Supernatants were transferred to a clean microcentrifuge tube and stored at 280uC until further analysis

well as to a lack of technical precision in the previous determination. Internal peptide sequences of peak 18 and 16 exactly match residues 208-224 and 277291, respectively, thus further confirming that the cloned cpd gene encodes for Delta toxin. By comparison with protein sequences available in the data bank, Delta toxin displays significant homology with C. perfringens Beta toxin . Beta toxin is produced by C. perfringens type B and C and is involved in SR 2516 site necrotic enteritis in young animals and in humans, as well as in sheep enterotoxemia. Beta toxin is synthesized as a 336 amino acid protein, the first 27 25833960 residues of which constitute a signal peptide. The secreted protein has a predicted molecular mass of 34861 Da and a pI of 5.5. Delta toxin is also significantly related to C. perfringens necrotic enteritis toxin B-like, which has been recently identified in C. perfringens strains responsible for avian necrotic enteritis. As found for Beta toxin and NetB, Delta toxin is closely related, at the amino acid level, to pore forming cytolysins produced by other bacteria such as Staphylococcus aureus alpha-toxin, subunit F and subunit D , lukS from S. aureus leukotoxin, PantonValentine leukocidin subunit F , components B and C from S. aureus gamma hemolysin . In addition, Delta toxin is related to the hemolysin II from Bacillus cereus and Bacillus thuringiensis . However, Delta toxin shows no significant similarity with C. perfringens Beta2 toxin, and cholesterol-dependent pore-forming toxins such as perfringolysin O and streptolysin O. Recombinant Delta toxin Recombinant Delta toxin without the signal peptide and with a N-terminal extension containing a six His-tag motif from the pET28a vector was produced in E. coli and purified on a cobalt column with elution buffer containing 100 mM imidazole. Processed recombinant Delta toxin was obtained after thrombin treatment. prDelta migrated at about 3536 kDa on SDS-PAGE with a slightly higher molecular mass than that predicted. rDelta and prDelta were recognized by antibodies raised against native Delta toxin as visualized by Western blotting, but not with anti-Beta antibodies. However, immunopurified anti-Delta 10980276 toxin antibodies interacted also with Beta toxin, although to a lesser extent than that with prDelta. This indicates that Delta and Beta toxins share a low level of crossed immunological reactions. containing the six His motif impaired the hemolytic activity of Delta toxin. The 50% hemolytic concentration of prDelta toxin with sheep red blood cells was estimated to 10 ng/ml , which is very close to that determined using native Delta toxin . prDelta was much less active on red blood cells from human, rabbit and horse in agreement with that already found with native Delta toxin. As Delta toxin was reported to specifically bind to ganglioside GM2, we tested the inhibition of Delta toxin hemolytic activity with various gangliosides. prDelta was incubated with gangliosides for 5 min and then tested for hemolytic activity with sheep red blood cells. GM2 efficiently inhibited the hemolytic activity of Delta toxin on sheep red blood cells, whereas GM1 was slightly less inhibitory . In contrast, GM3 in the same range of concentrations than those of GM1 or GM2, which were inhibitory, did not modify the hemolytic activity of prDelta. Since Delta toxin shows a significant sequence homology with Beta toxin, we checked whether both toxins competed for the same cell surface receptor. Beta toxin prepar

Leave a Reply