Fast protein purification of Clostridium difficile cytotoxin

Rihn, Bertrand; Bisseret, Françoise; Girardot, Raymonde; Scheftel, J.M.; Nguyen, Vu Khue; Monteil, H.

doi:10.1016/s0378-4347(00)83936-5

Cited by 13 publications

(2 citation statements)

References 18 publications

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“…Further purification of pooled fractions containing cytotoxicity on a Sephacryl $300 gel filtration column also showed co-elution of toxicity and enolase activity. This is in agreement with literature where it has been reported that enolase activity copurifies with toxin B in a wide variety of purification methods including (NH4)2SO4 precipitation, Biogel A5m gelfiltration, phenylboronate column chromatography, ultracentrifugation [4], FPLC anion exchange [7], and DEAESepharose ion-exchange chromatography. Neutralization of cytotoxicity by C sordellii antitoxin did not result in inhibition of enolase activity.…”

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confidence: 93%

Comment to Knoop et al. (1990) FEBS Letters 267, 9‐12, Toxin B of Clostridium difficile does not have enolase activity

et al. 1993

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Ctostridium difficile, recognized as a major cause of antibiotic-associated pseudomembranous enterocolitis~ produces two toxins: toxin A and B. Toxin B is an extremely potent cytotoxin (for review see [1]). The toxin B gene has been sequenced [2,3]. Knoop et al. [4] showed that toxin B has enolase activity as measured by degradation of 2-phosphoglycerate. Here we show that toxin B does not have enolase activity.Amino acid sequences of three tryptic peptides obtained from presumed toxin B showed homology to ~-enolase of the rat and enolase of Saccharon~vces cerevisiae [5]. These sequences were compared with the amino acid sequence of toxin B deduced from the DNA sequence and showed no homology. The tryptic peptides were obtained from a protein with a relative molecular mass of 52 kDa under denaturing conditions. The Toxin B preparation (MW 163 kDa on SDS-PAGE) showed enolase activity [12]. However, the expected molecular weight is 270 kDa [3].These discrepancies led us to study the presence of enolase activity in culture supernatants of toxigenic and nontoxigenic strains of C difficile (obtained from the American Type Culture Collection (ATCC 9689, 43593, 43594, and 43596 through 43603)). Culture supernarants from six C. difficile strains which produced toxin and contained the genes for both toxin A and B, and five strains which possessed neither the toxin A nor the toxin B gene and were not toxigenic [6], were assayed for enolase activity. Both toxigenic and nontoxigenic strains of C. difficile produced enolase activity in the culture supernatant.Partial purification of culture supernatant over DEAE-Sepharose CL-6B showed co-elution of cytotoxicity and enolase activity. However, enolase activity was also demonstrated in the fraction containing unbound proteins. When culture supernatants of nontoxigenic strains were used enolase activity was only recovered in the fraction containing unbound proteins. Further purification of pooled fractions containing cytotoxicity on a Sephacryl $300 gel filtration column also showed co-elution of toxicity and enolase activity. This is in agreement with literature where it has been reported that enolase activity copurifies with toxin B in a wide variety of purification methods including (NH4)2SO4 precipitation, Biogel A5m gelfiltration, phenylboronate column chromatography, ultracentrifugation [4], FPLC anion exchange [7], and DEAESepharose ion-exchange chromatography. Neutralization of cytotoxicity by C sordellii antitoxin did not result in inhibition of enolase activity. However, a goat antiserum against C. dijficile filtrate abolished enolase activity [4], but when the enolase activity and toxin B are two closely linked entities as suggested by purification of culture supernatants over DEAE-Sepharose, it is to he expected that the antiserum contained neutralizing antibodies directed against C difficite enolase activity and toxin B.From these data we conclude that enolase activity is not an integral part of the toxin B gene product, but toxin B and enolase form a stable compl...

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confidence: 93%

Comment to Knoop et al. (1990) FEBS Letters 267, 9‐12, Toxin B of Clostridium difficile does not have enolase activity

et al. 1993

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“…Preparation of toxin B. Toxin B was obtained as described elsewhere (37). Briefly, C. difficile (strain 79685) was incubated for 72 h at 37°C in brain-heart medium (Difco, OSI, Strasbourg, France) under anaerobic conditions.…”

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confidence: 99%

Effects of Clostridium difficile toxin B on human monocytes and macrophages: possible relationship with cytoskeletal rearrangement

Siffert¹,

Baldacini²,

Kuhry³

et al. 1993

Infect Immun

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Toxin B from Clostridium difficile is cytopathic in vitro for various types of cells, including polymorphonuclear cells, lymphocytes, and monocytes. Since intestine lamina propria is rich in macrophages, we studied the effect of toxin B on human monocytes and on human macrophages generated in vitro by long-term culture of purified circulating blood monocytes. Upon addition of toxin B, human monocytes exhibited few modifications whereas macrophages adopted a stellate morphology, with rounding up of the perikaryon. Toxin B made microfilaments of actin disappear and induced an important reorganization of vimentin and a redistribution of tubulin. Membrane area increased by approximately 16%. Toxin B did not affect the viability of human mononuclear phagocytes and did not exert any significant lytic effect. It profoundly altered the phagocytic function of macrophages. When activated by gamma interferon in the presence of toxin B, monocytes were more cytotoxic for U-937 target cells than control monocytes activated in absence of toxin. Finally, the combined treatment of monocytes with gamma interferon and toxin B increased significantly the secretion of tumor necrosis factor alpha, whereas toxin B alone was unable to induce tumor necrosis factor production. These results suggest that morphological and functional alterations induced in human mononuclear phagocytes by toxin B from C. difficile are due to the disorganization of the cytoskeleton and the resulting impairment of the membrane traffic equilibrium.

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Effect of okadaic acid on the cytotoxic activity of Clostridium difficile toxin B and Clostridium sordellii toxin L

et al. 1993

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Clostridium difficile toxin B and Clostridium sordellii toxin L, which are immunologically related toxins, possess a cytotoxic activity inducing depolymerization of microfilaments and cellular retraction of cell bodies that are different for toxin B- and toxin-L-treated cells. The biological mechanisms responsible for these effects are unknown, but a previous study revealed that both toxins induce modification of phosphorylation of cellular proteins extracted from toxin B- and toxin L-treated cells without changes in protein kinase C activity or cAMP concentration. In the present study, we have investigated the effect of okadaic acid, an inhibitor of protein phosphatases, on the cytotoxic activity of toxins B and L in MacCoy cells. Firstly, we reveal by cytotoxic assay and staining of F-actin that okadaic acid (1 microM or higher) induces depolymerization of microfilaments and cellular morphological modifications which are similar to that of cells treated with toxin L. Secondly, we show that 1 microM okadaic acid potentials the cytotoxic activity of toxin L but not of toxin B. These observations suggest that the cytotoxic mechanisms induced by okadaic acid and toxin treatment are partly common, indicating that an increase in phosphorylation favors the cytotoxicity of toxin L. Since okadaic acid had no influence on the cytotoxicity of toxin B, we suggest that toxin B and L, alter the cells by different cellular biological mechanisms.

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Fast protein purification of Clostridium difficile cytotoxin

Cited by 13 publications

References 18 publications

Comment to Knoop et al. (1990) FEBS Letters 267, 9‐12, Toxin B of Clostridium difficile does not have enolase activity

Comment to Knoop et al. (1990) FEBS Letters 267, 9‐12, Toxin B of Clostridium difficile does not have enolase activity

Effects of Clostridium difficile toxin B on human monocytes and macrophages: possible relationship with cytoskeletal rearrangement

Effect of okadaic acid on the cytotoxic activity of Clostridium difficile toxin B and Clostridium sordellii toxin L

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