1 and in B subunits are readily iodinated, but the intact B assembly does not react with iodine. Upon reaction with the cross-linking reagent dimethyl suberimidate, B subunits may be covalently connected to each other, to A1 and to A2. A1 and A2 may also be cross-linked. The B subunits are probably arranged in a ring with A on the axis. A2 is required for the re-assembly of toxin from its subunits and may serve to hold A1 on the B ring. The maximum activity of cholera toxin in vitro is obtained only when the active peptide, A1, is separated from the rest of the molecule. Such separation, and the insertion of A1 into the cytosol, must follow the binding of the complete toxin, through component B, to the exterior of intact cells. This binding increases the effective concentration of the toxin in the vicinity of the plasma membrane. Possible ways in which A1 then crosses the membrane are considered in the Discussion.     

Identification of a novel 4 kDa immunoglobulin-A-binding peptide obtained by the limited proteolysis of jacalin

Jacalin, an IgA-binding lectin from jackfruit (Artocarpus heterophyllus) seeds, was isolated by the passage of PBS extracts of seeds over an affinity matrix containing IgA-Sepharose-4B. It was further purified by HPLC. When analyzed by SDS-PAGE under both reducing and nonreducing conditions, the native jacalin was dissociated into two subunits of 12 and 15.4 kDa. Both the subunits could bind IgA. Peptide mapping performed with radioiodinated jacalin indicated that both the subunits were susceptible to proteolysis by Staphyloccous aureus V8 proteinase. One degradation product was a small peptide of 4 kDa. This small proteolytic fragment also bound IgA. The amino-termini of the two major IgA binding subunits, 12 and 15.4 kDa, were identical. The 4 kDa IgA-binding proteolytic fragment of jacalin had a different amino-terminal sequence, suggesting that the region of jacalin which binds IgA does not remain close to the amino-terminus of the peptide.     

Effects of LTB4 and Ca++ ionophore A23187 on the release by human alveolar macrophages of factors controlling fibroblast functions

Cultured human alveolar macrophages from smokers with lung cancer produced spontaneously variable amounts of factors stimulating fibroblast proliferation and production of prostaglandin E2 and collagenase by fibroblasts. These biological activities belong to molecules similar or identical to interleukin 1. Exogenous leukotriene B4 added to alveolar macrophage cultures increased the production of these factors. The Ca++ ionophore A23187 was found to have similar effects. By the control of monokine production, leukotriene B4 locally released by inflammatory cells may modulate lung fibroblast functions.