Abstract
Fibronectin-binding protein A (FnBPA), a protein displayed on the outer surface of Staphylococcus aureus, has a structured A-domain that binds fibrinogen (Fg) and a disordered repeat-region that binds fibronectin (Fn). Amino acid substitutions in Fn-binding repeats (FnBRs) have previously been linked to cardiovascular infection in humans. Here we used microtiter and atomic force microscopy (AFM) to investigate adhesion by variants of full-length FnBPA covalently anchored in the outer cell wall of Lactococcus lactis, a Gram-positive surrogate that otherwise lacks adhesins to mammalian ligands. Fn adhesion increased in five of seven FnBPA variants under static conditions. The bond targeting Fn increased its strength with load under mechanical dissociation. Substitutions extended bond lifetime (1/koff) up to 2.1 times for FnBPA-Fn. Weaker adhesion was observed for Fg in all FnBPA variants tested with microtiter. However, mechanical dissociation with AFM showed significantly increased tensile strength for Fg interacting with the E652D/H782Q variant. This is consistent with a force-induced mechanism and suggests that the dock, lock, and latch (DLL) mechanism is favored for Fg-binding under mechanical stress. Collectively, these experiments reveal that FnBPA exhibits bimodal, ligand-dependent adhesive behavior. Amino acid substitutions in the repeat-region of FnBPA impact binding to both ligands. This was unexpected for Fg since all variants have the same A-domain sequence, and the Fg-binding site is distant from the repeat region. This indicates that FnBRs may fold back on the A-domain in a way that impacts the DLL binding mechanism for Fg.
Highlights
Staphylococcus aureus is an opportunistic pathogen that can cause a wide range of afflictions from superficial and self-limiting infections to more serious endovascular infections, such as infective endocarditis[1,2,3]
We examined three amino acid substitutions in Fn-binding repeats (FnBRs) that were associated with disease (E652D, H782Q, K786N; see locations in Fig. 1a) to determine their impact, if any, on binding to Fn when expressed in Lactococcus lactis
A microtiter assay and a more advanced atomic force microscopy (AFM) technique were used to evaluate binding between Fn or Fg and each of seven fibronectin-binding protein A (FnBPA) variants with amino acid substitutions in FnBR-5 (E652D) and/or FnBR-9 (H782Q, K786N)
Summary
Staphylococcus aureus is an opportunistic pathogen that can cause a wide range of afflictions from superficial and self-limiting infections to more serious endovascular infections, such as infective endocarditis[1,2,3] These infections are facilitated by cell-wall anchored adhesins of the MSCRAMM (microbial surface components recognizing adhesive matrix molecules) type that bind to host blood proteins, including fibronectin (Fn) and fibrinogen (Fg). Previous clinical studies have identified nonsynonymous, single nucleotide polymorphisms in the fnbA gene associated with cardiovascular infections in human patients[14,15,16] These prior studies found amino acid substitutions in FnBR-5 and FnBR-9 of FnBPA in S. aureus isolated from patients with infected cardiac implants and infective endocarditis. Other S. aureus adhesins (i.e., clumping factor A and B, ClfA and ClfB, respectively) have shown force-enhanced adhesion when subjected to mechanical stress[21,22,23]
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