Streptokinase variants from Streptococcus pyogenes isolates display altered plasminogen activation characteristics – implications for pathogenesis

Abstract

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.

Full Time Course Kinetics of the Streptokinase-Plasminogen Activation Pathway

“…Whether the Pm generated by the SK⅐Pg*⅐Fbg⅐M-protein quaternary complex remains bound through the picomolar affinity of formation of the SK⅐Pm catalytic complex and many other unanswered questions will require much further investigation. The assays developed here may be similarly extended to investigate the mechanisms of allelic variants of SK (3,4,62) and other pathogenic effectors of SK-Pg activation.…”

“…SK from Streptococcus equisimilis used in the present studies is a group C streptococcal protein responsible for the potentially fatal infection in horses called strangles (2). S. equisimilis SK is most homologous (ϳ90%) to phylogenetic cluster 1 SKs from S. pyogenes (3,4). SK subverts the fibrinolytic system by specific binding to the catalytic domain of plasminogen (Pg) and by induction of non-proteolytic activation of the Pg zymogen by the molecular sexuality mechanism (5-9).…”

“…K C , the dissociation constant for Pg binding to Fbg, was fixed at its determined value of 19 M for the analysis. 3 K D, calc. , the dissociation constant for SK binding to the Pg⅐Fbg binary complex, forming the ternary SK⅐Pg*⅐Fbg complex, was calculated from detailed balance.…”

Plasmin(ogen) Acquisition by Group A <b><i>Streptococcus</i></b> Protects against C3b-Mediated Neutrophil Killing

“…The isogenic derivative NS88.2 prp , which expresses a mutated M protein attenuated for plasminogen binding, has been described elsewhere [5]. A precise, in-frame allelic replacement of the streptokinase ( ska ) gene with cat encoding chloramphenicol transferase was created in GAS wild-type strain NS88.2 using established methods [13], and the resulting strain was designated NS88.2Δ ska . Phenotypic properties of this isogenic mutant strain (i.e.…”

Direct Host Plasminogen Binding to Bacterial Surface M-protein in Pattern D Strains of Streptococcus pyogenes Is Required for Activation by Its Natural Coinherited SK2b Protein

“…Apart from different modes of interaction with SK, hPm shows differential ability to be inhibited by its major physiological inhibitor, ␣ 2 -antiplasmin (AP), depending on the manner in which it is bound to proteins on the GAS surface (30). Thus, it was important to determine whether targeted alterations of the SK subtype in the genomes of GAS would affect hPg activation, the result of which would be expected to alter bacterial virulence and may in fact be an evolutionary mechanism for GAS survival.…”