Degradation of 2‐phosphoglycerate by cytotoxin B of <i>Clostridium difficile</i>

Knoop, Floyd C.; Martig, Robert J.; Owens, M.J.

doi:10.1016/0014-5793(90)80274-m

Cited by 11 publications

(7 citation statements)

References 23 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.…”

supporting

confidence: 93%

“…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.…”

Section: Received 30 October 1992mentioning

confidence: 99%

“…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 complex. …”

mentioning

confidence: 94%

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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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supporting

confidence: 93%

Section: Received 30 October 1992mentioning

confidence: 99%

mentioning

confidence: 94%

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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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“…These functions include its identi ed role as Hsp48, a heat shock protein in Saccharomyces cerevisiae, its structural role in crystallin formation in the turtle eye lens and its possible identity as cytotoxin B of Clostridium dif cile, in which it acts to disrupt the micro lament cytoskeleton by inhibiting adenosine diphosphate (ADP)-ribosylation of the guanosine 5 0 -triphosphate (GTP)-binding protein Rho [1][2][3][4][5][6]. It exhibits additional, nonglycolytic functions in some organisms.…”

Section: Introductionmentioning

confidence: 99%

Plasminogen-binding activity of enolase in the opportunistic pathogenPneumocystis carinii

Fox

Smulian²

2001

Med Mycol

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The glycolytic enzyme enolase is one of the most abundant proteins expressed in fungi and has been shown to be an immunodominant cell-wall-associated antigen of the pathogenic fungus, Candida albicans. Enolase has also been found on the surface of some mammalian cells where it functions as a plasminogen-binding motif and facilitator of plasminogen activation to plasmin. To investigate the immunogenicity of enolase in the opportunistic pathogen, Pneumocystis carinii, the genomic and complementary DNA (cDNA) enolase were cloned and characterized. The predicted protein comprises 433 amino-acid residues and shows extensive homology to other fungal enolases, including those of C. albicans (76%), Aspergillus oryzae (79%) and Saccharomyces cerevisiae (77%). The purified recombinant P. carinii enolase was immunogenic, and may be an important antigen and indicator of P. carinii infection. The active site and conformation metal ion-binding site residues necessary for dimerization and enzyme function are conserved in the predicted P. carinii enolase protein. Enolase of P. carinii is unique among the fungal enolases in that it possesses a catalytic carboxyl-terminal lysyl residue that was necessary and sufficient for the plasminogen-binding activity of the enolase of P. carinii. The activity of the plasminogen binding suggests its involvement in the local regulation of fibrinolysis within the alveolar space.

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“…The reported molecular weights for native toxins A and B have ranged from 440,000 to 600,000 and 107,000 to 550,000, respectively (10,14,15,129,130,148,235,259,265,290 (234) reported that toxin A had a native molecular weight of 52,000 and could be dissociated with 1% sodium VOL. 6,1993 on April 27, 2019 by guest http://cmr.asm.org/ Downloaded from dodecyl sulfate into two subunits with molecular weights of 41,500 and 16,000.…”

Section: Introductionmentioning

confidence: 99%

Clostridium difficile: clinical disease and diagnosis

1993

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Clostridium difficile is an opportunistic pathogen that causes a spectrum of disease ranging from antibiotic-associated diarrhea to pseudomembranous colitis. Although the disease was first described in 1893, the etiologic agent was not isolated and identified until 1978. Since clinical and pathological features of C. difficile-associated disease are not easily distinguished from those of other gastrointestinal diseases, including ulcerative colitis, chronic inflammatory bowel disease, and Crohn's disease, diagnostic methods have relied on either isolation and identification of the microorganism or direct detection of bacterial antigens or toxins in stool specimens. The current review focuses on the sensitivity, specificity, and practical use of several diagnostic tests, including methods for culture of the etiologic agent, cellular cytotoxicity assays, latex agglutination tests, enzyme immunoassay systems, counterimmunoelectrophoresis, fluorescent-antibody assays, and polymerase chain reactions.

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Degradation of 2‐phosphoglycerate by cytotoxin B of Clostridium difficile

Cited by 11 publications

References 23 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

Plasminogen-binding activity of enolase in the opportunistic pathogenPneumocystis carinii

Clostridium difficile: clinical disease and diagnosis

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