Antibodies to coagulase of Staphylococcus aureus crossreact to Efb and reveal different binding of shared fibrinogen binding repeats

Abstract

Staphylococcus aureus pathology is caused by a plethora of virulence factors able to combat multiple host defence mechanisms. Fibrinogen (Fg), a critical component in the host coagulation cascade, plays an important role in the pathogenesis of this bacterium, as it is the target of numerous staphylococcal virulence proteins. Amongst its secreted virulence factors, coagulase (Coa) and Extracellular fibrinogen-binding protein (Efb) share common Fg binding motives and have been described to form a Fg shield around staphylococcal cells, thereby allowing efficient bacterial spreading, phagocytosis escape and evasion of host immune system responses. Targeting these proteins with monoclonal antibodies thus represents a new therapeutic option against S. aureus. To this end, here we report the selection and characterization of fully human, sequence-defined, monoclonal antibodies selected against the C-terminal of coagulase. Given the functional homology between Coa and Efb, we also investigated if the generated antibodies bound the two virulence factors. Thirteen unique antibodies were isolated from naïve antibodies gene libraries by antibody phage display. As anticipated, most of the selected antibodies showed cross-recognition of these two proteins and among them, four were able to block the interaction between Coa/Efb and Fg. Furthermore, our monoclonal antibodies could interact with the two main Fg binding repeats present at the C-terminal of Coa and distinguish them, suggesting the presence of two functionally different Fg-binding epitopes.

Keywords: Efb; Staphylococcus aureus; coagulase; fibrinogen-binding repeats; monoclonal antibodies; phage display.

Figure 1

Domain organization and recombinant constructs of Coagulase and Efb. Domains and recombinant constructs of Coagulase (A) and Efb (B) derived from full length protein of S. aureus Newman strain are shown. Residues, fibrinogen (Fg)- and prothrombin-binding regions are indicated, Signal peptide (S) is necessary for extracellular release. Gluthation-S-Transferase (GST) tag is in red (not to scale), 6 His tag in grey. Images were prepared with DOG2.0 (32). Panel (C) shows an alignment, generated with Geneious, of CoaR0 and R repeats of Coa with a sequence logo and identity to highlight the most conserved amino acids. Amino acids in the single repeats are highlighted with Clustal colour scheme if they are present in more than 50% of the sequences.

Figure 2

FBE5 antibodies dose-dependently bind CoaC fragment. Titration ELISA to evaluate the binding of FBE5 scFv-Fcs to the antigen CoaC (200ng/well immobilized protein). BSA binding curves were always below 0,1 and are not reported for clarity. Isotype control is an unrelated scFv-Fc with human Fc moiety. Data ± SEM are representative of two independent experiments.

Figure 3

Antibodies generated against CoaC bind CoaR0 repeat, but not CoaRI-RV repeats, and cross react with EfbN. Single point ELISA to evaluate the binding of FBE5 scFv-Fc to fragments of Coa and Efb (200ng/well immobilized protein, [scFv-Fc] = 0,5μg/ml). Represented is the average ± SEM.

Figure 4

Dose-dependent binding of anti-CoaR mAbs to Coa and Efb recombinant proteins. Titration ELISA to investigate binding of LIG40-A11 (A) and LIG40-D8 (B) to CoaC, CoaR, CoaR0, CoaF, EfbN, EfbA, EfbO recombinant fragments and determine EC₅₀. BSA and an unrelated human scFv-Fc (both not represented for clarity) were used as negative and isotype controls, respectively, and showed no binding.

Figure 5

Anti-CoaC antibodies inhibit both Coa and Efb fibrinogen binding activity. Antibodies FBE5-A12 (A), FBE5-D10 (B), FBE5-F9 (C) and FBE5-F11 (D) were pre-incubated at the indicated amounts with Coa or Efb recombinant constructs (CoaF, CoaR0, EfbN and EfbO at final concentration of 10nM, EfbA at 750µM) and then transferred to a Fg-coated ELISA plate. The remaining Fg-bound antigens were detected through their tags (GST, except for EfbN which harbours a 6xHis tag). Control wells with no antibody were set to 100% and the residual binding of Coa and Efb constructs was determined by comparing control wells with the ones where indicated amounts of mAbs were added. Average ± SEM of two independent experiments is represented.

Figure 6

Binding of FBE 5 mAbs to Coa C is inhibited by CoaR0 peptide but not by CoaRI peptide. CoaC was immobilized on an ELISA plate and a fixed quantity of each mAb was added (0.5μg/ml) with different dilutions of either CoaR0 (A, B) or CoaRI (C, D) peptide. Detection of mAb was performed through anti-human HRP (HorseRadish Peroxidase)-conjugated secondary antibody. Data ± SEM are reported and are representative of two independent experiments.

Figure 7

Anti-CoaR antibodies are inhibited differently by CoaR0 and CoaRI peptides. CoaC and CoaR were immobilized on an ELISA plate. 0.5μg/ml of LIG40-A11 (A, C) or LIG40-D8 (B, D) were incubated with different dilutions of either CoaR0 (A, B) or CoaRI (C, D) peptides. Detection of mAb was performed through anti-human HRP-conjugated secondary antibody. Data ± SEM are reported and are representative of two independent experiments.