Targeting Staphylococcus aureus α-toxin as a novel approach to reduce severity of recurrent skin and soft-tissue infections

Abstract

Staphyococcus aureus frequently causes recurrent skin and soft-tissue infection (SSTI). In the pediatric population, elevated serum antibody targeting S. aureus α-toxin is correlated with a reduced incidence of recurrent SSTI. Using a novel model of recurrent SSTI, we demonstrated that expression of α-toxin during primary infection increases the severity of recurrent disease. Antagonism of α-toxin by either a dominant-negative toxin mutant or a small molecule inhibitor of the toxin receptor ADAM10 during primary infection reduces reinfection abscess severity. Early neutralization of α-toxin activity during S. aureus SSTI therefore offers a new therapeutic strategy to mitigate primary and recurrent disease.

Keywords: Staphylococcus aureus; novel therapeutics; recurrent infection; skin and soft-tissue infection; α-toxin.

Figure 1.

α-Toxin (Hla) modulates Staphylococcus aureus abscess formation during recurrent skin and soft-tissue infection (SSTI). A, Timeline for recurrent SSTI mouse model demonstrating primary infection of mice at 4 weeks of age, with abscess scoring and infection recovery, and secondary infection, with abscess scoring. CFU, colony-forming units. B, Abscess mean area recordings in mice after primary infection with wild-type (WT; open circles) or Hla-deficient (Δhla; open triangles) S. aureus USA300 and in the same groups of mice then subjected to reinfection with WT S. aureus (filled symbols). Error bars represent standard error of the mean for each time point, calculated from recordings in groups of 20 mice. C, Recovery of WT S. aureus from the tissues of mice reinfected as in B, harvested 1 and 3 days after primary (open symbols) or reinfection (closed symbols); values are given as means with standard errors of the mean (error bars). D, Hematoxylin-eosin–stained sections of skin lesions harvested from mice after primary infection with WT or Δhla S. aureus (left panels) or mice receiving primary infection with WT or Δhla S. aureus followed by reinfection with WT S. aureus (right panels). Histopathologic samples were harvested 3 days after infection. Arrows demarcate extent of epidermal injury overlying the abscess lesion. All experiments were repeated for reproducibility, with displayed results representative of 2 or 3 independent analyses.

Figure 2.

Contribution of active α-toxin (Hla) in modulating the host response to recurrent Staphylococcus aureus skin and soft-tissue infection (SSTI). A, B, Analysis of abscess formation in mice subjected to primary infection (1°) with wild-type (WT; circles), Δhla (triangles), Δhla/phla (squares), or Δhla/phla_H35L (diamonds) strains (A) or (B) after WT reinfection of mice infected as in A; 2°, secondary infection. Abscess mean area (n = 15) with plus standard error of the mean (SEM) over 14 days. For the inset in A, overnight culture supernatants of S. aureus WT, Δhla, Δhla/phla, or Δhla/phla_H35L were probed byα-Hla quantitative immunoblotting with relative expression level noted in comparison with the WT strain. C, Hematoxylin-eosin–stained sections of WT-reinfected skin lesions harvested from mice subjected to primary infection with Δhla/phla (upper panels) or Δhla/phla_H35L (lower panels). Histopathologic samples were harvested 1 and 3 days after infection. Arrows demarcate extent of epidermal injury overlying the abscess lesion. D, Reciprocal end point titer analysis of the anti-Hla response calculated from enzyme-linked immunosorbent assay (ELISA) measurements performed on serum samples collected from mice on day 14 after primary infections (A) and secondary infection (B); 6–9 mice were analyzed in each group. Error bars represent mean ± SEM. IgG, immunoglobulin G. E, Percentage of red blood cell hemolysis observed in an in vitro assay assessing the ability of serum harvested from reinfected mice to protect against Hla-mediated lysis of rabbit red blood cells. The percentage of hemolysis is calculated relative to 100% hemolysis obtained in detergent-lysed wells. Dotted line represent mean hemolysis in wells incubated with preinfection serum; error bars, mean + SEM; mAb, monoclonal antibody. *P < .05 (sample vs preinfected serum control). Red dots denote percent red blood cell hemolysis in the presence of serum derived from mice that received passive immunization with a neutralizing anti-Hla monoclonal antibody 24 hours prior to serum harvest. F, G, SSTI mean abscess areas in mice reinfected with WT S. aureus after being treated with either purified, recombinant Hla_H35L or a control protein 6–8 hours after primary infection (F) or being treated with either the ADAM10 inhibitor (GI254023X) or the dimethyl sulfoxide (DMSO) vehicle 6–8 hours after primary infection (G). *P < .05 (Student t test). All experiments were repeated for reproducibility; results are representative of 2–3 independent analyses.