(113f) The Antagonistic Effect of Membrane Oxysterols on Bacterial Pore Forming Toxins

Pore-forming toxins are a class of proteins secreted by various bacteria that form nanopores on target cell membranes, causing leakage of cellular components, resulting in cell death. Upon encountering the plasma membrane, the water-soluble monomeric form of the protein undergoes significant conformational changes necessary to oligomerize and form pores. Previous experimental and all-atom MD simulations have emphasized the intricate and supportive role of cholesterols in increasing the pore formation activity of Cytolysin A (ClyA), a bacterial pore forming toxin expressed by E coli. On the contrary, oxidized cholesterol derivatives such as 25-hydroxycholesterol produced naturally from cholesterol by the cell have shown antiviral effects against various viruses and can provide innate immunity against bacterial infections by altering the accessible cholesterol content in the cell. Here, we report the effect of 25-hydroxycholesterol (oxysterol) on the pore formation by ClyA. Dye leakage experiments with small unilamellar vesicles demonstrates reduced pore forming activity in concentration dependent manner. We observe that replacement of about one third of cholesterol by 25-hydroxycholesterol negates the enhancement in ClyA activity observed in presence of cholesterol. Using all-atom MD simulations, we observe a distinct tendency for oxysterols to replace cholesterols which binds in the β-tongue pockets to stabilize the membrane inserted oligomeric complex of ClyA. Oxysterols also induce large structural deviations in the membrane inserted N-terminus protein domain compared ClyA in purely cholesterol membranes. We used enhanced string based free energy sampling to map the free energy of transition between the membrane inserted monomer to protomer states of ClyA and probe the change due to the presence of oxysterols. These all-atom and free-energy MD analyses shed light on the molecular action of oxysterols and to reveal the mitigating influence of oxysterols in the prevention of pore formation by bacterial toxins. Our study has implications in understanding bacterial infection pathways in aged and senescent cells where oxidative stress levels are elevated.