OC 75.4 - Assessing the contribution of (staphylo)coagulases to neutrophil-mediated immunothrombosis

Background: Staphylococcus aureus (S. aureus) is a frequent cause of cardiovascular infections, with virulence factors targeting the coagulation and the immune systems through interaction with plasma proteins. Coagulases from S. aureus bind to prothrombin to form staphylothrombin which generates fibrin. The formation of a fibrin shield around the bacteria can prevent neutrophil-mediated defense mechanisms and hence aids immune evasion. We recently showed that S. aureus induces neutrophil extracellular traps (NETs) formation, whereas the coagulase-negative strain Staphylococcus epidermidis lacks this ability.

Aims: We hypothesized that coagulases may play a role in NET generation and potentially impact other neutrophil-mediated defense mechanisms. Herein, we investigated the impact of coagulases on NET release and myeloperoxidase (MPO) production, in vitro.

Methods: Neutrophils were isolated from healthy donors and incubated with wild-type S. aureus (laboratory strain USA300) or a selective coagulase-deficient mutant (USA300 ΔcoaΔvwb). Neutrophil activation was evaluated using adherent neutrophils or neutrophils in suspension, in the presence or absence of staphylothrombin. For this, NET generation and degranulation were quantified in cell culture supernatants by measuring citrullinated histone H3 (H3Cit)-DNA complexes and myeloperoxidase (MPO), respectively.

Results: Selective deletion of coagulase activity did not affect the ability of S. aureus to stimulate neutrophils to form NETs; however, coagulases’ impact on NET release was dependent on neutrophil adherence. Coagulases promoted NET release from adherent neutrophils yet reduced NET generation from neutrophils in suspension. However, staphylothrombin enhanced NET release from neutrophils in suspension compared to conditions lacking staphylothrombin but with coagulases. Finally, coagulases were observed to promote MPO release from non-adherent neutrophils.

Conclusion(s): Our results suggest that staphylococcal coagulases are important for neutrophil-mediated immunity, hence implying possible contribution for coagulases in infectious context beyond their role in homeostasis.