Auto-Assembling Detoxified Staphylococcus aureus Alpha-Hemolysin Mimicking the Wild-Type Cytolytic Toxin

Abstract

Staphylococcus aureus alpha-hemolysin (Hla) assembles into heptameric pores on the host cell membrane, causing lysis, apoptosis, and junction disruption. Herein, we present the design of a newly engineered S. aureus alpha-toxin, HlaPSGS, which lacks the predicted membrane-spanning stem domain. This protein is able to form heptamers in aqueous solution in the absence of lipophilic substrata, and its structure, obtained by transmission electron microscopy and single-particle reconstruction analysis, resembles the cap of the wild-type cytolytic Hla pore. HlaPSGS was found to be impaired in binding to host cells and to its receptor ADAM10 and to lack hemolytic and cytotoxic activity. Immunological studies using human sera as well as sera from mice convalescent from S. aureus infection suggested that the heptameric conformation of HlaPSGS mimics epitopes exposed by the cytolytic Hla pore during infection. Finally, immunization with this newly engineered Hla generated high protective immunity against staphylococcal infection in mice. Overall, this study provides unprecedented data on the natural immune response against Hla and suggests that the heptameric HlaPSGS is a highly valuable vaccine candidate against S. aureus.

Deciphering the Pathological Role of Staphylococcal α-Toxin and Panton–Valentine Leukocidin Using a Novel Ex Vivo Human Skin Model

“…Equal molar concentrations of recombinant LukS-PV tag-free (code: 0540-001, IBT Bioservices) and recombinant LukF-PV tag-free (code: 0540-002, IBT Bioservices) were premixed to generate a working concentration of PVL. α-toxin (Hla) and its detoxified mutant Hla H35L were produced as previously described ( 20 ). Lipopolysaccharide contamination in purified proteins was assessed using the LAL test (from Charles River).…”

α-Hemolysin-Aided Oligomerization of the Spike Protein RBD Resulted in Improved Immunogenicity and Neutralization Against SARS-CoV-2 Variants

“…The sequence encoding the signal peptide (residues 1-26) of Hla was removed. The sequence encoding the two b sheets involved in forming the heptameric pore was replaced by a sequence encoding "PSGS", as reported previously (18). Then, the two sequences were linked together by a "GGGGS" linker, with sequences encoding an Nterminal TPA signal peptide and a C-terminal 8×His tag.…”

“…a-Hemolysin (Hla) is a pore-forming toxin secreted by Staphylococcus aureus that can self-assemble into a heptameric pore structure on host cell membranes, resulting in cell lysis and death (17). A previous study revealed that an Hla mutant (mHla) lacking the two b-sheets that form the pore-like structure retains the ability to assemble into a heptamer but loses its biological activity (18). We have demonstrated that mHla could serve as a carrier protein to aid the assembly of IC43, a candidate antigen of Pseudomonas aeruginosa (19), resulting in improved immunogenicity and protective efficacy of the latter.…”

Vaccination with VLPs Presenting a Linear Neutralizing Domain of S. aureus Hla Elicits Protective Immunity

“…Several Hla vaccines have been tested in preclinical animal models including (i) a full length nontoxigenic Hla (Hla H35L ), (ii) the N-terminal 50 amino acids of Hla fused to glutathione S-transferase (GST) (GST-Hla 1-50 ), (iii) a structurally designed vaccine consisting of 62 non-contiguous Hla amino acids, and (iv) Hla engineered to lack the predicted membrane-spanning stem domain (HlaPSGS) [ 10 , 12 , 13 , 14 ]. Despite some successes in animal models, no S. aureus or Hla vaccine has been successful in clinical trials.…”