Analysis of Plasminogen-Binding M Proteins of Streptococcus pyogenes

METHODS 21, 143–150 (2000) doi:10.1006/meth.2000.0985, available online at http://www.idealibrary.com on

Analysis of Plasminogen-Binding M Proteins of Streptococcus pyogenes Ulrika Ringdahl 1 and Ulf Sjo¨bring Institute for Laboratory Medicine, Section for Microbiology, Immunology and Glycobiology, Lund University, So¨lvegatan 23, S-223 62 Lund, Sweden

Group A streptococci are common human pathogens that cause a variety of infections. They express M proteins which are important cell wall-bound type-specific virulence factors. We have found that a set of strains, associated primarily with skin infections, express M proteins that bind plasminogen and plasmin with high affinity. The binding is mediated by a 13amino-acid internal repeated sequence located in the N-terminal surface-exposed portion of these M proteins. This sequence binds to kringle 2 in plasminogen, a domain that is not involved in the interaction with streptokinase, a potent group A streptococcal activator of plasminogen. It could be demonstrated that plasminogen, absorbed from plasma by growing group A streptococci expressing the plasminogenbinding M proteins, could be activated by exogenous and endogenous streptokinase, thereby providing the bacteria with a surface-associated enzyme that could act on the tissue barriers in the infected host. © 2000 Academic Press

The group A streptococcus Streptococcus pyogenes is an important pathogen that can cause a variety of infections at several different locations. The organism is highly adapted to its human host and rarely, if at all, infects species other than humans. The infections caused by group A streptococci include both uncomplicated conditions, such as tonsillitis and impetigo, and life-threatening diseases, such as the toxic shock-like syndrome and necrotizing fasciTo whom correspondence should be addressed. Fax: ⫹46-46189117. E-mail: [email protected]. 1

1046-2023/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

itis. A striking increase in the frequency and severity of the invasive manifestations of group A streptococcal infections has occurred in recent years (1). Group A streptococci produce an array of secreted and surface-exposed virulence factors. One of the major virulence determinants is the cell wall-bound M protein (2). The M proteins are variable proteins that provide the streptococci with a type-specific capacity to resist phagocytosis (3, 4). Despite their variability the different M proteins all have a common organization in that they form elongated ␣-helical coiled coil dimers. The proteins are covalently linked to the cell wall through a conserved C-terminal region, whereas the highly variable NH 2terminal part protrudes out from the cell surface. Presently more than 100 different M types have been defined. Many group A streptococcal strains express two or even three proteins that have the structural features typical of M proteins. These proteins, designated Mrp, M/Emm, and Enn, respectively, are encoded by adjacent genes. The relative contributions of the different proteins belonging to the M protein family to virulence are not well known, but it is considered that each of them can contribute to the antiphagocytic properties of group A streptococci. The molecules in the M protein family specifically bind to a number of host proteins with high affinity. Different members have affinity for different ligands. Known ligands include fibrinogen (5), IgG (6), IgA (7), and albumin (8), as well as the complement regulatory proteins factor H (9), factor H-like protein 1 (10), and C4B-binding protein (11). These and 143

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other interactions are likely to contribute to the pathogenicity of group A streptococci. The focus of the present discussion is on the interaction between a set of M proteins and human plasminogen (12). Plasminogen is a single-chain glycoprotein present in plasma and extracellular fluids. It can be converted to the broad-spectrum protease plasmin by cleavage of a single peptide bond by one of the two physiological activators, tissue-type plasminogen activator and urokinase. Plasmin plays a key role in the fibrinolytic system by cleaving fibrin but is also believed to participate in several other physiological and pathophysiological processes. The active serine protease domain is located in the C-terminal part whereas the N-terminal region contains five kringle domains (K1 through K5). The kringles mediate binding to various ligands including the major target, fibrin, and the principal regulator, ␣ 2-plasmin inhibitor. The interactions depend on lysine binding sites in the kringle structures that mediate binding to C-terminal lysines in the ligand or to less welldefined sequences located within proteins. Many virulent bacterial species express structures capable of binding and/or activating plasminogen (13), and some of them have been shown to contribute to bacterial dissemination (14, 15). The ability of group A streptococci to secrete a highly potent plasminogen activator, streptokinase, has been recognized for decades. In contrast, the fact that these pathogens also express surface structures capable of binding plasminogen, directly or indirectly, is a more recent finding (16). During the last 12 years at least four different plasminogen-binding surface receptors have been identified. These receptors include glyceraldehyde-3-phosphate dehydrogenase (17), ␣-enolase (18), fibrinogen-binding M proteins that capture plasminogen indirectly (19, 20), and, finally, M proteins that bind plasminogen and plasmin directly and with high affinity (13, 21, 22). The procedures used for identification, isolation, and characterization of this latter group of proteins are described in the following section.

DESCRIPTION OF METHODS A Set of Group A Streptococcal Strains Efficiently Absorb Plasminogen from Human Plasma Group A streptococci can interact with many different plasma proteins. To determine the binding pattern of streptococci expressing different M pro-

teins, 1 ⫻ 10 10 bacterial cells were incubated with 1.0 ml human plasma for 60 min at 20°C. The cells were washed three times with phosphate-buffered saline (PBS). To elute bound proteins the bacteria were incubated with 0.1 M glycine, pH 2.0, for 15 min at 20°C. The bacteria were pelleted and the proteins in the supernatant were analyzed by SDS– PAGE. The strains could be divided into distinct groups with respect to their protein absorption pattern. The majority of the strains bound fibrinogen and some of them also simultaneously bound IgG. In contrast, some strains bound IgA, IgG, and albumin but not fibrinogen. A set of strains, including isolates expressing the 33, 41, 52, 53, 56, and 80 M serotypes specifically absorbed plasminogen from plasma. The binding of plasminogen to these strains was confirmed by binding experiments with radiolabeled plasminogen. Thus, bacteria expressing the M33, M41, M52, M53, M56, and M80 proteins bound significant amounts of the labeled zymogen whereas all other strains analyzed showed little affinity for the radiolabeled probe. Interestingly, these strains are associated primarily with skin infections and only rarely cause tonsillitis (23). Isolation of Plasminogen-Binding M Proteins We considered the possibility that a member of the M protein family could be responsible for the binding to plasminogen. Type M53 expressing group A streptococci were therefore treated with hot acid, a treatment known to solubilize M proteins (3). Streptococci were grown in Todd–Hewitt medium at 37°C, 5% (v/v) CO 2, for 16 h. The cells were harvested by centrifugation and washed twice with 0.9 M NaCl. The bacteria were resuspended in 0.9 M NaCl, pH 2.0 (HCl), and incubated at 95°C for 12 min. When the mixture had cooled to 20°C, the pH was adjusted to 7.2 with 1 M NaOH and the bacteria were pelleted by centrifugation. The supernatant was recovered. To precipitate the proteins in the solution, the pH was adjusted to 2.0 with 6 M HCl and the mixture was incubated for 2 h at 4°C. The precipitate was recovered by centrifugation and resuspended in 0.1 M NaPO 4, pH 8.0. Ribonuclease was added to a final concentration of 1 ␮g/ml and the proteins were dialyzed against 0.1 M NaPO 4. The proteins in the extract were separated by SDS– PAGE and analyzed by Western blot experiments using 125I-labeled plasminogen as probe. The probe reacted specifically with a 43-kDa protein present in

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the extract. The 43-kDa protein could be purified from the extract on a plasminogen–agarose affinity column. The protein was designated PAM for plasminogen-binding group A streptococcal M protein. Cloning and Expression of the PAM Gene in Escherichia coli The assumption that PAM was an M protein was supported by the fact that it could be solubilized by the hot acid hydrolysis method. However, it could not be excluded that the 43-kDa protein represented a cleavage product exposing a C-terminal lysine generated by the acid treatment. Since the C-terminal region of M proteins is anchored in the bacterial envelope such a cleavage product would hardly exist in nature. To further verify that the M53 protein was responsible for the interaction, the corresponding gene was cloned and expressed in Escherichia coli. To this end, oligonucleotide primers were constructed from highly conserved sequences in the M protein encoding genes. MI (5⬘-AGAAAATTAAAAACAGGTACGGCATCA-3⬘) is a sequence located in the 5⬘ region that encodes the signal sequence, whereas MII (5⬘-AGTTGTTTCACCTGTTGATGGTAA-3⬘) is located in the 3⬘ region that encodes the part of the M protein linked to the cell wall. When used together in polymerase chain reaction (PCR), using chromosomal DNA from AP53 (M53) as a template, these primers amplified a 1.2 kb DNA fragment. The resulting fragment was cloned into an expression plasmid (pHD389). Lysates from E. coli, containing this construct, were analyzed by SDS–PAGE and Western blot using 125 I-labeled plasminogen as a probe. The probe bound to a 43-kDa protein in the lysate. The 43-kDa protein could be purified from the lysate by affinity chromatography on a plasminogen agarose. The protein was coupled to 1 ml N-hydroxysuccinimide ester-preactivated agarose (Hitrap, Pharmacia). One milliliter plasma was run over the column. The agarose was washed and bound plasma proteins were eluted with 0.1 M glycine, pH 2.0 . The eluate contained almost pure plasminogen (Fig. 1). The affinity constant for the interaction between PAM and plasminogen was determined by a competitive binding assay in microtiter plate format whereby unlabeled and 125I-labeled PAM were allowed to compete for the binding to immobilized plasminogen. The dissociation equilibrium constant (K d ) was calculated to be 1.25 nM.

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Together, these experiments strongly support the notion that the plasminogen-binding protein on the streptococcal surface is the M53/PAM protein. However, it could not be completely ruled out that the expression and purification in E. coli had not generated a protein with a C-terminal lysine. To entirely exclude that this was the case we proceeded to fully characterize the binding site in PAM. Definition of Plasminogen-Binding Site in M Proteins Preliminary experiments indicated that the variable N-terminal part of PAM was involved in the binding. Amino acid sequence comparison between the N-terminal thirds of the M proteins expressed by five different plasminogen-binding strains, M33, M41, M52, M53, and M56, showed only a minor identity in the absolute N-terminal region. In contrast, the region comprising 40 –50 residues in the middle contains two conserved 13- to 16-amino-acid repeated regions, designated the a-repeats. The a-repeats were followed by a highly conserved 45amino acid region. Fusion Proteins To identify the region in PAM responsible for plasminogen binding, polypeptides constituting the different regions mentioned above were expressed as

FIG. 1. Specific binding of plasminogen to the group A streptococcal PAM/M53 protein. One milliliter of human plasma was passed over an agarose column coupled with the recombinant PAM protein. Following washing of the column with 5 ml of bufffer, bound proteins were eluted with 0.1 M glycine–HCl, pH 2.0. The eluate was analyzed by SDS–PAGE and Western blot experiments usning antiplasminogen as a probe. Lane A, eluate: lane B, plasminogen.

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N-terminal fusion protein linked to one of the immunoglobulin-binding domains (B1) of protein L (24). The construct was made partly to allow purification on IgG–agarose, but in addition to avoid plasminogen binding through C-terminal exposed lysine residues generated during expression or purification. Thus, fusion proteins containing such lysines would not be expected to interact with IgG–agarose, since exposure of lysines from the B1 module would not be compatible with maintenance of the structure required for Ig binding (25). A fragment spanning the two a-repeats, residues 42– 89, inhibited the binding between [ 125I]PAM and immobilized plasminogen in the competitive binding assay whereas fragments spanning residues 1– 48 and 86 –133 failed to inhibit the binding. Synthetic Peptide Further evidence for the importance of the a-repeats in the interaction with plasminogen was provided by experiments with a synthetic peptide (VEK30) comprising the amino acid residues 56 – 84 of PAM and equipped with an additional tyrosine residue at the C terminus. The purpose of the tyrosine was to make it possible to radiolabel the peptide using the chloramine-T method, which only allows incorporation of iodine in tyrosines, but again also to demonstrate that binding was independent of C-terminal lysine residues. Thus, when the peptide was labeled with 125I, the radiolabeled probe bound strongly to immobilized plasminogen and a K1–K3containing fragment thereof (Fig. 2). Construction of Arp/PAM Chimeric Proteins The amino acids of M proteins are arranged in a seven-residue (heptad) repeat pattern giving rise to the ␣-helical coiled-coil dimeric structure typical for M proteins. To enable further mapping of the binding region in PAM and also to compare the binding strength of the motif with that of other putative streptococcal surface receptors, we transferred the a-repeats of PAM to another M protein, Arp4 (Emm4), which does not bind plasminogen, but instead binds IgAFc through a well-defined sequence (26) (Fig. 3). The reasons for choosing Arp4 as a recipient are that both proteins are anchored to the cell wall, they both contain three conserved C repeats in the C-terminal part, and they both have a surface exposed variable N-terminal region that contains the binding motif. Their binding patterns are also similar; in addition to a unique binding prop-

erty, they both bind C4BP but not fibrinogen. Manipulations were done in an E. coli–Streptococcus shuttle vector, pJRS264, that encodes Arp4 and that supports expression of Arp4 on the streptococcal surface (27). pJRS264 contains unique SacI and BstBI sites at positions 315 and 403 of the arp4 gene, respectively. For construction of the Arp/a1a2 chimera, a 110-bp DNA fragment encoding the a1a2 repeat of PAM was generated by PCR using chromosomal DNA from AP53 as template and the synthetic oligonucleotides 5⬘-GAGGAGCTCAAAGATGATGTTGAGAAGCTTACC-3⬘ and 5⬘-GCTCCGGAGATCATGTCTCTCGCTTTTAAGTCG-3⬘ as primers. The oligonucleotides constitute the recognition sequence for SacI and HpaII, respectively. For construction of the Arp/a1 chimera, two synthetic oligonucleotides, 5⬘-CAAGGTAGATGCTGAGTTGCAACGACTTAAAAACGAGAGACATGAAGGTGAAAATCAAGATCTT-3⬘ and 5⬘-CGAAGATCTTGATTTTCACCTTCATGTCTCTCGTTTTTAAGTCGTTGCAACTCACATCTACCTTGAGCT-3⬘, were annealed. The fragments were ligated to SacI/ BstBI-digested pJRS264 and the constructs were used to transform E. coli DH5␣. The purified chimeric proteins bound plasminogen with high affinity. The dissociation equilibrium constants, K d, , for the interactions were calculated to be 1.67 nM for both chimeras, which is a value close to

FIG. 2. A 30-amino-acid-long PAM-derived polypeptide binds to the kringle domains of plasminogen. The indicated proteins were spotted onto a nitrocellulose membrane. Following blocking, the membrane was probed with the 125I-labeled synthetic polypeptide VEK30 (VEKLTADAELQRLKNERHEEAELERLKSEY). Following washes the membrane was autradiographed.

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that previously determined for the interaction between PAM and plasminogen (1.25 nM). Similar values were obtained for the interaction with plasmin. The group A streptococcal strain JRS 145, an M6 protein-deficient derivative of the strain JRS4 (28), was then transformed with the constructs. Arp/ a1a2- and Arp/a1-expressing JRS4 and streptococci bound 125I-labeled plasminogen. The experiment shows that replacement of the IgA binding domain with a single a-repeat of 13 amino acids is necessary and sufficient to endow group A streptococci with the capacity to bind plasminogen. Definition of the PAM Binding Site in Plasminogen Kringles mediate interactions with C-terminal lysines through the omega amino group and the free carboxyl group of the residue. The kringles also bind to free lysine as well as to lysine analogs such as 6-aminohexanoic acid (6-AHA). Some kringles, particularly K1 and K5, also bind to lysine analogs or proteins lacking the carboxyl group. The molecular requirements for these latter interactions are not known. Moreover, the internal kringle-binding regions in ligands, including fibrinogen, have not been well defined. With a long-term objective to analyze the requirements for interactions between kringles and internal kringle-binding sequences, using the PAM–plasminogen interaction as a model, we wished to map the region in plasminogen that mediates the binding to PAM.

FIG. 3.

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First we measured the ability of radiolabeled proteolytic fragments of plasminogen to bind PAMexpressing streptococci. The K1–K3 fragments bound PAM- and Arp/PAM-expressing streptococci, whereas K4 and miniplasminogen (holding K5 and the protease domain) showed very little binding (29). The same proteolytic plasminogen fragments were then immobilized on a polyvinylidene difluoride (PVDF) membrane. The membranes were blocked with PBS containing 0.25% gelatin and 0.25% Tween 20 (blocking buffer). Following incubation for 3 h at 20°C with 125I-labeled VEK30 the membranes were washed with blocking buffer containing 0.5 M NaCl. The membranes were air-dried and autoradiographed. Again PAM interacted with K1–K3 but not with the two other fragments. To further localize the binding site in plasminogen we used recombinant kringles (rK1, rK2, rK3, rK1–K2 and rK2–K3). Various amounts of the fragments were immobilized in microtiter plates overnight at 4°C. After blocking with PBS containing 0.1% bovine serum albumin (BSA) for 4 h at 20°C the immobilized proteins were incubated with the radiolabeled synthetic oligopeptide VEK30 or with a radiolabeled recombinant fusion protein in which the VEK30 sequence had been fused to protein L (VEK30B1). Binding of the 125I-labeled proteins was monitored in a gamma counter. Binding of both these PAM-derived peptides occurred to rK2, rK1–K2 and K2–K3, but not to rK1 or rK3. In com-

Construction of a Arp/a1a2 chimeric gene.

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petitive binding experiments rK2 was as efficient an inhibitor of the VEK30 –plasminogen interaction as plasminogen itself, whereas rK1 and rK3 possessed only a minor inhibitory capacity in this system. The strength of the interactions was quantified using surface plasmon resonance analysis, using VEK30B1 as the ligand. Again no binding of the ligand occurred to immobilized rK1 and rK3 whereas binding occurred to rK2 with an affinity of 22 nM. 6-AHA completely blocked the binding of [ 125I]VEK30 to rK2, demonstrating an involvement of the relatively weak lysine binding site of K2. The involvement of the lysine binding site in K2 was further supported by experiments with rhesus plasminogen. VEK30 failed to bind rhesus plasminogen in the direct binding asssay and rhesus plasminogen failed to block the interaction between VEK30 and human plasminogen even at high concentrations. K2 of rhesus plasminogen differs from its human counterpart in only two positions (30, 31), both of which are immediately adjacent to two conserved negatively charged amino acids that are an essential part of the lysine binding site (32). The K2/VEK30 interaction provides a unique model for a structural analysis of the molecular requirements of interactions between kringles and internal binding sites in proteins. From an evolutionary perspective it is likely that the remarkable specificity of PAM for human plasminogen is likely to reflect a molecular adaptation to the only natural host of group A streptococci. This notion is supported by the findings that the binding of different M proteins to other ligands, such as IgG, IgA, and the C4b-binding proteins shows a similar species restriction. Cooperation between Plasminogen and Streptokinase Group A streptococci secrete streptokinase, which when bound to plasminogen in a 1:1 molar ratio is a very efficient activator of plasminogen. To test the hypothesis that the plasminogen bound by PAM can be activated by streptokinase at the streptococcal surface, 1 ⫻ 10 10 streptococci expressing the chimeric Arp/a1a2 protein were grown in Todd–Hewitt medium containing 30% human plasma. The cultures were terminated at various time points. The bacteria were pelleted and washed three times in PBS containing 0.1% Tween 20. To analyze the surface-bound plasminogen/plasmin two methods were used. For one of these methods the plasma absorption type of experiment described above was

used. Thus, the bacteria were incubated in 0.1 M glycine buffer, pH 2.0, for 15 min at 20°C. The bacteria were pelleted and the proteins in the supernatant were analyzed by SDS–PAGE and Western blots. An 125I-labeled antibody recognizing both plasmin and plasminogen was used as a probe in the Western blots. The plasmin/plasminogen ratio clearly increased with time (Fig. 4A, top). In the second experiment we wished to test if the surface-bound plasmin was active. The washed bacteria were therefore incubated with an 80 ␮M concentration of the chromogenic substrate H-D-Val– Leu–Lys-p-nitroanilide (S2251) in 2.5 ml of PBS containing 0.4 M NaCl for 1 h at 37°C. The bacteria were pelleted and the absorbance of the supernatant was measured at 405 nm. The results clearly showed that there was a time-dependent generation of surface-associated functional plasmin (Fig. 4A, bottom). When similar experiments were performed with heat-killed streptococci, plasmin formation failed to occur. However, small amounts of streptokinase added to the growth medium restored the plasmin formation on the bacterial surface. The role of endogenous streptokinase for generation of plasminogen was analyzed in the same experimental setup using strains in which the streptokinase gene (ska) had been inactivated on the chromosome. For the inactivation experiments a suicide plasmid containing a streptokinase gene in which a part of the central region had been replaced by a kanamycin resistance gene (⍀Km-2) was used. To construct the plasmid the streptococcal strains AP53 and JRS4 were used as templates together with the oligonucleotides SK3 (5⬘-GACGTCGACACTTGCATCTCTGGAAAATAGTC-3⬘) and SK4 (5⬘-GACGGATCCATGAGTGACGATTGAGGAGTCAC-3⬘) to amplify an 1180-bp fragment of ska with PCR. Similarly, SK6 (5⬘-GACGGATCCTTTCTGAGAAATATTACGTCC-3⬘) and SK7 (5-GACGAGCTCGGTACCTTTCTATTGATGGGAAAATTGC-3⬘) were used to amplify a 950-bp fragment. The two fragments were digested with SalI/BamHI and BamHI/SacI, respectively, and cloned into SalI/ SacI-digested pUC18. The resulting plasmid was digested with BamHI and ⍀Km-2 was introduced between the two ska fragments. The construct was digested with SalI and SacI, blunted, and cloned into the linearized and blunted plasmid pJRS233 (33). pJRS233 contains an erythromycin resistance gene (erm), a temperature-insensitive E.coli replicon, and a promiscuous replicon that is sensitive to temperatures above 35°C in both E.coli and strepto-

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cocci. The plasmid was used to transform S. pyogenes. The transformants were grown at 30°C and subsequently at 37°C to select for clones with integrated constructs. Integration of ⍀Km-2 into the ska gene was monitored by PCR. The streptokinase activity was determined by incubating 5 ␮g of plasminogen with streptococcal cell lysates in the presence of the chromogenic substrate H-D-Val–Leu– Lys-p-nitroanilide. PCR-positive clones failed to activate plasminogen in this assay. In contrast Arp/a1a2-expressing but ska-negative group A streptococci failed to aquire surface-bound plasmin when grown in plasma (Fig. 4A, top). Similarly, the PAM-expressing AP53 ska-negative

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strain failed to acquire surface-associated plasmin with time as opposed to the wild-type AP53 strain.

CONCLUSIONS Together these data demonstrate that a set of group A streptococcal strains express M proteins able to bind plasminogen with specificity and affinity. The binding is mediated through a well-defined 13-amino-acid residue repeated sequence in the N-terminal surface-exposed end of these M proteins. The interaction site in the plasminogen is located to

FIG. 4. Cooperation between the plasminogen-binding sequence of PAM and streptokinase in the generation of streptococcal-bound plasmin. Streptococci expressing the chimeric Arp/a1a2 protein were cultured in the presence of plasma. The cultures were stopped at the indicated time points and the bacteria were washed repeatedly. Half of the bacteria were incubated in a buffer with the chromogenic plasmin-sensitive substrate H-D-Val–Leu–Lys-p-nitroanilide. The plasmin associated with the bacteria was measured as a function of the increase in absorbance, at 405 nm, of the buffer after incubation with the bacteria for 1 h at 37°C (A and B, bottom). The remaining half of the bacteria were incubated with 0.1 M glycine–HCl, pH 2.0. Eluted proteins were analyzed by Western blot experiments, using antiplasminogen as a probe (A and B, top). (A) JRS145(Arp/a1a2); (B) JRS145(Arp/a1a2) in which the streptokinase gene has been inactivated. (C) Insertional inactivation of the ska gene in AP53 and in JRS 145 was accomplished with the kanamycin cassette ⍀Km-2, flanked by ska fragments.

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the kringle domains, specifically to kringle 2 which is located in the N-terminal part of the zymogen, suggesting that the interaction does not interfere with the complex formation that occurs between the C-terminal region of plasminogen and streptokinase. In fact, multiple evidence suggests that plasminogen bound by streptococci expressing these M proteins can be activated by streptokinase, thereby generating surface-associated plasmin that can target proteins in the bacterial environment. Alternatively, plasminogen can form a binary complex with streptokinase, thereby providing the bacteria with an efficient cell-bound plasminogen activator. The finding that these processes can occur in plasma also suggests that the plasmin or the plasmin(ogen)/ streptokinase complex is protected from the host inhibitors of plasmin generation when bound to the surface of group A streptococci. Plasmin plays a key role in the control of coagulation by binding and degrading fibrin. However, plasmin has a broad substrate specificity and it appears to play a role in several biological processes. For example, plasmin can also degrade soft tissue proteins and activate latent matrix metalloproteinases. It is therefore possible that cells that are able to generate plasmin on their surface may use it to degrade physiological barriers other than those formed by formed by fibrin. In fact, data derived from eukaryotic systems suggest that this is the case. Multiple evidence, including the findings reported here, suggest that that some bacteria may use similar strategies to facilitate their invasion or dissemination. This notwithstanding, the observations will have to be substantiated by in vivo experiments. Because of the limitation in binding spectrum of both streptococcal products, such experiments will require the design of novel animal models. Such experiments are currently underway.

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