Synthetic short peptide mimicking IsdB and IsdH conserved motifs selectively bind and enable detection of...

Staphylococcus aureus (S. aureus) is the most common pathogen in musculoskeletal infections (MSKI). Application of antibody responses to S. aureus virulence factors have suggested to improve diagnostic specificity. Iron acquisition plays a crucial role in S. aureus pathogenesis. The bacterium scavenges iron from the host by expressing cell surface receptors that bind heme and hemoglobin through the iron-regulated surface determinant (Isd) system. These heme and hemoglobin binding motifs are essential for bacterial survival and virulence. In particular, the highly conserved region of the near iron transport (NEAT) domain may serve as a target for diagnostic applications.

Based on our review of multiple IsdB and IsdH amino acids sequences, we identified a highly conserved near iron transport (NEAT) domains that are unique to S. aureus. We designed and synthesized two short peptides: IsdB-NEAT2, containing the heme binding motifs, and IsdH-NEAT2, containing the hemoglobin binding motif. Each represents the most highly conserved region of the IsdB and IsdH. We utilized infected tissue culture and Staphylococcal complement inhibitor (SCIN) as an adjunct reference. We used enzyme-linked immunosorbent assay (ELISA) with three antigens; IsdB-NEAT2, IsdH-NEAT2, and SCIN to measure reactivity of sera from three groups: patients with S. aureus MSKI (N = 20), patients with MSKI due to other microbial etiologies (N = 20), and healthy controls (N = 21). Circulating immunoglobulins IgM and IgG were measured. Cytokines production by peripheral blood mononuclear cells (PBMC) following stimulation with IsdB-NEAT 2 and IsdH-NEAT 2 antigens were comparatively analyzed against PBMCs from a control group not challenged with S. aureus-specific antigens.

Sera from S. aureus-infected patients showed significantly elevated ELISA optical density (OD) reactivity with IsdB-N2, IsdH-N2, and SCIN (p <0.0001; AUC at 95% confidence interval [CI] > 0.99) respectively compared to healthy controls. Comparisons with other MSKI, sera from S. aureus-associated MSKI yielded significant differences for all three antigens IsdB-N2, IsdH-N2, and SCIN (p <0.0001; AUC at 95% CI were = 0.995, = 0.885 and = 0.920) respectively. Total serum IgM and IgG were markedly higher in S. aureus patients vs. healthy controls (both p = 0.0001). Antigen-specific IgM and IgG levels were significantly elevated in the S. aureus group vs. healthy controls for all three antigens (IgM: IsdB-N2, p = 0.0057; IsdH-N2, p = 0.0040; SCIN, p = 0.0011; IgG: IsdB-N2, p = 0.0499; IsdH-N2, p = 0.0207; SCIN, p = 0.0068). Both peptides stimulated significant cytokine production by PBMCs compared with the untreated PBMCs, inducing IL-6, IL-8, IL-10, IL-1β, MCP-1, TNF-α, EGF, and RANTES.

Our findings indicate that humoral responses to the novel S. aureus antigens (IsdB-NEAT2, IsdH-NEAT2) were able to differentiate S. aureus associated MSKI from other infections, and from healthy controls. Both IsdB-NEAT2 and IsdH-NEAT2 stimulated pro-inflammatory and chemotactic cytokines, confirming that these short-sequenced antigenic motifs activated the innate immune system. This study presents a novel, shortened, and highly conserved component of synthetic Isd peptides, that mimics the functional heme and hemoglobin binding motifs of S. aureus Isd proteins, which could be utilized for diagnosis of S. aureus MSKI.