Proteolytic processing of the Cyt1Ab1 toxin produced by Bacillus thuringiensis subsp medellin

Bacillus thuringiensis produces delta-endotoxins that require proteolytic p rocessing to become active. The activation of the B. thuringiensis subsp. m edellin 28 kDa (Cyt1Ab1) cytolytic toxin by trypsin, chymotrypsin and gut e xtract from Culex quinquefasciatus larvae was analyzed. The Cyt1Ab1 toxin o f B. thuringiensis subsp. medellin was processed by all proteases tested to fragments between 23 and 25 kDa, while processing of the Cyt1Aa1 toxin pro duce fragments between 22.5 and 24.5 kDa. The Cyt1Ab1 toxin was preferentia lly processed at the alkaline pH of 12. The in vitro proteolytic processing of the Cyt1Ab1 toxin by C. quinquefasciatus larvae midgut extract showed a 25 kDa fragment; a similar result was observed when the activation was per formed in the in vivo experiments. The solubilized Cyt1Ab1 toxin and the pr otease resistant cores generated by in vitro processing showed hemolytic ac tivity but not mosquitocidal activity. Amino terminal sequence of the C. qu inquefasciatus gut extract resistant fragment indicated that the cutting si te was located between Lys(31) and Asp(32), with a sequence DDPNEKNNHNS; wh ile for the trypsin-resistant fragment the cutting site was, determined bet ween Leu(29) and Arg(30), and for the chymotrypsin-resistant fragment betwe en Arg(30) and Lys(31).