Journal of Medical Bacteriology 2014. 3(3-4):8-13.

A New Purification Method for Beta-Toxin of Clostridium perfringens Type C Vaccinal Strain
Ehsan Zayerzadeh, Azadeh Fardipour, AhmadReza Jabbari

Abstract


Background: Clostridium perfringens type C strains cause severe necrotizing enteritis in sheep, calves, goats, pigs and humans. Beta-toxin is introduced to be the essential virulence factor of this microorganism. In the present study, a new method was established for the purification of
beta toxin from culture supernatant fluid of C. perfringens type C vaccinal strain.
Methods: The four steps of the purification scheme involved ammonium sulfate precipitation, cation exchange chromatography (CM- Sepharose), anoion exchange chromatography (DEAECellulose) and gel filtration (Sephadex G-100).
Results: Beta toxin was purified about 78-fold from the Sephadex G-100 column with a yield of about 16.7% in terms of lethality of the toxin. The molecular weight of the beta toxin as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was approximately 37
KD. The LD50 for adult mice was 2.21 μg/kg. Human umbilical vein endothelial cells (HUVEC) exposed to CPB showed a cell border retraction, cytoplasmic blebbing, cell shrinkage and cell rounding. The toxin was heat labile and was inactivated by trypsin.
Conclusions: In conclusion, the results of this study showed the new protocol is suitable for purification of beta toxin of C. perfringens type C regarding good purity, good yields and high activity of beta toxin.

Keywords


Clostridium, perfringens, type C, Beta-toxin, Enterotoxemias, chromatography

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References


Songer JG. Clostridial enteric diseases of domestic animals. Clin Microbio Rev 1996; 9:216–234.

Uzal FA, Vidal JE, McClane BA, et al.Clostridium Perfringens Toxins Involved in Mammalian Veterinary Diseases The Open Toxinology Journal 2010; 3, 24-42.

Songer JG. Clostridial diseases of small ruminants. Vet Res 1998; 29: 219-32.

Schumacher VL, Martel A, Pasmans F, et al.Endothelial binding of beta toxin to small intestinal mucosal endothelial cells in early stages of experimentally induced Clostridium perfringens type C enteritis in pigs. Vet Pathol 2013; 50:626-9.

Uzal FA. Diagnosis of Clostridium perfringens intestinal infections in sheep and goats. Anaerobe 2004; 10:135–143.

Steinthorsdottir V, Halldorsson H, Andresson OS. Clostridium perfringens beta-toxin forms multimeric transmembrane pores in human endothelial cells. Microb Pathog 2000; 28:45–50.

Sakurai J, Duncan CL. Some properties of beta- toxin produced by Clostridium perfringens type C. Infect Immun 1978; 21: 678-80.

Worthington RW, Mulders MSG. The partial purification of Clostridium perfringens beta toxin. Onderstepoort J Vet Res 1975; 42, 91-98.

Uzal FA, Saputo J, Sayeed S, et al.Development and application of new mouse models to study the pathogenesis of Clostridium perfringens type C enterotoxemias. Infect Immun 2009; 77: 5291-9.

Lowry OH, Rosenbrough NJ, Farr AL et al.Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 194: 265–275.

Reed LJ, Muench H. A simple method of estimating fifty percent endpoints. Am J Hyg 1938; 27:493–497.

Sayeed S, Uzal FA, Fisher DJ, et al. Beta toxin is essential for the intestinal virulence of

Clostridium perfringens type C disease isolate CN3685 in a rabbit ileal loop model. Mol Microbiol 2008; 67:15-30.

Ma M, Gurjar A, Theoret JR, et al. Synergistic effects of Clostridium perfringens enterotoxin and beta toxin in rabbit small intestinal loops. Infect Immun 2014; 82:2958-70.

Fisher DJ, Fernandez-Miyakawa ME, Sayeed S, et al. Dissecting the contributions of Clostridium perfringens type C toxins to lethality in the mouse intravenous injection model. Infect Immun 2006; 74: 5200-10.

Shatursky O, Bayles R, Rogers M, et al.Clostridium perfringens beta-toxin forms potential-dependent, cation-selective channels in lipid bilayers. Infect Immun 2000; 68: 5546-51.

Vidal JE, McClane BA, J. Saputo J, et al.Effects of Clostridium perfringens beta-toxin on the rabbit small intestine and colon. Infect Immun 2008; 76:4396–4404.

Gurtner C, Popescu F, Wyder M, et al. Rapid Cytopathic Effects of Clostridium perfringens Beta-Toxin on Porcine Endothelial Cells. Infect Immun 2010; 2966–2973.

Nagahama MS, Hayashi S, Morimitsu J, et al.Biological activities and pore formation of Clostridium perfringens beta toxin in HL 60 cells. J Biol Chem 2003; 278:36934–36941.


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