Infections refractory to standard antibiotic therapy are contributing to adverse treatment outcomes in patients suffering from deep-seated bacterial infections caused by increasingly resistant pathogens. Adjunctive strategies targeting bacterial virulence factors have been considered to supplement the host immune response in fighting the infection. Previous studies suggest that the FDA-approved anti-inflammatory drug diflunisal inhibits Staphylococcus aureus (SA) α-toxin expression by its interaction with the response regulator AgrA. We investigated the broader anti-virulence properties of diflunisal against pathogenic strains of SA and established proof-of-concept for its efficacy in blocking SA virulence. Our studies reveal that diflunisal inhibits α-toxin production, sensitizes SA to cationic antibiotics and human antimicrobial peptides, inhibits the production of the golden pigment staphyloxanthin, and reduces biofilm formation. Molecular docking simulations revealed potential interactions between diflunisal and AgrA binding sites. In addition, sequence alignment of the SA AgrA response regulator demonstrated similarities to other response regulators involved in controlling virulence factor expression. Appreciation of the antivirulence properties of diflunisal supports a therapeutic strategy distinct from structurally similar compounds, such as salicylic acid. The repurposing of diflunisal may mitigate disease severity and provide a unique adjunctive tool in the treatment of SA infection.

Development of an effective vaccine against the leading human bacterial pathogen group A Streptococcus (GAS) is a public health priority. The species defining group A cell wall carbohydrate (GAC, Lancefield antigen) can be engineered to remove its immunodominant N-acetylglucosamine (GlcNAc) side chain, implicated in provoking autoimmune cross-reactivity in rheumatic heart disease, leaving its polyrhamnose core (GACPR). Here we generate a novel protein conjugate of the GACPR and test the utility of this conjugate antigen in active immunization. Instead of conjugation to a standard carrier protein, we selected SpyAD, a highly conserved GAS surface protein containing both B-cell and T-cell epitopes relevant to the bacterium that itself shows promise as a vaccine antigen. SpyAD was synthesized using the XpressTM cell-free protein expression system, incorporating a non-natural amino acid to which GACPR was conjugated by site-specific click chemistry to yield high molecular mass SpyAD-GACPR conjugates and avoid disruption of important T-cell and B-cell immunological epitopes. The conjugated SpyAD-GACPR elicited antibodies that bound the surface of multiple GAS strains of diverse M types and promoted opsonophagocytic killing by human neutrophils. Active immunization of mice with a multivalent vaccine consisting of SpyAD-GACPR, together with candidate vaccine antigens streptolysin O and C5a peptidase, protected against GAS challenge in a systemic infection model and localized skin infection model, without evidence of cross reactivity to human heart or brain tissue epitopes. This general approach may allow GAC to be safely and effectively included in future GAS subunit vaccine formulations with the goal of broad protection without autoreactivity.