SummaryStreptococcus pyogenes (group A streptococcus, GAS) secretes streptokinase, a potent plasminogen activating protein. Among GAS isolates, streptokinase gene sequences (ska) are polymorphic and can be grouped into two distinct sequence clusters (termed cluster type-1 and cluster type-2) with cluster type-2 being further divided into sub-clusters type-2a and type-2b. In this study, far-UV circular dichroism spectroscopy indicated that purified streptokinase variants of each type displayed similar secondary structure. Type-2b streptokinase variants could not generate an active site in Glu-plasminogen through nonproteolytic mechanisms while all other variants had this capability. Furthermore, when compared with other streptokinase variants, type-2b variants displayed a 29-to 35-fold reduction in affinity for Glu-plasminogen. All SK variants could activate Gluplasminogen when an activator complex was preformed with plasmin; however, type-2b and type-1 complexes were inhibited by a 2-antiplasmin. Exchanging skatype-2a in the M1T1 GAS strain 5448 with skatype-2b caused a reduction in virulence while exchanging skatype-2a with skatype-1 into 5448 produced an increase in virulence when using a mouse model of invasive disease. These findings suggest that streptokinase variants produced by GAS isolates utilize distinct plasminogen activation pathways, which directly affects the pathogenesis of this organism.

The globally significant human pathogen group A Streptococcus (GAS) sequesters the host protease plasmin to the cell surface during invasive disease initiation. Recent evidence has shown that localized plasmin activity prevents opsonization of several bacterial species by key components of the innate immune system in vitro. Here we demonstrate that plasmin at the GAS cell surface resulted in degradation of complement factor C3b, and that plasminogen acquisition is associated with a decrease in C3b opsonization and neutrophil-mediated killing in vitro. Furthermore, the ability to acquire cell surface plasmin(ogen) correlates directly with a decrease in C3b opsonization, neutrophil phagocytosis, and increased bacterial survival in a humanized plasminogen mouse model of infection. These findings demonstrate that localized plasmin(ogen) plays an important role in facilitating GAS escape from the host innate immune response and increases bacterial virulence in the early stages of infection.

SK (streptokinase) is a secreted plasminogen activator and virulence factor of GAS (group A Streptococcus). Among GAS isolates, SK gene sequences are polymorphic and are grouped into two sequence clusters (cluster type-1 and cluster type-2) with cluster type-2 being further classified into subclusters (type-2a and type-2b). In the present study, we examined the role of bacterial and host-derived cofactors in SK-mediated plasminogen activation. All SK variants, apart from type-2b, can form an activator complex with Glu-Plg (Glu-plasminogen). Specific ligand-binding-induced conformational changes in Glu-Plg mediated by fibrinogen, PAM (plasminogen-binding group A streptococcal M protein), fibrinogen fragment D or fibrin, were required for type-2b SK to form a functional activator complex with Glu-Plg. In contrast with type-1 and type-2a SK, type-2b SK activator complexes were inhibited by α2-antiplasmin unless bound to fibrin or to the GAS cell-surface via PAM in combination with fibrinogen. Taken together, these data suggest that type-2b SK plasminogen activation may be restricted to specific microenvironments within the host such as fibrin deposits or the bacterial cell surface through the action of α2-antiplasmin. We conclude that phenotypic SK variation functionally underpins a pathogenic mechanism whereby SK variants differentially focus plasminogen activation, leading to specific niche adaption within the host.