Abstract
Staphylococcus aureus infection is a major public health threat in part due to the spread of antibiotic resistance and repeated failures to develop a protective vaccine. Infection is associated with production of virulence factors that include exotoxins that attack host barriers and cellular defenses, such as the leukocidin (Luk) family of bicomponent pore-forming toxins. To investigate the structural basis of antibody-mediated functional inactivation of Luk toxins, we generated a panel of murine monoclonal antibodies (MAbs) that neutralize host cell killing by the γ-hemolysin HlgCB. By biopanning these MAbs against a phage-display library of random Luk peptide fragments, we identified a small subregion within the rim domain of HlgC as the epitope for all the MAbs. Within the native holotoxin, this subregion folds into a conserved β-hairpin structure, with exposed key residues, His252 and Tyr253, required for antibody binding. On the basis of the phage-display results and molecular modeling, a 15-amino-acid synthetic peptide representing the minimal epitope on HlgC (HlgC241-255) was designed, and preincubation with this peptide blocked antibody-mediated HIgCB neutralization. Immunization of mice with HlgC241-255 or the homologous LukS246-260 subregion peptide elicited serum antibodies that specifically recognized the native holotoxin subunits. Furthermore, serum IgG from patients who were convalescent for invasive S. aureus infection showed neutralization of HlgCB toxin activity ex vivo, which recognized the immunodominant HlgC241-255 peptide and was dependent on His252 and Tyr253 residues. We have thus validated an efficient, rapid, and scalable experimental workflow for identification of immunodominant and immunogenic leukotoxin-neutralizing B-cell epitopes that can be exploited for new S. aureus-protective vaccines and immunotherapies.
Highlights
Staphylococcus aureus infection is a major public health threat in part due to the spread of antibiotic resistance and repeated failures to develop a protective vaccine
To investigate the molecular basis for antibody recognition of the HlgC subunit, naive mice were immunized with recombinant HlgC subunit protein, and immunoassays of postimmune sera were performed to confirm the induction of an anti-HlgC IgG antibody response
These IgG1- monoclonal antibodies (MAbs) were designated anti-HIgC1 MAb, anti-HIgC2 MAb, anti-HIgC3 MAb, and anti-HIgC4 MAb. Each of these four MAbs displayed strong binding activity with the immunizing HlgC recombinant protein as well as high-level cross-reactivity with LukS, a structurally homologous leukocidin subunit that naturally pairs with the LukF subunit to form the Panton-Valentin leukocidin (LukSF) holotoxin
Summary
Staphylococcus aureus infection is a major public health threat in part due to the spread of antibiotic resistance and repeated failures to develop a protective vaccine. We have validated an efficient, rapid, and scalable experimental workflow for identification of immunodominant and immunogenic leukotoxin-neutralizing B-cell epitopes that can be exploited for new S. aureus-protective vaccines and immunotherapies Staphylococcus aureus is both a ubiquitous commensal microbe and a leading cause of community-acquired and hospital-acquired bone, joint, lung, and bloodstream infections. Adults commonly have circulating IgG antibodies (Abs) to hundreds of S. aureus proteins with high reactivity for exotoxins that include the members of the bicomponent pore-forming toxin (PFT) family [9], and memory B-cell responses to PFT members are common in both healthy adults and those recovering from S. aureus infection [3]. The characteristics of the epitopes that are targeted by naturally occurring and immunization-induced neutralizing anti-S. aureus antibodies are unknown
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