Purification and characterization of alpha-toxin from vaccinal strain of Clostridium novyi.

The genus Clostridium represents a heterogeneous group of anaerobic spore-forming bacteria, comprising prominent toxin-producing species that the most potent biological toxins known to affect humans. Thus it is important to know characters of each toxin when we want to use. In this study clostridium novyi vaccinal strain was growth in proper media and its alpha toxin production was controlled by Vero cell according to percentage of cell rounding. The purification was performed by precipitation using %12 PEG 6000 follow with centrifugation. The toxin was purified up to 80-85% purity by using sephadex G-100 and ion exchange (DEAE- cellulose) chromatography. Molecular weight of alpha toxin was estimated by SDSPAGE and it was near 170 KDa. Glycosyltransferase activity of the toxin was tested by using UDP-N-acetylglucosamine and UDP-glucose as unique substrates. The toxin pH stability was in ringed of 7.5-9 and pI was estimated near 5.5. With respect to this study none of the used metal ions could stimulate the toxin activity, in contrast Ag and Hg could inhibit the toxin activity 95 and 100 percent at 5 mM concentration. EDTA as metal ion chelator did not inhibit the toxin activity but in some concentration EDTA could induce its activity. Effect of alphatoxin on different cells (Vero, MCF-7 and BHK21) was shown that this toxin is able to affect on cell cytoskeleton and finally kill the cells. These results were confirmed by FITC staining according to specific antibody against the toxin. These data were cross checked by dot blot, western blot and ELIZA too. After FITC staining it was shown that fluoresce antibody was more on cell membrane and nucleus membrane. According to the different reports, alpha toxin could act on Rho/Ras proteins and inhibit their activity. Since Rho/Ras families are important proteins for cell signaling and effective on cytoskeleton stability, therefore any inhibition or inactivation of these proteins could guide the cells to apoptosis. It is interesting that the most of cell lines (cancerous cells) have much more Rho/Ras protein than normal cells, suggesting alpha-toxin could be useful for cancerous cells treatment. With respect of capability of this toxin, it could be suggested for cancer therapy, detection kits production and apoptosis study. Keywords: clostridium novyi, alpha-toxin, purification, Rho/Ras proteins.