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Published as doi: 10.1096/fj.08-109348.
 (The FASEB Journal. 2008;22:3146-3153.)
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Allelic variants of streptokinase from Streptococcus pyogenes display functional differences in plasminogen activation

Jason D. McArthur*, Fiona C. McKay*, Vidiya Ramachandran*, Priya Shyam*, Amanda J. Cork*, Martina L. Sanderson-Smith*, Jason N. Cole*, Ulrika Ringdahl{dagger}, Ulf Sjöbring{dagger}, Marie Ranson* and Mark J. Walker*,1

* School of Biological Sciences, University of Wollongong, Wollongong, Australia, and

{dagger} Department of Laboratory Medicine, Section for Microbiology, Immunology and Glycobiology, Lund University, Lund, Sweden

1Correspondence: School of Biological Sciences, University of Wollongong, Wollongong, NSW, 2522, Australia. E-mail: mwalker{at}uow.edu.au


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A common mammalian defense mechanism employed to prevent systemic dissemination of invasive bacteria involves occlusion of local microvasculature and encapsulation of bacteria within fibrin networks. Acquisition of plasmin activity at the bacterial cell surface circumvents this defense mechanism, allowing invasive disease initiation. To facilitate this process, S. pyogenes secretes streptokinase, a plasminogen-activating protein. Streptokinase polymorphism exhibited by S. pyogenes isolates is well characterized. However, the functional differences displayed by these variants and the biological significance of this variation has not been elucidated. Phylogenetic analysis of ska sequences from 28 S. pyogenes isolates revealed 2 main sequence clusters (clusters 1 and 2). All strains secreted streptokinase, as determined by Western blotting, and were capable of acquiring cell surface plasmin activity after incubation in human plasma. Whereas culture supernatants from strains containing cluster 1 ska alleles also displayed soluble plasminogen activation activity, supernatants from strains containing cluster 2 ska alleles did not. Furthermore, plasminogen activation activity in culture supernatants from strains containing cluster 2 ska alleles could only be detected when plasminogen was prebound with fibrinogen. This study indicates that variant streptokinase proteins secreted by S. pyogenes isolates display differing plasminogen activation characteristics and may therefore play distinct roles in disease pathogenesis.—McArthur, J. D., McKay, F. C., Ramachandran, V., Shyam, P., Cork, A. J., Sanderson-Smith, M. L., Cole, J. N., Ringdahl, U., Sjöbring, U., Ranson, M., Walker, M. J. Allelic variants of streptokinase from Streptococcus pyogenes display functional differences in plasminogen activation.


Key Words: bacterial pathogenesis • virulence • invasive disease


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
GROUP A STREPTOCOCCUS (GAS; Streptococcus pyogenes) is a common bacterial pathogen whose virulence has been honed through the process of evolution with its singular human host. GAS employs numerous pathogenic mechanisms that interact specifically with human proteins that enable evasion of host defenses and promote bacterial colonization, proliferation, and dissemination. As an early mammalian defense mechanism, invading bacterial pathogens such as GAS are confined at sites of infection through the deposition of fibrin networks, thereby allowing a directed inflammatory immune response to specifically target this area (1) . To circumvent this defense mechanism, GAS subverts components of the host fibrinolytic system to degrade fibrin and thus spread to other areas of the body (2) .

A key component of the fibrinolytic system is plasminogen. Plasminogen is a 92 kDa glycoprotein found in plasma and extracellular fluids (3) . Cleavage of the plasminogen activation bond (i.e., Arg561-Val562) by the specific host plasminogen activators, urokinase (uPA) and tissue plasminogen activator (tPA), results in the formation of the serine protease, plasmin (4) . Plasmin has a broad substrate spectrum that includes fibrin clots and the extracellular matrix (4) . GAS secretes streptokinase, a human-specific plasminogen-activating protein. Unlike host plasminogen activators, streptokinase lacks any intrinsic enzymatic activity and activates plasminogen by forming a stable 1:1 stoichiometric complex with either plasminogen or plasmin (5) . Both complexes display the broad-spectrum protease activity of plasmin, but this activity is unique in that it cannot be regulated by the normal plasma inhibitors (i.e., {alpha}2-antiplasmin and {alpha}2-macroglobulin) and can also activate other molecules of plasminogen (6) .

Plasminogen contains several distinct structural domains, consisting of the amino-terminal peptide, followed by Kringle domains 1–5 (K1–5) and the carboxy-terminal serine protease domain. K1, K4, and K5 contain lysine-binding motifs that are responsible for binding to fibrinogen and to plasminogen receptors (7) . The circulating, soluble form of plasminogen (glu-plasminogen) is maintained in a closed form through lysine-dependent interaction between the amino-terminal peptide and K5. On binding to mammalian or bacterial receptors, a conformational change is induced in glu-plasminogen, producing an open, activation-susceptible form (6 , 7) .

GAS captures and facilitates plasminogen activation to plasmin, conferring cell-bound proteolytic activity, by several pathways. Plasminogen bound directly to GAS via cell surface receptors such as the plasminogen-binding group A streptococcal M-like proteins (PAMs) (8 9 10) , glyceraldehye-3-phosphate dehydrogenase (GAPDH) (11) , or streptococcal enolase (SEN) (12) can be activated by streptokinase:plasminogen/plasmin complexes, or by host activators such as tPA (13) . Alternatively, plasmin formed in solution may subsequently bind to GAS cell surface receptors (14 , 15) . GAS may also acquire cell surface plasmin activity by binding a trimolecular complex of plasminogen, human fibrinogen, and streptokinase (16 , 17) .

Streptokinase alleles from GAS are polymorphic (18) . Previous studies have linked streptokinase polymorphism with strains associated with acute poststreptococcal glomerulonephritis (19 , 20) and with strains that exhibit skin tissue tropism (21) . However, the functional significance of streptokinase polymorphism has not been elucidated. With the majority of structural and functional studies on streptokinase having been performed using the therapeutic streptokinase from group C streptococcus, the phenotypic differences displayed by GAS streptokinase proteins have yet to be explored. This study investigates streptokinase polymorphism in a number of clinical GAS isolates and demonstrates that variant streptokinase proteins display differing plasminogen activation capacities.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
GAS isolates and culture
The 28 GAS isolates from the Northern Territory (NT) of Australia used in this study were collected from patients between 1990 and 1998, and their clinical source, emm type, pam genotype, and plasminogen-binding characteristics have been described in detail (17) . This strain set was selected because the region suffers endemic GAS infections, and the strains examined were epidemiologically diverse. Pam-positive isolates were defined as those positive for pam by Southern hybridization and containing pam encoding A1 and A2 repeat regions with greater than 50% amino acid sequence homology to that of the prototype PAM sequence (8) . The invasive GAS isolate 5448 (M type 1), the North American emm53 impetigo isolate ALAB49, and its isogenic streptokinase deletion mutant have been described previously (22 23 24 25 26) . GAS was cultured on defibrinated horse blood agar plates (Biomerieux, Sydney, NSW, Australia) or in Todd Hewitt broth (BD, Sydney, NSW, Australia) supplemented with 1% (w/v) yeast extract (THY; Oxoid, Adelaide, SA, Australia) at 37°C as stationary cultures.

Streptokinase expression
For analysis of streptokinase expression, aliquots of overnight GAS cultures were washed twice by centrifugation, followed by subsequent resuspension with equal volumes of fresh THY before being used to inoculate new cultures. When GAS cultures had reached midlog phase (A600=0.6), supernatants were harvested by centrifugation, filtered using a 0.2 µm polyvinylidene difluoride (PVDF) syringe filter (Millipore, Sydney, NSW, Australia) and stored at –70°C until analysis.

Thawed GAS culture supernatants were then concentrated 18-fold by 10% trichloroacetic acid precipitation before SDS-PAGE and Western transfer to PVDF membrane (Millipore). For detection of streptokinase, the membranes were immunoblotted using rabbit immune serum raised against commercial group C streptokinase (Sigma-Aldrich, Sydney, NSW, Australia) as described previously (27) .

Streptokinase activity
An indirect plasminogen activation assay was used to measure streptokinase activity in GAS culture supernatants using the plasmin amidolytic substrate Spectrozyme® PL (American Diagnostica, Stamford, CT, USA), as described previously (28) . Briefly, supernatants were defrosted on ice and a 20 µl aliquot was incubated at 37°C with 100 µl 50 mM Tris, pH 7.5, containing 20 µg/ml glu-plasminogen (Hematologic Technologies, Essex Junction, VT, USA) for 15 min. Spectrozyme PL (20 µl of 2.5 mM) was added, and absorbance at 405 nm was measured every 3 min for 60 min. Absorbance was plotted against time, and activity rates were determined from the linear portion of the curve. The amount of streptokinase activity in each bacterial culture supernatant was converted to units per milliliter using a standard curve of group C streptokinase (Sigma-Aldrich) serially diluted in THY medium, using Softmax® Pro software (Molecular Devices, Sunnyvale, CA, USA). All reactions were performed in duplicate in the presence and absence of plasminogen to confirm that proteolytic activity of supernatants was attributable to plasminogen activation. Interassay variation was corrected for using an internal positive control sample (250 U/ml of purified group C streptokinase; Sigma-Aldrich) in each assay. In assays examining the effects of plasminogen-binding ligands on streptokinase-mediated plasminogen activation, human fibrinogen (Sigma), recombinant SEN (29) , and recombinant PAM (30) were each mixed in a 1:1 stoichiometric ratio with plasminogen and preincubated at 37°C for 15 min before the addition of bacterial culture supernatants.

Cell surface plasmin activity
Plasmin acquisition by S. pyogenes isolates after incubation in human plasma was determined essentially as described previously (27) . Frozen plasma was purchased from the Red Cross Blood Bank (Sydney, NSW, Australia), defrosted on ice, and pooled. Aliquots of pooled plasma were depleted of plasminogen by incubation at 4°C on ice with excess lysine-sepharose® 4B (Amersham Biosciences, Sydney, NSW, Australia) for 1–2 h with gentle agitation. GAS was cultured overnight, washed once in PBS, pH 7.4, and resuspended to A600 = 0.7. Aliquots of this suspension were pelleted by centrifugation (1500 g for 10 min) and resuspended in an equal volume of 100% human plasma or plasminogen-depleted plasma at 37°C. GAS was incubated in plasma for 3 h at 37°C, pelleted by centrifugation, and washed twice with 1 volume of ice-cold 0.01 M EDTA, 0.1% gelatin in PBS, pH 7.4. GAS was resuspended in 0.1% gelatin in PBS, pH 7.4, to A600 = 0.75. Aliquots (100 µl) of this suspension were incubated in triplicate in the presence and absence of 20 µl Spectrozyme PL (2.5 mM) at 37°C for 60 min in a 96-well plate. The reaction was quenched with 80 µl of 1.75 M acetic acid, the plates were centrifuged, and A405 of supernatants was determined.

Plasmin activity was determined as the difference between A405 in the presence and absence of substrate. Isolate NS931 was included as an internal control in every experiment. Each isolate was assayed in at least 3 independent experiments. Plasmin equivalents and the linear range of the assay (A405=0–0.6) were determined using a standard curve of purified plasmin (Roche Diagnostics, Sydney, NSW, Australia).

Streptokinase genotype
Chromosomal DNA was extracted from GAS strains using a commercially available system (DNeasy Kit; Qiagen, Melbourne, VIC, Australia). The variable region of the streptokinase gene was PCR amplified using previously described primers (31) , sequenced using the BigDye Terminator cycle sequencing kit, and analyzed using ABI Prism Autoassembler software (Applied Biosystems, Melbourne, VIC, Australia). A phylogenetic tree was constructed for the 423 bp nucleotide sequence encoding the variable β-domain of the streptokinase protein of the 30 GAS isolates using molecular evolutionary genetics analysis (MEGA; ref. 32 ) as described previously (21) . The Genbank accession numbers for the 29 partial ska sequences are EU352612 to EU352641.

Statistical analyses
Differences of enzyme activities between isolates were determined using unpaired, 2-tailed Student’s t test with 95% confidence intervals. The effect of prebinding ligands to plasminogen on streptokinase activation was analyzed using a 1-way ANOVA and a Newman-Keuls multicomparison posttest. All statistical analyses were carried out using GraphPad Prism (GraphPad Software Inc., San Diego, CA, USA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Phylogenetic analysis of streptokinase β-domain gene sequences
The relatedness of streptokinase alleles from 5448, ALAB49, and 28 NT GAS isolates were investigated by phylogenetic analysis of nucleotide sequences encoding the highly variable β-domain of streptokinase (Fig. 1 ). The β-domain sequences were found to cluster into 2 major groups (clusters 1 and 2) with evidence of several smaller subclusters present. This phylogeny is similar to that seen in previous studies (21) . The ska alleles of all pam-negative isolates fell within cluster 1, with the exceptions of the 2 emm1 isolates NS696 and 5448, which fell within a smaller subcluster of cluster 2 alleles (termed cluster 2a). The majority of pam-positive isolates (12/13) possessed a cluster 2 allele (termed cluster 2b). A notable exception was the pam-positive isolate NS53, which possessed a cluster 1 ska allele. This association between cluster 2b ska alleles and pam genotype has been previously observed (21) .


Figure 1
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  		Figure 1. Phylogenetic tree for a 423 bp variable region encoding the β-domain of streptokinase for the 30 isolates examined in this study. The DNA sequence of the ska allele from ALAB49 (AY234134) was obtained from a previous study (21) . Bootstrap values of ≥90% (500 replicates) are indicated. Scale bar = 0.05 substitution per site. Pam-positive GAS isolates are in italics. The Genbank accession numbers for the 29 partial ska sequences are EU352612 to EU352641.

Activity and expression of streptokinase allelic variants
Streptokinase activity was determined in supernatants collected from cultures at midlog phase for the 31 GAS isolates. The results demonstrate a striking difference in soluble plasminogen activation capacities displayed by the different streptokinase variants produced by each isolate. For all strains harboring a cluster 1 ska allele, high levels of streptokinase activity were present in culture supernatants (Fig. 2 A). These ranged from 9 to 132 U/ml. For strains harboring a cluster 2 ska allele, no streptokinase activity was detected in the culture supernatants (Fig. 2A ). The pam-positive strain, NS53, which contains a cluster 1 ska allele, produced measurable streptokinase activity (33 U/ml) in culture supernatants (Fig. 2A ).


Figure 2
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  		Figure 2. Solution-phase streptokinase activity and expression by 31 clinical isolates of GAS. A) Streptokinase activity in GAS culture supernatants at midlog phase (A600=0.6) measured by an indirect assay of plasmin activity in the presence of human plasminogen using the plasmin-specific chromogenic substrate Spectrozyme PL. Data represent mean streptokinase activity (U/ml) from duplicate assays, using a standard curve of group C streptokinase and corrected for an internal positive control sample in each assay. Error bars = range for each point. B) Western blot of midlog phase GAS culture supernatants using rabbit immune serum raised against group C streptokinase. Solid horizontal bar = GAS isolates containing a cluster 1 ska allele; dashed horizontal bar = isolates containing a cluster 2 ska allele; underscore = GAS isolates containing a subcluster 2a ska allele; italics = PAM-positive GAS isolates.

We next investigated whether the lack of soluble streptokinase activity seen in culture supernatants from strains containing a cluster 2 ska allele was the result of a lack of streptokinase protein in culture supernatants. Western immunoblotting experiments using anti-SpeB polyclonal sera demonstrated that the cysteine protease SpeB, which is known to degrade streptokinase (33) , was not present in supernatants harvested at this phase of growth (data not shown). This finding was corroborated by Western immunoblotting performed using antistreptokinase polyclonal sera because it detected an immunoreactive band ranging from ~46 to 49 kDa in culture supernatants for all GAS isolates, including those harboring a cluster 2 ska allele (Fig. 2B ). This corresponds to the approximate size of streptokinase (28) . The ALAB49{Delta}ska mutant strain lacked this 46 to 49 kDa immunoreactive band, instead producing a 27 kDa band consistent with the expected size of the truncated streptokinase protein produced by this strain (25) . These results clearly show that the lack of soluble streptokinase activity in culture supernatants from isolates with a cluster 2 ska allele was not attributable to a lack of streptokinase protein being present in these samples.

Acquisition of cell surface plasmin by GAS isolates in plasma
To determine whether the secreted streptokinase protein encoded by cluster 2 ska alleles was inactive, an indirect assay measuring the acquisition of cell surface-bound plasmin after incubation in human plasma was performed for all isolates (Fig. 3 ). Despite the apparent lack of streptokinase activity in solution, isolates containing a cluster 2 ska allele acquired significantly higher levels of plasmin activity at the cell surface compared with the isolates containing a cluster 1 ska allele (Fig. 3 ; P=0.0018). As might be expected, the isogenic streptokinase-negative mutant ALAB49{Delta}ska acquired significantly less cell surface plasmin activity than wild-type ALAB49 (Fig. 3 ; P=0.019). Because secreted cluster 2 type streptokinase appears to contribute to cell surface plasmin acquisition, these observations suggest that this type of streptokinase is active but only in the presence of cell surface receptors or human plasma.


Figure 3
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  		Figure 3. Cell surface plasmin activity of GAS isolates. Cell surface plasmin activity of 31 clinical GAS isolates after incubation in human plasma (filled bars) or plasminogen-depleted plasma (unfilled bars) determined by cleavage of Spectrozyme PL. Data represent mean ± SE of triplicate cultures of GAS, expressed as A405 of cleaved substrate in solution. Overall, strains containing cluster 2 ska alleles acquire more cell surface plasmin activity compared with strains containing cluster 1 ska alleles (P=0.0018). Solid horizontal bar = GAS isolates containing a cluster 1 ska allele; dashed horizontal bar = isolates containing a cluster 2 ska allele; underscore = GAS isolates containing a subcluster 2a ska allele; italics = PAM-positive GAS isolates.

Effects of plasminogen-binding receptors on plasminogen activation by streptokinase
Fibrinogen can contribute to the acquisition of cell surface plasmin activity by binding streptokinase-plasminogen complexes to M-proteins located on the bacterial cell surface (16) . The effect of prebinding plasminogen to fibrinogen on plasminogen activation by group A streptococcal streptokinase variants was thus examined (Fig. 4 ). Fibrinogen significantly increased the streptokinase activity detected in GAS culture supernatants from isolates harboring cluster 1 ska alleles when compared with activities detected in the absence of fibrinogen (Fig. 4A ; P<0.001). Notably, streptokinase activity was also detected in GAS culture supernatants from isolates harboring the cluster 2 ska alleles when plasminogen was prebound with fibrinogen (Fig. 4A ).


Figure 4
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  		Figure 4. Streptokinase activity in GAS culture supernatants at midlog phase (A600=0.6) measured by an indirect assay of plasmin activity in the presence of human plasminogen prebound with fibrinogen (A), PAM (B), SEN (C), fibrinogen and PAM (D), or fibrinogen and SEN (E). Data represent mean streptokinase activity (U/ml) from duplicate assays, using a standard curve of group C streptokinase and corrected for an internal positive control sample in each assay. Error bars = range for each point. Solid horizontal bar = GAS isolates containing a cluster 1 ska allele; dashed horizontal bar = isolates containing a cluster 2 ska allele; underscore = GAS isolates containing a subcluster 2a ska allele; italics = PAM-positive GAS isolates.

The binding of glu-plasminogen to mammalian or bacterial receptors induces a conformational change that can affect the activation of this zymogen (6 , 7) . When plasminogen was prebound to PAM, a significant increase in streptokinase activity in GAS culture supernatants from isolates harboring cluster 1 and cluster 2a ska alleles was observed when compared with activities detected in the absence of PAM (Fig. 4B ; P<0.01). However, this enhanced streptokinase activity is not biologically relevant, because the majority of cluster 1 ska allele GAS strains do not express PAM. GAS culture supernatants from pam-positive isolates harboring a cluster 2b ska allele did not produce detectable streptokinase activity when plasminogen was prebound to PAM (Fig. 4B ). When plasminogen was prebound to SEN, streptokinase activity in all GAS culture supernatants was similar to levels detected in the absence of SEN (Fig. 4C ; P>0.05). When plasminogen was prebound to a combination of fibrinogen and PAM or fibrinogen and SEN, streptokinase activity was increased to levels similar to those seen for fibrinogen alone, suggesting that the presence of PAM or SEN does not confer an additional benefit (Fig. 4D, E ). For all reaction conditions, ALAB49{Delta}ska did not exhibit streptokinase activity in culture supernatants (Fig. 4) . These data demonstrate that, for cluster 2 ska alleles, the presence of fibrinogen is a prerequisite for the cluster 2 streptokinase-plasminogen complex to gain functional activity.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Activating plasminogen and acquiring plasmin activity to the bacterial cell surface has been recognized as a pathogenic mechanism for a variety of bacterial species that can cause invasive infections (34 , 35) . A longstanding question in group A streptococcal research has been the correlation of high-level cell surface plasminogen-binding capacity with the absence of detectable streptokinase activity (2 , 28) . In this study, we demonstrate for the first time that allelic variants of streptokinase produced by different GAS isolates display unique plasminogen activation properties that confer on all GAS isolates the potential to harness plasmin activity onto the bacterial cell surface.

Plasminogen receptors interact with different regions of the plasminogen protein. The GAS plasminogen-binding M protein, PAM, binds to plasminogen via interaction with K2 (36) , whereas fibrinogen and the GAS plasminogen receptors SEN and GAPDH bind via lysine-dependent interactions with K1, K4, and K5 (7) . Streptokinase encoded by cluster 2b ska alleles failed to produce any soluble plasminogen activation activity in the absence of fibrinogen. Prebinding of the plasminogen receptors PAM or SEN to plasminogen did not enhance plasminogen activation by cluster 2b allelic variants of streptokinase. This finding is consistent with previous studies (28) that have noted that specific isolates of S. pyogenes fail to activate plasminogen. All the GAS isolates used in the current study expressed and secreted streptokinase into culture supernatants, indicating that the lack of plasminogen activation activity was not caused by the lack of ska expression. Streptokinase encoded by cluster 2b ska alleles was able to activate plasminogen when prebound to fibrinogen or to cells preincubated in human plasma. Cluster 2b streptokinase expression by the PAM-positive strain ALAB49 was required for cell surface plasmin acquisition during incubation in human plasma, because deletion of the ska gene in ALAB49{Delta}ska abolished surface plasmin acquisition. Streptokinase encoded by cluster 2a ska alleles also readily activated plasminogen to produce plasmin activity in the presence of fibrinogen. Taken together, these data suggest that cluster 2 streptokinase requires the formation of a trimolecular complex with plasminogen and fibrinogen to display plasminogen-activating capability.

Streptokinase encoded by cluster 1 ska alleles readily activated soluble plasminogen to produce plasmin activity. The addition of fibrinogen to cluster 1 ska supernatants resulted in significantly higher levels of streptokinase activity (Fig. 4) . Similarly, activation of glu-plasminogen by group C streptokinase can be enhanced when plasminogen is prebound with fibrinogen (37) . Although SEN has been shown to bind to plasminogen (38) , this interaction did not affect plasminogen activation mediated by any of the streptokinase variants examined in this study.

Kalia and Bessen (21) demonstrated a strong linkage disequilibrium between skin-tropic GAS strains, cluster 2b ska alleles, and PAM, suggesting that the resultant phenotype may contribute to increased bacterial fitness during skin infection. In this study, 13 of the 14 pam-positive strains contained a cluster 2b ska allele. Although PAM was shown to have no effect on plasminogen activation by streptokinase encoded by cluster 2b allele types, we hypothesize that PAM, rather than playing a direct role in plasminogen activation, has evolved to bind to K2 of plasminogen, allowing fibrinogen to interact with K1, K4, and K5 in a noncompetitive manner. This process allows the plasminogen-fibrinogen-streptokinase trimolecular complex to bind to the surface of PAM-positive GAS isolates expressing cluster 2b ska (Fig. 5 A). Supporting this hypothesis, Svensson et al. (25) showed that an isogenic pam knockout mutant of ALAB49 failed to acquire and activate plasminogen after incubation in human plasma. Other GAS plasminogen-binding proteins such as SEN cannot bind this trimolecular complex, because the plasminogen domains (K1, K4, and K5) required for interaction with SEN are involved in the interaction with fibrinogen. This hypothesis accounts for the coselection of PAM and cluster 2b ska observed by Kalia and Bessen (21) . For cluster 2a GAS, the fibrinogen-binding capacity of M1 protein (39 40 41) allows attachment of the trimolecular complex to the bacterial surface (Fig. 5B ). In contrast, cluster 1 GAS produces a streptokinase molecule that does not require the participation of fibrinogen to activate plasminogen. We hypothesize that anchoring of the cluster 1 streptokinase-plasminogen complex to the GAS cell surface can occur via the interaction of K4 and K5 with SEN or other plasminogen receptors, or alternatively via the interaction of the cluster 1 streptokinase-plasminogen complex with fibrinogen bound to GAS fibrinogen receptors (Fig. 5C ).


Figure 5
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  		Figure 5. Schematic diagram summarizing the hypothesized pathways of cell surface plasmin acquisition by GAS strains expressing different alleles of streptokinase. A) Cluster 2b streptokinase must combine with plasminogen and fibrinogen to form a trimolecular complex that exhibits plasmin activity. The trimolecular complex is bound to the cell surface via the interaction between PAM and kringle 2 of plasminogen. Other GAS plasminogen-binding proteins such as SEN cannot bind this trimolecular complex, because the domains K1, K4, and K5 required for interaction are involved in the interaction with fibrinogen. B) Cluster 2a streptokinase also must combine with plasminogen and fibrinogen to form a trimolecular complex that exhibits plasmin activity. The trimolecular complex is bound to the cell surface via the interaction between fibrinogen-binding receptors (FgR) such as M1 protein and fibrinogen. C) Cluster 1-type streptokinase will combine with plasminogen to form a complex with plasmin activity. This complex can be bound directly to the bacterial cell surface via plasminogen receptors (PLRs) or through an interaction with fibrinogen and fibrinogen receptors.

Our results show that allelic variants of streptokinase produced by different GAS strains exhibit differing plasminogen activation capacities. This finding suggests that variation within the β domain of these proteins may be responsible for the observed differences in plasminogen activation activities. Lizano and Johnston (42) have suggested that sequence polymorphism in the β-domain has little effect on plasminogen activation. The recombinant streptokinase produced from SF13013 (containing a cluster 2a ska allele) in that study displayed soluble plasminogen activation activity. In our study, streptokinase produced by 2 serotype M1 isolates containing cluster 2a alleles displayed no soluble plasminogen activation activity. This finding indicates that sequence differences outside the β-domain region may also contribute to the variable plasminogen activation activities observed for streptokinase variants produced by GAS.

In this study, we demonstrate that activation of plasminogen by cluster 2-type streptokinase requires the presence of fibrinogen and the formation of a trimolecular complex between streptokinase, plasminogen, and fibrinogen. This requirement underpins the previously observed linkage disequilibrium between PAM and cluster 2b alleles (21) , because PAM is critical for harnessing plasmin activity to the bacterial cell surface via binding of the trimolecular complex. The sequence diversity of streptokinase displayed by GAS may be the result of selection pressures acting on ska during the infection process, thereby producing streptokinase proteins that are better adapted to interact with the human fibrinolytic system while avoiding a functional immune system. Therefore, understanding the molecular basis for the phenotypic variations observed in this study could assist the rational design of second-generation thrombolytic therapeutics (43) with improved efficacy and safety.


   ACKNOWLEDGMENTS
 
This work was supported by grants from the National Health and Medical Research Council of Australia and from the University Research Committee at the University of Wollongong. A.C. and M.S-S. were recipients of Australian postgraduate awards. F.C.M. was the recipient of a National Health and Medical Research Council biomedical postgraduate scholarship.

Received for publication March 25, 2008. Accepted for publication April 24, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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