IISc researchers found cholesterol present in cell membrane plays a central role in the two-step process of stabilising and binding the pore-forming toxin Cytolysin A. On binding to cholesterol, the structure of the toxin protein that is bound to the cell membrane undergoes a change and is also stabilised. Cholesterol is also essential in holding together the molecules before the toxin forms the pore.
Researchers at the Indian Institute of Science (IISc), Bangalore have found that cholesterol present in cell membrane plays a crucial role in stabilising and binding together the pore-forming toxin Cytolysin A. The pore-forming toxins form the largest class of bacterial proteins causing virulence that kills human cells. The Cytolysin A toxin is secreted by E. coli, Shigella and Salmonella.
The toxin secreted by E. coli bacteria is water-soluble and binds to the cell membrane. The binding of the water-soluble toxin to the cell membrane does not depend on cholesterol present in the membrane surface. In fact, the binding drops in cholesterol-containing membranes. “Binding is only a part of the pore forming process and cholesterol has no role to play,” says Dr. Rahul Roy from the Department of Chemical Engineering at IISc who led the team.
Once the toxin gets bound to the cell membrane it does not stay in one place. Instead, it keeps moving around the cell membrane surface. Using a powerful microscope that allows them to look at single molecules tagged with a fluorescent tag, the researchers could actually see the toxin proteins moving around.
Unlike the usually observed Brownian movement, these proteins tend to move around fast and then slow down before picking up speed and moving fast again. This happens even when no cholesterol is present. The structure capable of puncturing the cell membrane is supposed to slow down the protein movement. So this suggested that the structure of the bound protein is similar to the water-soluble protein and different from the structure that pierces the cell membrane.
The structure of the toxin is stabilised in the presence of cholesterol and that is essential for pore formation.
In the presence of cholesterol, the protein stops moving quickly. “Using molecular dynamic simulations, we found cholesterol interacting with the protein just as we suspected. The interaction was with the region on the protein that is responsible for forming the pore,” says Dr. Roy.
Binding to cholesterol per se does not stop the motion of the protein. But on binding to cholesterol, the structure of the protein undergoes a change resulting in slowing down of the motion.
The change in the speed of motion happens even in the absence of cholesterol due to the change in structure of the protein. But in the absence of cholesterol, the protein is unable to maintain the structure required for pore formation.
“The structure of the toxin is stabilised in the presence of cholesterol and that is essential for pore formation,” says Pradeep Sathyanarayana from the Centre for BioSystems Science and Engineering at IISc and first author of a paper published in the Proceedings of the National Academy of Sciences. “This is a clever strategy by the bacteria to use the toxin to specifically target only human/animal cells while the bacteria themselves are protected from the toxicity since cholesterol is absent in bacterial membranes.”
To be able to rupture the cell membrane, the proteins bound to cholesterol have to come together to form a ring-like structure comprising of 12 molecules. The coming together of the molecules to form the ring-like structure is also enhanced in the presence of cholesterol.
“Computer simulations showed that when two pore-forming protein molecules come together there is a small pocket where the cholesterol goes and interacts with the proteins. So cholesterol provides additional support to hold the two molecules together,” says Dr. Roy.
Studies by other groups have shown that cancer cells in mice can be reduced dramatically by using cytolysin A toxin. “Based on our study, we can work on making the toxin target only the cancer cells. We can also use cholesterol-like molecules to prevent the toxin protein from changing its structure thereby prevent cell destruction,” says Dr. Roy.