Mechanism of action of Cyt1A a delta-endotoxin from Bacillus thuringiensis var. israelensis: Detergent or pore-former?

摘要

Cyt1A is a delta-endotoxin produced by Bacillus thuringiensis var. israelenisis (Bti). This protein has been used in the preparation of environmentally safe insecticides, due to it is toxicity toward Diptera species, especially black flies and mosquitoes. Its mode of action is a subject of controversy. The current hypothesis suggests that Cyt1A forms cation selective channels in the cell membranes, resulting in the equilibration of ions across the membrane followed by osmotically driven influxes of water, leading to cell swelling and lysis. However, there is a sufficient amount of data that does not support this hypothesis. On the basis of the available data we put forward a new hypothesis, suggesting that Cyt1A may act in a detergent-like manner where the soluble toxin diffuses in the extracellular phase and due to the negative membrane surface charge, the toxin experiences lower local pH and changes its conformation, thereby increasing its hydrophobicity and, consequently its affinity to the lipid. On the membrane it aggregates in a "carpet" like manner, thereby disturbing the lipid packing. Through the introduced faults, intracellular molecules leak, leading to cell death. In this work we compare and contrast this two existing hypotheses using a variety of biophysical and biochemical techniques.; Our data suggest that when bound to the lipid, the toxin exhibits a molten-globule state, which is induced by the low pH. This was determined using fluorescence spectroscopy of the environmentally sensitive probes, bis-ANS, Nile red and the intrinsic tryptophan at the conditions close to that of the membrane (lower pH and increased ionic strength). Furthermore, combination of SDS PAGE, and fluorescence lifetime and anisotropy decay measurements, suggest that Cyt1A in the presence of lipid forms nonspecific aggregates, rather than self-assembles into a well-organized proteinaceous pore. In the protein-lipid interactions, electrostatic forces bring the toxin close to the membrane surface and hydrophobic forces keep the toxin bound to the lipid. The effect of the toxin on the size and morphology of the osmotically stable model membranes was studied by the means of fluorescence photobleaching recovery and epifluorescence microscopy. These experiments show that the model membranes (LUV and GUV) with an average diameter of 0.1 and 10 mum were broken into 100 times smaller faster diffusing objects, which is not expected if the toxin had formed a pore. All of these results suggest that Cyt1A acts in a detergent-like manner, rather than the pore-forming manner.