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.     

Degradation of 2-phosphoglycerate by cytotoxin B of Clostridium difficile

Cytotoxin B of C. difficile was highly purified by selective ammonium sulfate precipitation, Biogel A5m chromatography, phenyl boronate hydrophobic interaction chromatography and ultracentrifugation. The final cytotoxic product had a specific activity of 7.8 X 10(8) units/mg protein and showed a single protein band with an estimated molecular weight of 163,000 when subjected to SDS-PAGE. Immunoelectrophoresis of the final product showed a single precipitin arc. The addition of cytotoxin B to imidazole-HCl buffer (pH 7.4) containing MgSO4, KCl and the substrate 2-phosphoglycerate resulted in the formation of phosphoenolpyruvate as demonstrated by spectrophotometric analysis. Phosphoglycerate conversion was absent when the cytotoxin was heat-inactivated of reacted with specific antitoxin prior to assay.     

Effect of the cytotoxin of Clostridium difficile on cultured hepatoma cells

Clostridium difficile is the major etiologic agent of human pseudomembranous colitis. It produces two toxins: an enterotoxin and a cytotoxin. In cultured hepatoma cells, at very low doses, the cytotoxin inhibits the incorporation of precursors into biological macromolecules. Protein synthesis is more affected than RNA and DNA synthesis. The toxin also induces severe alterations of the cell morphology consisting in damages to the cytoskeleton and to the cell shape.     

Nutritional aspects of cytotoxin production by Clostridium difficile.

Arginine was the only amino acid used by Clostridium difficile that permitted cytotoxin synthesis in a peptone-based medium. Synthesis of cytotoxin was delayed when glucose was used as the substrate. Addition of rifampin or puromycin to cultures prior to release of cytotoxin inhibited the release of cytotoxin, suggesting that a protein essential for cytotoxin release is synthesized after cytotoxin is synthesized.     

Internalization of Clostridium difficile cytotoxin into cultured human lung fibroblasts

In cultured human lung fibroblasts treated with Clostridium difficile cytotoxin, the latency before appearance of the cytopathogenic effect was dose-related with a minimum of 45 min. At 37 degrees C, the toxin was accessible on all cells to inactivation with trypsin or neutralization with antitoxin during the first tenth of the latency. At 0 degrees C, the toxin was accessible considerably longer. The cytopathogenic effect was reversibly prevented by the lysosomotropic agents chloroquine and ammonium chloride, which had to be added within one-fifth of the latency to protect all cells. In the presence of chloroquine, but not of ammonium chloride, the time period during which the toxin remained amenable to neutralization with antitoxin was prolonged. The protective effect of ammonium chloride was not influenced by dropping the extracellular pH to 4.5, but that of chloroquine was abolished. The expression of the intoxication was not affected by inhibitors of the DNA, RNA or protein synthesis. Inhibitors of the energy metabolism prevented the cytopathogenic effect when added before the last phase of the latency. The results suggest that expression of the cytopathogenic effect requires internalization of the toxin, and that metabolic energy but no macromolecular synthesis is needed for the action of the toxin after this internalization.     

Purification and some properties of cytotoxin produced by Clostridium difficile

The cytotoxin produced by Clostridium difficile was highly purified by using ammonium sulfate fractionation and successive column chromatographies of DEAE-Sephadex A-25, hydroxyapatite, Bio-Gel A-0.5m, Phenyl-Sepharose CL-4B, and Mono Q. The purified cytotoxin gave a single band on conventional and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of dithiothreitol. Its molecular weight was estimated to be 260,000 and 50,000 by gel filtration and sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis in the presence of dithiothreitol, respectively. Thus it was supposed that the toxin consists of 5 subunits having molecular weight of approximately 50,000. It had an isoelectric point of 6.6. The toxin was heat-labile (60 C for 10 min) and inactivated by treatment with trypsin and pronase, or at pH below 4 or over 10. The minimum cytotoxic dose of the cytotoxin against Chinese hamster ovary cells was 3 ng. It was also demonstrated that the toxin is antigenically different from enterotoxin of C. difficile.     

艰难梭菌细胞毒素B功能区的克隆及序列分析

目的克隆艰难梭菌(Clostridium difficile,C.d)细胞毒素B羧基末端功能区(CDB3)基因,并对其进行测序及生物信息学分析。方法利用PCR技术扩增CDB3基因,并将其定向插入pET-22b( )载体中,以DNA自动分析仪进行序列测定,并以生物信息学软件分析其生物学特性。结果成功克隆了艰难梭菌CDB3基因,经测序表明与GenBank中分布的Clostridium difficile VPI10463的ToxinB3基因序列完全一致。DNAstar软件预测其蛋白质的相对分子量(Mr)约为71.3 kD,并显示出良好的抗原性。结论研究获得了序列正确的CDB3基因,为其重组表达及其相关研究奠定了良好基础。     

Comparative sequence analysis of the Clostridium difficile toxins A and B.

Summary The six clones pTB112, pTB324, pTBs12, pCd122, pCd14 and pCdl3 cover thetox locus ofClostridium difficile VPI 10463. This region of 19 kb of chromosomal DNA contains four open reading frames including the completetoxB andtoxA genes. The two toxins show 63% amino acid (aa) homology, a relatedness that had been predicted by the cross-reactivity of some monoclonal antibodies (mAb) but that is in contrast to the toxin specificity of polyclonal antisera. A special feature of ToxA and ToxB is their repetitive C-termini. We define herein 19 individual CROPS (combinedrepetitiveoligopeptides of 20–50 as length) in the ToxB C-terminus, which are separable into five homologous groups. Comparison of the as sequences of the N-terminal two-thirds of ToxA and ToxB revealed three marked structures, a cluster of 172 hydrophobic, highly conserved as in the centre of both toxins, a sequence of 120 residues with an accumulation of highly conserved arginine, cysteine, histidine, methionine, and tryptophan residues, and a stretch of 248 less conserved aa. The probable function of these domains is discussed. Structural and functional homologies of ToxA and ToxB indicate that both genes have a common ancestor and may have evolved by gene duplication, with subsequent recombination and mutation, as has been reported for streptococcal glucosyltransferases (Gtf).     

Toxins A and B of Clostridium difficile

Abstract: The toxins produced by Clostridium difficile share several functional properties with other bacterial toxins, like the heat-labile enterotoxin of Escherichia coli and cholera toxin. However, functional and structural differences also exist. Like cholera toxin, their main target is the disruption of the microfilaments in the cell. However, since these effects are not reversible, as found with cholera toxin, additional mechanisms add to the cytotoxic potential of these toxins. Unlike most bacterial toxins, which are built from two structurally and functionally different small polypeptide chains, the functional and binding properties of the toxins of C. difficile are confined within one large polypeptide chain, making them the largest bacterial toxins known so far.     

Clostridium difficile and its cytotoxin in infants admitted to hospital with infectious gastroenteritis.

During a prospective study of infectious gastroenteritis in children under 2 years, 19 out of 390 patients (4.9%) were found to have Clostridium difficile cytotoxin in the faeces. In several there was no history of use of antibiotics. The symptoms of many infants with toxin settled spontaneously, but one child became acutely and severely ill and developed a toxic megacolon and five others required, and responded to, vancomycin. Cl difficile was cultured from the stools in 191 (49%) of the children. The highly significant increased prevalence of past use of antibiotics in 118 control patients was not associated with an increased incidence of either isolation of Cl difficile or presence of faecal cytotoxin. Cl difficile should not be overlooked as a cause of acute diarrhoea and vomiting in children under 2 years.     

Secretome analysis of Clostridium difficile strains

Clostridium difficile causes infections ranging from mild C. difficile-associated diarrhea to severe pseudomembranous colitis. Since 2003 new hypervirulent C. difficile strains (PCR ribotype 027) emerged characterized by a dramatically increased mortality. The secretomes of the three C. difficile strains CDR20291, CD196, and CD630 were analyzed and compared. Proteins were separated and analyzed by means of SDS--PAGE and LC-MS. MS data were analyzed using Mascot and proteins were checked for export signals with SecretomeP and SignalP. LC-MS analysis revealed 158 different proteins in the supernatant of C. difficile. Most of the identified proteins originate from the cytoplasm. Thirty-two proteins in CDR20291, 36 in CD196 and 26 in CD630 were identified to be secreted by C. difficile strains. Those were mainly S-layer proteins, substrate-binding proteins of ABC-transporters, cell wall hydrolases, pilin and unknown hypothetical proteins. Toxin A and toxin B were identified after growth in brain heart infusion medium using immunological techniques. The ADP-ribosyltransferase-binding component protein, which is a part of the binary toxin CDT, was only identified in the hypervirulent ribotype 027 strains. Further proteins that are secreted specifically by hypervirulent strains were identified.     

Enhanced fermentation of mannitol and release of cytotoxin by Clostridium difficile in alkaline culture media.

Clostridium difficile ATCC 43255 fermented less than 10% of the mannitol in a medium at pH 7; however, when the initial pH of the medium was adjusted to 8.5 or 9, about 80% of the mannitol was fermented. Cell extracts of C. difficile phosphorylated mannitol with phosphoenolpyruvate, not ATP, indicating a phosphoenolpyruvate phosphotransferase system transport phosphorylation of mannitol. The phosphorylation product was dehydrogenated by D-mannitol-1-phosphate:NAD oxidoreductase. Growth at an initial pH of 8.5 yielded cytotoxin titers of 10(7) to 10(8) in Trypticase-yeast extract-mannitol medium, wit a titer of 10(8) as early as 13 h.     

Effect of Streptococcus parvulus and Peptostreptococcus magnus on cytotoxin levels of Clostridium difficile in anaerobic continuous flow culture

An anaerobic continuous flow (CF) culture method was used in order to study the effect of Peptostreptococcus magnus and Streptococcus parvulus, anaerobic gram-positive cocci which are members of intestinal bacterial flora, on growth and cytotoxin-activity of Clostridium difficile. The growth- and the cytotoxin activity-patterns of C. difficile in an established CF culture of P. magnus were similar to those of C. difficile alone. On the other hand, in the mixed culture system of C. difficile and S. parvulus, the cytotoxin levels were significantly lower as compared with C. difficile alone in spite of the fact that no differences existed between growth of C. difficile in mixed and single culture systems. The culture filtrate of P. magnus did not influence the growth and cytotoxin production of C. difficile, nor did that of S. parvulus have any effect on growth of C. difficile in static culture. The cytotoxin activity of C. difficile was, however, suppressed by the culture filtrate of S. parvulus. Furthermore, when P. magnus or S. parvulus was statically cultured in a medium containing cytotoxic culture filtrate of C. difficile, the toxin in the medium was not inactivated.     

中药复方对艰难梭菌毒素基因tcd A/tcd B体外表达的影响

目的探讨中药复方治疗抗生素相关性腹泻(antibiotic associated diarrhea,AAD)的药理机制。方法应用荧光定量PCR技术检测2种中药复方(参苓白术散和理中汤)对2株艰难梭菌临床分离株(A+B+和A-B+)体外培养下毒素基因表达的影响。结果在A+B+艰难梭菌中,参苓白术散组对tcd A和tcd B基因的表达均有显著或是完全抑制,理中汤也有显著的抑制;而对于A-B+艰难梭菌,2种中药复方则完全抑制tcd B基因的表达。结论通过影响毒素基因的表达可能是中药复方治疗AAD的药理学机制之一。     

Structural basis for the function of Clostridium difficile toxin B

Toxin B is a member of the family of large clostridial cytotoxins which are of great medical importance. Its catalytic fragment was crystallized in the presence of UDP-glucose and Mn2+. The structure was determined at 2.2 A resolution, showing that toxin B belongs to the glycosyltransferase type A family. However, toxin B contains as many as 309 residues in addition to the common chainfold, which most likely contribute to the target specificity. A superposition with other glycosyltransferases shows the expected positions of the acceptor oxygen atom during glucosyl transfer and indicates further that the reaction proceeds probably along a single-displacement pathway. The C1' donor carbon atom position is defined by the bound UDP and glucose. It assigns the surface area of toxin B that forms the interface to the target protein during the modifying reaction. A docking attempt brought the known acceptor atom, Thr37 O(gamma1) of the switch I region of the RhoA:GDP target structure, near the expected position. The relative orientation of the two proteins was consistent with both being attached to a membrane. Sequence comparisons between toxin B variants revealed that the highest exchange rate occurs around the active center at the putative docking interface, presumably due to a continuous hit-and-evasion struggle between Clostridia and their eukaryotic hosts.     

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.     

A novel cytotoxin from Clostridium difficile serogroup F is a functional hybrid between two other large clostridial cytotoxins

The large clostridial cytotoxins (LCTs) constitute a group of high molecular weight clostridial cytotoxins that inactivate cellular small GTP-binding proteins. We demonstrate that a novel LCT (TcdB-1470) from Clostridium difficile strain 1470 is a functional hybrid between "reference" TcdB-10463 and Clostridium sordellii TcsL-1522. It bound to the same specific receptor as TcdB-10463 but glucosylated the same GTP-binding proteins as TcsL-1522. All three toxins had equal enzymatic potencies but were equally cytotoxic only when microinjected. When applied extracellularly TcdB-1470 and TcdB-10463 were considerably more potent cytotoxins than TcsL-1522. The small GTP-binding protein R-Ras was identified as a target for TcdB-1470 and also for TcsL-1522 but not for TcdB-10463. R-Ras is known to control integrin-extracellular matrix interactions from inside the cell. Its glucosylation may be a major determinant for the cell rounding and detachment induced by the two R-Ras-attacking toxins. In contrast, fibroblasts treated with TcdB-10463 were arborized and remained attached, with phosphotyrosine containing structures located at the cell-to-cell contacts and beta3-integrin remaining at the tips of cellular protrusions. These components were absent from cells treated with the R-Ras-inactivating toxins. The novel hybrid toxin will broaden the utility of the LCTs for clarifying the functions of several small GTPases, now including also R-Ras.