Date Published: January 26, 2017
Publisher: Public Library of Science
Author(s): Sian Huish, Craig Thelwell, Colin Longstaff, Catherine A. Brissette.
Streptokinase is a virulence factor of streptococci and acts as a plasminogen activator to generate the serine protease plasmin which promotes bacterial metastasis. Streptokinase isolated from group C streptococci has been used therapeutically as a thrombolytic agent for many years and its mechanism of action has been extensively studied. However, group A streptococci are associated with invasive and potentially fatal infections, but less detail is available on the mechanism of action of streptokinase from these bacteria. We have expressed recombinant streptokinase from a group C strain to investigate the therapeutic molecule (here termed rSK-H46A) and a molecule isolated from a cluster 2a strain from group A (rSK-M1GAS) which is known to produce the fibrinogen binding, M1 protein, and is associated with life-threatening disease. Detailed enzyme kinetic models have been prepared which show how fibrinogen-streptokinase-plasminogen complexes regulate plasmin generation, and also the effect of fibrin interactions. As is the case with rSK-H46A our data with rSK-M1GAS support a “trigger and bullet” mechanism requiring the initial formation of SK•plasminogen complexes which are replaced by more active SK•plasmin as plasmin becomes available. This model includes the important fibrinogen interactions that stimulate plasmin generation. In a fibrin matrix rSK-M1GAS has a 24 fold higher specific activity than the fibrin-specific thrombolytic agent, tissue plasminogen activator, and 15 fold higher specific activity than rSK-H46A. However, in vivo fibrin specificity would be undermined by fibrinogen stimulation. Given the observed importance of M1 surface receptors or released M1 protein to virulence of cluster 2a strain streptococci, studies on streptokinase activity regulation by fibrin and fibrinogen may provide additional routes to addressing bacterial invasion and infectious diseases.
Streptokinase (SK) is a bacterial plasminogen (Pgn) binding and activating protein widely used as a therapeutic thrombolytic agent . The commercial molecule is isolated from Group C streptococci and is able to activate Pgn in solution and is not fibrin specific . Subsequent development of Pgn activator drugs, so-called second and third generation thrombolytics highlighted the desirability of fibrin specificity and led to the development of the tissue Pgn activator (tPA) family of drugs . However, other microbial Pgn activators have a preference for activating fibrinogen (Fgn) or fibrin-bound Pgn which is associated with pathogenesis [4, 5]. Thus studying the mechanisms of Pgn activation by microbial activators is helpful in better understanding infection  and has been applied to the development of fibrin specific thrombolytics, such as staphylokinase and engineered versions of streptokinase [8, 9].
S. pyogenes GAS causes a range of infections from mild to life threatening and it is estimated that globally 18 million people harbour serious infections, with disease burden disproportionately affecting poor, disadvantaged or weakened individuals . S. pyogenes binds Fgn via M protein, which is a known virulence factor and is a target for vaccine development . The ability M protein-Fgn complex to provide a template for assembly of SK•Pgn complex generation is a key regulatory feature for Pm generation [20, 42]. By this mechanism, bacterial SK hijacks the host fibrinolytic system to generate Pm which can overcome defensive fibrin barriers produced by the host. A range of bacteria have evolved systems to arm themselves with surface Pm to optimise invasiveness and pathogenicity . Understanding the regulation of Pgn activation by GAS is a possible route to the development of improved antimicrobial therapies, increasingly important as traditional antibiotics become less effective . Other bacterial proteins are also associated with pathogenesis/invasiveness, including Pgn receptors such as alpha-enolase and Pgn-binding group A streptococcal M-like protein (PAM) [19, 36]. In this case, Pgn activation and Pm binding to the cell surface can be achieved without the involvement of Fgn and Fgn binding proteins. PAM has a high affinity for Pgn and it has been suggested that PAM-expressing GAS strains are adapted for environments where Pgn concentrations are low, such as skin, rather than tissues bathed in plasma or fluids rich in Pgn . Naturally, SK is a key virulence marker and SK structure function studies have identified the beta domain of SK as an important factor in accounting for differences between strains . However, pathogenesis is also affected by other host Pgn activators such as urokinase , and coagulation proteins affecting thrombin generation also have a role .