Chimeric mutants of staphylococcal hemolysin, which act as both one-component and two-component hemolysin, created by grafting the stem domain

Staphylococcus aureus expresses several hemolytic pore-forming toxins (PFTs), which are all commonly composed of three domains: cap, rim and stem. PFTs are expressed as soluble monomers and assemble to form a transmembrane beta-barrel pore in the erythrocyte cell membrane. The stem domain undergoes dramatic conformational changes to form a pore. Staphylococcal PFTs are classified into two groups: one-component alpha-hemolysin (alpha-HL) and two-component gamma-hemolysin (gamma-HL). The alpha-HL forms a homo-heptamer, whereas gamma-HL is an octamer composed of F-component (LukF) and S-component (Hlg2). Because PFTs are used as materials for nanopore-based sensors, knowledge of the functional properties of PFTs is used to develop new, engineered PFTs. However, it remains challenging to design PFTs with a beta-barrel pore because their formation as transmembrane protein assemblies requires large conformational changes. In the present study, aiming to investigate the design principles of the beta-barrel formed as a consequence of the conformational change, chimeric mutants composed of the cap/rim domains of alpha-HL and the stem of LukF or Hlg2 were prepared. Biochemical characterization and electron microscopy showed that one of them assembles as a heptameric one-component PFT, whereas another participates as both a heptameric one- and heptameric/octameric two-component PFT. All chimeric mutants intrinsically assemble into SDS-resistant oligomers. Based on these observations, the role of the stem domain of these PFTs is discussed. These findings provide clues for the engineering of staphylococcal PFT beta-barrels for use in further promising applications.