The Molecular Mechanisms of Bacterial Virulence Factors that Modulate Host Haemostatic Factors

Group A Streptococcus (GAS) express an arsenal of virulence factors that interact with the host haemostatic system. Streptokinase (SK) from GAS activates human plasminogen, which degrades fibrin clots to facilitate bacterial dissemination. GAS strains have been divided into evolutionary clusters based upon SK sequence variation. Unlike Cluster 1 SK, Cluster 2 variants have very little activity against plasminogen alone and depend on stimulation by co-factors (e.g., fibrin(ogen)). Cluster 2 SK variants also appear to correlate with cell-surface M-like proteins: SK2a with M1 (fibrinogen-binding) and SK2b with PAM (plasminogen-binding). We have shown that plasminogen activation rates using rSK2b are stimulated by rPAM following a template model, suggesting rPAM binding sites are present on rSK2b. Maximum stimulation was found using immobilised rPAM indicating an important role for cell-surface plasmin generation by rSK2b. M1 is known to be cleaved from GAS cell-surface and form a supramolecular complex with fibrinogen, capable of activating neutrophils and platelets creating a hypercoagulable state. Fibrin was the most potent stimulator of rSK2a activity, and fibrin(ogen) stimulation of rSK2a was independent of rM1. The impact of rM1-bound fibrinogen on fibrin clot formation was investigated using microscopy, permeation assays and thromboelastometry, which revealed increasing rM1 produces heterogeneous clots with irregular fibre bundles, compacted fibrin with increased porosity and susceptibility to lysis. rM1 also disrupted the formation of the protective fibrin film and reduced mechanical clot strength. M1-bound fibrinogen at infection sites may contribute to the severity of infection by forming fibrin clots with a compromised protective film, that are mechanically weaker, more porous, and less resistant to lysis by plasmin. Currently no vaccine exists for GAS and reduced susceptibility to antibiotics has been observed. Targeted therapies against M1 and SK (or to counter the haemostatic processes they interrupt) could provide a novel therapeutic strategy for treating GAS diseases and sequalae