Intracellular damage and death caused by protease inhibitors on Alexandrium catenella natural cysts and vegetative cells

Proteases from a Chilean clone of Alexandrium catenella were studied using gelatin–zymogram gel and protease fluorescent substrate to facilitate their visual identification in vitro and in vivo, respectively. Proteolytic activity bands were grouped arbitrarily according to their molecular weight as P1 (150 and 120 kDa), P2 (100 kDa), P3 (70 and 65 kDa), P4 (60, 55 and 50 kDa) and P5 (25 kDa). Protease inhibitors affect differentially P2 and P3 proteases. Only P2 activity increased in the presence of 1–10 phenanthroline (σPhe), pepstatin A (pepA), leupeptin (leup) and phenylmethanesulfonyl fluoride (PMSF), while P2 and P3 become inactivated with ρ-aminobenzamidine. The protease inhibitors lethal dose was determined by incubating cells with different concentration of the protease inhibitor and evaluating their effect on cell viability. Furthermore, cells treated for 4 h with one lethal dose of 1–10 phenanthroline and ρ-aminobenzamidine, caused serious damage to the intracellular vacuolar system and nuclear material. Live cysts also die when treated independently with these two protease inhibitors. Future work will be aimed at chemically designing species-specific inhibitors for their potential use in killing cysts transported within the sediment of ship ballast water before washing them off to the environment.