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

Abstract

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 β‐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 α‐hemolysin (α‐HL) and two‐component γ‐hemolysin (γ‐HL). The α‐HL forms a homo‐heptamer, whereas γ‐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 β‐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 β‐barrel formed as a consequence of the conformational change, chimeric mutants composed of the cap/rim domains of α‐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 β‐barrels for use in further promising applications.

A High-Homology Region in Bacterial β-Barrel Pore-Forming Toxins

“…Production and purification of homologous peptide were achieved by cloning this region into an expression vector (see Materials and Methods). To increase immunogenicity, the sequence of homologous peptide was expanded in the N-terminal part to the region involved in the formation of the oligomeric spatial structure of the stem or transmembrane channel and the triangle region corresponding to the PFT region inserted into the membrane [13]. Homologous peptide at the Cterminus used to obtain mAbs includes the HlyIILCTD region described previously [14] (Figure 3a).…”

A High-Homology Region Provides the Possibility of Detecting β-Barrel Pore-Forming Toxins from Various Bacterial Species

“…Production and purification of the HP were achieved by cloning this region into an expression vector (see Section 4). To increase immunogenicity, the sequence of the HP was expanded in the N-terminal part to the region involved in the formation of the oligomeric spatial structure of the stem part or transmembrane channel and the triangle region corresponding to the PFT region inserted into the membrane [14]. The HP at the C-terminus includes the HlyII Met225-Gly250 region, which is a part of the HlyIILCTD described previously [15] (Figure 3).…”

“…The thrombin recognition site, linker, and six histidine residues are highlighted in bold. For the synthetic peptide against which the SHP-series mAbs were obtained, part of the stem domain and triangle region [14] are noted with signatures. [15] region is underlined.…”

Gamma-Hemolysin Components: Computational Strategies for LukF-Hlg2 Dimer Reconstruction on a Model Membrane

“…Two observations suggest that some local rearrangements are required during the formation of the dimer: first, the 15-residue-long N-terminal domain of LukF (N-ter, also called the amino-latch) is folded in the structure of the monomer, but unfolded in the pore [ 10 ]. Second, the same contact interface between the proteins, as observed in the final pore, is not possible due to the steric hindrance between the folded prestem domains, meaning that the protomers can readjust to form the final interface observed in the pore only after the release of the prestem; at the same time, the presence of a folded prestem does not impair oligomerization [ 13 ].…”