Discovery And Mechanism Study Of Natural Blockers Of α-hemolysin Nanopore

Staphylococcus aureus(S. aureus) is a gram-positive zoonotic pathogen that many cause severe human diseases, such as superficial skin suppurations, wound infections, life-threatening septicemias, bacteraemia and necrotising pneumonia. To date, antibiotic is still the only effective therapy for S. aureus infections. However, the evolution of the bacteria and the abuse of antibiotics always induce the drug resistant effect of bacteria which result in the therapy against the S. aureus infections difficult to achieve. Consequently, the target therapy against virulence factor has attracted more and more attention, and the virulence factor inhibition has gradually become an important strategy for the treatment of S. aureus infections. As an important virulence factor of pathogenic S. aureus, α-hemolysin plays a crucial role in the process of bacterial disease in sepsis.α-hemolysin constituted with 293 amino acids, is a hydrosoluble protein about 33.2 k Da. α-hemolysin is a membrane-inserted heptamer, self-assembled in the host cell membranes or synthetic phospholipid bilayer. The heptamer pore is about 1.5 nm, which can transfer water, ions and/or small molecular, causing red blood cell lysis and sepsis. In vitro, blocking the channel can reduce the red blood cells and blood platelet lysis, and it also shows good efficacy in the pneumonia models induced by S. aureus. α-hemolysin is proposed to be a potential drug target for S. aureus infections.In the current study we applied Planar Lipid Bilayer Workstation(BLM) to record the single channel current of α-hemolysin, and studied single channel’s characteristics under different voltages. We found that the single channel current is most stable under the voltage of-40 m V. Then we investigated the effects of a series of natural compounds on the α-hemolysin nanopore under the-40 m V testing potentials. The results indicated that SS-88 can inhibit the α-hemolysin nanopore current for a long time. Further study by BLM demonstrated that SS-88 could inhibit the single channel currents from both sides of the pore, which distinguish SS-88 from previous reported blockers. Next, the potential protective effects of SS-88 on the injuries caused by S. aureus were examined using red blood cells in vitro and the acute intravascular hemolysis mice model in vivo. We found that the newly identified natural resin, SS-88, effectively suppressed the hemolysis in the both assays. Notably, SS-88 significantly prolonged the mice survival time and reduced the mortality. Finally the molecular mechanism of SS-88 inhibiting α-hemolysin nanopore was investigate by combing residue mutation, protein purification and BLM.In summary, a natural resin SS-88 was identified to be a novel type of inhibitor of α-hemolysin nanopore. Different from previous reported inhibitors, SS-88 could suppress the single channel currents from both sides of the pore. Particularly, SS-88 exhibited significantly protective effects against the hemolysis induced by S. aureus in vitro and in vivo. Our study may provide a novel therapeutic strategy for the S. aureus bacteraemia or sepsis.