Biological Characterization And Hemolysis Mechanism Of Hemolysin Produced By Synechocystis Sp. PCC 6803

Hemolysin is a member of cyanobacterial toxins, and it is harmful to aquaculture security and environment. Studying the biological characterization and hemolysis mechanism of cyanobacterial hemolysins can facilitate the evaluation of their impact on water quality and the death of fish or seashell, and shed light on the research of the biological and ecological impact of cyanobacterial toxin. In the support of National Natural Science Fund and Natural Science Fund of Shandong Province, the thesis studied the effects of different conditions on the stability and hemolytic activity of the hemolysin produced by the wild type strain of Synechocytstis sp. PCC6803. Moreover, the interaction between hemolysin and erythrocytes and the morphological alteration of erythrocytes treated with hemolysin were also studied as an attempt to explore the hemolysis mechanism of the hemolysin.Keeping in vitro biological activity of hemolysin is the bases of further research. So the effects of different physical and chemical storage conditions on stability of hemolysin were studied at first. The results showed that the hemolysin only had a low hemolytic activity when it was freshly extracted, but the hemolytic activity increased drastically after 3 to 6 days of conservation, then decreased gradually with the time. Low temperature of–20℃and light favored the sustaining of hemolytic activity. Hemolysin of Synechocytstis sp. PCC6803 is not light-dependent because it showed hemolytic activity under dark condition, however, illumination enhances its hemolytic activity significantly. The addition of cysteine monohydrochloride, bovine serum albumin or hemoglobin as stabilizers enhanced the hemolytic activity. Therefore, the hemolysin of Synechocytstis sp. PCC6803 is conserved at -20℃, and used for further research after 3 to 6 days of extraction.The effects of different physical and chemical factors on hemolytic activity of hemolysin were then studied. The effect of temperature and pH showed that hemolysis was enhanced with the increase of temperature and the decrease of pH. The hemolytic activity is facilitated in the presence of lower concentration of hemoglobin than control, but inhibited in the presence of higher concentration of hemoglobin. The determination that the hemolytic activity was enhanced by oxidant of hydrogen peroxide and inhibited by reducers indicated that the hemolysin was a non thiol-activated toxin. In addition, supplement of metal ions of Ca²⁺, Co²⁺, Fe³⁺, Ni²⁺, Cu²⁺, Mn²⁺ and Mg²⁺ decreased the hemolytic activity, whereas Zn²⁺ and EDTA increased it significantly.The spectrum of erythrocytes sensitivity indicated that mouse, sheep and rabbit erythrocytes were highly sensitive to this hemolysin, whereas goldfish, human and chicken erythrocytes were insensitive. The fact that sphingomyelin inhibited the hemolysis significantly while cholesterol and lecithin showed no inhibition suggested that sphingomyelin might be acting as potential receptors of the hemolysin on the erythrocyte membrane. The interaction between hemolysin and rabbit erythrocytes was further studied as an attempt to determine the initial characteristics leading to hemolysis. The analysis of binding of hemolysin and erythrocytes at various temperatures also indicated that hemolysis contained at least two steps: a binding and a lytic stage, both of which were highly temperature-dependent. Hemolytic activity decreased with the increase of the ratio between hemolysin and erythrocytes, this implied that one erythrocyte required a certain number of hits by hemolysin to induce lysis. Thus, the pattern of hemolysis by the hemolysin of Synechocystis sp. PCC 6803 conformed to the multi-hit phenomenon. The finding that none of the osmotic protectants with different hydrodynamic diffusion diameter inhibited hemolysis effectively implied that the hemolysin might not act as a pore-forming toxin.The results of scanning electron micrographs of erythrocytes showed that morphology of rabbit erythrocytes changed greatly after treated by hemolysin of Synechocystis sp. PCC 6803. The occurring of metamorphic became more and much serious with incubating time. Many cells metamorphosed after 0.5 h exposure to the hemolysin and many echinocytes appeared after 1 h. After 2 or 3 h, many cells were completely disrupted. The results of transmitting electron micrographs showed that some erythrocytes contained agglutinated hemoglobin and many cell membranes lysed after 1 h and 3 h of treatment. The characteristics of hemolysis of the hemolysin differ markedly from that of pore-forming toxins suggests that the hemolysin produced by wild type strain of Synechocystis sp. PCC 6803 might act through a detergent or phospholipase mechanism.