Imaging oligomers of alpha-toxin (Hla) variants using high-speed AFM and neutralizing Hla hemolytic activity with their antisera

Abstract

Alpha-toxin (Hla) variants, such as the toxoids HlaH35A, HlaH35L, and HlaH35LH48L, have been shown to lack hemolytic activity and present promising antigen sources for vaccine development against S. aureus. The His35 site is critical in the oligomerization process of Hla during transmembrane pore formation, leading to cell lysis. This study employed high-speed atomic force microscopy (HS-AFM) to image the structures of HlaH35A, HlaH35L, and HlaH35LH48L proteins on POPC/Chol lipid membranes. Single-site His35 mutations (HlaH35A, HlaH35L) could form oligomer structures, whereas the double-site HlaH35LH48L mutation resulted in the monomer state. These HS-AFM findings confirm that the region between His35 and His48 is crucial for protomer-protomer interactions essential for oligomerization and pore formation. Hemolytic activity of wild-type Hla on red blood cells (RBCs) was significantly reduced when mixed with HlaH35A, HlaH35L, or HlaH35LH48L at weight ratios 1:5 (HlaWT:toxoid) or higher. However, these toxoids exhibited weak neutralization activities at lower mixing ratios with HlaWT. The increased anti-Hla antibodies (IgG) in mice treated with these Hla toxoids have emerged as a potential treatment avenue to neutralize the hemolytic activity of the HlaWT toxin on RBCs. Serum analysis from mice injected with HlaH35A, HlaH35L, and HlaH35LH48L toxoids showed that these sera could neutralize the hemolytic activity of the HlaWT toxin. Thus, these Hla variants are promising candidates for developing supportive treatments for S. aureus infections.