Pathogen Activators of Plasminogen

Summary

Group A streptococci (GAS) express soluble and surface-bound virulence factors. Secreted streptokinase (SK) allelic variants exhibit varying abilities to activate host plasminogen (Pg), and GAS pathogenicity is associated with Pg activation and localisation of the resulting plasmin (Pm) on the bacterial surface to promote dissemination. The various mechanisms by which GAS usurp the host proteolytic system are discussed, including the molecular sexuality mechanism of conformational activation of the Pg zymogen (Pg*), and subsequent proteolytic activation of substrate Pg by the S•KPg* and SK•Pm catalytic complexes. Substantial progress has been made to delineate both processes in a unified mechanism. Pm coats the bacteria by direct and indirect binding pathways involving plasminogen-binding group A streptococcal M-like protein (PAM), and host fibrin(ogen). Transgenic mouse models using human Pg are being optimised to mimic infections by SK variants in humans, and to define in vivo combined mechanisms of these variants and PAM.

Streptokinase from Group A Streptococcus Species

Group A, beta-hemolytic Streptococcus pathogens (GAS) are responsible for common throat and skin infections, but they are also the major cause of pneumonia, meningitis, sepsis, endocarditis, toxic shock syndrome, and necrotising fasciitis. These invasive infectious pathologies can trigger severe complications such as stroke and renal failure, and affect 18 million people globally, resulting in 517,000 deaths per year [1]. The human host-specific S. pyogenes enhances its pathogenicity by a multitude of cell wall-linked and secreted virulence factors [2–5], one of which is streptokinase (SK). SK usurps the host’s fibrinolytic system and uses unique molecular mechanisms to bind and ultimately activate the host zymogen, plasminogen (Pg) into plasmin (Pm). Phylogenetic studies of SK from GAS isolates classify sequence variants into clusters SK1, SK2a and SK2b [6]. The strains expressing these allelic variants exhibit varying pathogenicity, and different interpretations of their individual molecular mechanisms of Pg activation, fibrin(ogen) binding, and susceptibility to α2-antiplasmin (α2-AP) have been proposed [3, 4, 7]. Our mechanism studies reported here, have mainly focused on SK from the S. equisimilis strain H46A used to treat myocardial infarction [8]. SKH46A is 86% identical in sequence and ~90% homologous to SK1 variants. The SK allelic sequence variants most likely evolved under selective pressure for survival in the hostile host environment to encode critical differences in their mechanisms of Pg activation pathways to generate Pm. Long-term goals will include defining critical mechanism differences for these allelic variants.

Roles of Plasminogen Activation

Plasminogen is the zymogen precursor of plasmin, the central proteinase in the fibrinolytic system. Intravascular fibrinolysis, or fibrin clot dissolution by plasmin, occurs after wound repair, or can be pharmacologically induced by intravenous infusion of plasminogen activators after thrombosis and blood vessel occlusion. [Glu]Pg circulates as a compact α-form, with its spiral array of 5 kringle (K) domains stabilised by lysine-binding site (LBS) interactions of K2, K4 and K5 with basic residues and chloride ions [9]. The N-terminal PAN (Pg/Apple/Nematode) module binds to LBS of K4 and K5, and this interaction keeps [Glu]Pg in the spiral conformation. K1, the only kringle with an accessible LBS, extends from the structure and interacts with fibrin(ogen). Solving the crystal structure of compact [Glu]Pg provided a significant advance in understanding the conformational properties of circulating Pg [9, 10]. Small molecules and lysine analogues such as benzamidine and 6-aminohexanoic acid (6-AHA) can bind the kringles, and cause a conformational change to the partially extended β-conformation (benzamidine) or the fully extended γ-conformation (6-AHA) [11, 12]. The Pg activation cleavage site Arg⁵⁶¹-Val⁵⁶² in compact [Glu]Pg is shielded by the 350s loop, a K3-K4 linking segment. The Lys⁷⁷-Lys⁷⁸ bond, not readily accessible in compact [Glu]Pg, can be cleaved by plasmin, generating a more extended N-truncated Pg form, [Lys]Pg, which adopts a β-conformation and is more reactive with physiological and bacterial plasminogen activators [11]. In the absence of chloride ions [Glu]Pg adopts the γ-conformation.

In addition to degrading fibrin clots, plasmin also activates metalloproteinases and plays a role in tissue remodelling, angiogenesis, and processes of pathogen and tumor cell dissemination. Bacterial pathogens and cancer cells are capable of recruiting host plasmin to their cell surfaces, which facilitates spreading through tissues [13, 14]. Whereas tumor invasion and physiological fibrinolysis use proteolytic Pg activation, pathogens have developed unique mechanisms of host Pg binding to secreted and surface-bound virulence factors, resulting in conformational Pg activation without the need for proteolytic cleavage of the zymogen.

Proteolytic and Cofactor-Induced Activation Mechanisms

Physiological tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) use the classical cleavage mechanism to activate Pg, by proteolysis of the Arg⁵⁶¹-Val⁵⁶² bond (chymotrypsinogen numbering Arg¹⁵-Val¹⁶, or the P1–P1′ site) in the Pg activation loop that is sterically restrained by a disulphide bridge. This sterical restraint only allows the residues between P3 and P2′ of the activation loop to make contact with the specificity pocket of tPA and uPA [15]. The S2′ specificity pocket of these proteases is limited in size by the presence of a bulky tyrosine residue, which poses a restriction on the residues that can occupy this subsite. Upon cleavage of the Pg activation loop, the new N-terminal Val¹⁶ residue inserts into the Val¹⁶ binding pocket and engages the side chain carboxylate of Asp¹⁹⁴ (Pg numbering 740) to form the substrate recognition site and the oxyanion hole. This is the established mechanism for chymotrypsin(ogen)-like serine proteinase zymogens [16]. A model for the classic cleavage mechanism of such a zymogen is shown in Figures 1A and B.

FIGURE 1.

A,B. Proteolytic zymogen activation mechanism. Proteolytic cleavage of the Arg¹⁵-Ile¹⁶ of Pre2 generates a new N-terminal Ile¹⁶, which insets into the N-terminal binding pocket in the zymogen, forming a salt bridge with Asp¹⁹⁴. This induces a conformational change involving the folding of 3 segments of the catalytic domain. The conformational change forms the substrate binding site and oxyanion hole required for catalysis. C. Binding of the N-terminal ^SCIle¹-^SCTyr⁶ sequence to Pre2. The SC backbone is in yellow, side chains have coloured atoms, and the bound Pre2 is coloured according to its electrostatic surface potential. This figure was first published in Nature. Friedrich R, Panizzi P, Fuentes-Prior P, Richter K, Verhamme I, Anderson PJ, Kawabata S, Huber R, Bode W, Bock PE. Staphylocoagulase is a prototype for the mechanism of cofactor-induced zymogen activation. Nature. 2003; 425: 535–9.

By contrast, the secreted streptococcal virulence factor streptokinase (SK) forms a stoichiometric complex with host Pg, and activates it by a mechanism that does not involve proteolysis. The N-terminal sequence Ile¹-Ala-Gly of SK mimics the Ile¹-Val-Gly/Asn sequences of trypsin and chymotrypsin, and the plasmin Val¹-Val-Gly sequence, which insert into the activation pocket of the nascent proteases during proteolytic activation. Insertion of the SK N-terminal sequence is proposed to trigger zymogen activation, according to the ‘molecular sexuality’ hypothesis of zymogen activation by intrusion of N-terminal sequences of bacterial staphylocoagulase (SC) and SK into the activation pockets of prothrombin and Pg, respectively [17]. Upon insertion of the SK N-terminus in the Ile¹⁶ N-terminal binding cleft (chymotrypsinogen numbering) of Pg, Ile¹ of SK forms a salt bridge with Asp¹⁹⁴, resulting in a conformational change, and formation of the substrate-binding site and the oxyanion hole, required for catalytic activity. Loss-of-function studies with SK N-terminal deletion mutants, by us and others [18, 19] have supported this hypothesis, favouring it over an alternative mechanism in which SK binding was proposed to move the ε-amino group of the conserved Lys¹⁵⁶ (Lys⁶⁹⁸ in Pg) into the binding pocket, and trigger conformational activation [20]. The molecular sexuality mechanism of SK-induced conformational activation of Pg was postulated in 1976, when SK and SC were the only known conformational zymogen activators [17]. We showed clear structural evidence for such a molecular sexuality mechanism, by determining for the first time the high resolution crystal structure of the complex between a large N-terminal SC fragment, SC(1-325), and prethrombin 2 (Pre2), the prothrombin catalytic domain and immediate zymogen precursor of thrombin [21]. The N-terminus of SC(1-325) inserts into the Ile¹⁶ pocket and triggers folding of the zymogen activation domain (residues 142-152, 186-194 and 216-226) by forming a salt bridge with Asp¹⁹⁴, which generates substrate recognition, subsites and forms the oxyanion hole. The Ile¹-Val² amino-terminal residues of SC(1-325) inserting into the Pre2 binding pocket, and the critical salt bridge with Asp¹⁹⁴ are clearly resolved (Figure 1C). We also solved the structure of SC(1-325) bound to human thrombin, in which the Ile¹-Val² residues of the thrombin heavy chain occupy the specificity pocket. This study reported similar loss-of-function of SC N-terminal deletion and substitution mutants, further lending credence to the SK-triggered Pg conformational activation for which no crystal structure is yet available. A model of this activation is shown in Figure 2A. Based on this structure, we proposed a family of staphylococcal and streptococcal homologs named zymogen activator and adhesion proteins (ZAAPs) [22], that also includes the conformational prothrombin activator, von Willebrand factor-binding protein (VWbp), secreted by Staphylococcus aureus.

FIGURE 2.

A. Crystal structure of the SK•μPm complex, and model of Pg activation by SK. Top: The α (red), β (yellow) and γ (purple) domains of SK surround the catalytic domain of plasmin (green). The catalytic triad is shown in pink. The top and side views show the crater-like arrangement of the SK domains surrounding the catalytic site of μPm. The N-terminal 11 residues are disordered because the Pm N-terminus is inserted. This is the only structure available of a SK•Pm complex [29]. Bottom: A model was constructed of the SK complex with the Pg catalytic domain, based on the SK•μPm structure, showing the N-terminal insertion that activates Pg. Evidence for the molecular sexuality mechanism is that deletion of the N-terminal Ile residue of SK inactivates its ability to activate Pg conformationally. There is currently no structure of the SK•Pg* zymogen complex to support this. B. The trigger-and-bullet mechanism of Pg activation by SK. Rapid SK binding to the catalytic domain of Pg, and conformational Pg activation in the SK•Pg* catalytic complex form the first phase of the catalytic cycle (trigger). SK•Pg* binds a second Pg molecule in the substrate mode and proteolytically activates it to Pm. Pm displaces Pg from SK•Pg* because of its higher affinity compared to Pg. This causes transition of the catalytic complexes from SK•Pg* to SK•Pm as the sole catalyst, which initiates the bullet cycle. SK•Pm proteolytically converts the remaining free Pg to Pm. This figure was originally published in The Journal of Biological Chemistry. Boxrud PD, Bock PE. Coupling of conformational and proteolytic activation in the kinetic mechanism of plasminogen activation by streptokinase. J Biol Chem. 2004; 279: 36642–36649. © the American Society for Biochemistry and Molecular Biology. C. Specificity of SK(2-414) reactivation by Ala-substituted SK(1-10) peptides. The activities of SK(1-10) peptides with Ala substituted at the residues indicated were determined relative to the wild-type peptide (Ala at residue 2, 100% rescue). Peptide-initiated rescue of Pg activation by SK(2-414) is shown without (solid) and with (open) 6-AHA. This figure was originally published in The Journal of Biological Chemistry. Boxrud PD, Verhamme IM, Fay WP, Bock PE. Streptokinase triggers conformational activation of plasminogen through specific interactions of the amino-terminal sequence and stabilises the active zymogen conformation. J Biol Chem. 2001; 276: 26084–26089. © the American Society for Biochemistry and Molecular Biology. D. Rescue of Pg activation by Y⁴P substitution in the ALAB49 variant. ALAB49 shows weak conformational Pg activation (open circles) that is significantly enhanced by substitution of Tyr⁴ to Pro, the residue in wild-type SK (closed circles).

A Unified Mechanism Coupling Conformational and Proteolytic Activation

Our studies [23, 24] that couple conformational Pg activation to subsequent proteolytic Pm formation in a unified mechanism of SK action have clarified molecular events that were previously subject to different interpretations, such as Pm formation by intramolecular proteolytic activation within the complex with SK [25]. We demonstrated that SK binds rapidly to Pg and induces the activated Pg* conformation in the SK•Pg* catalytic complex by the molecular sexuality mechanism. This process is considered the trigger of the mechanism (Figure 2B). The SK•Pg* complex then binds another Pg molecule in the substrate mode, and converts this substrate Pg to Pm by the classical proteolytic mechanism. Pm binds SK with 500–900-fold higher affinity, thereby displacing Pg* from the complex. The resulting catalytic SK•Pm complex becomes the sole catalyst, which terminates the trigger cycle and initiates the bullet cycle (Figure 2B). The SK•Pm catalytic complex then proteolytically converts remaining free Pg to Pm. The very slow off-rate for the dissociation of Pm bound in the SK•Pm catalytic complex (t_1/2 ~800 s) suggests that the SK•Pm bullet cycle may turn without significant dissociation of SK•Pm [26]. Activation of Pg can be analysed by kinetic analysis of continuous hydrolysis of a plasmin-specific chromogenic substrate, D-VLK-pNA, that is also cleaved by the SK•Pg* and SK•Pm complexes. Progress curves of Pg activation are fit by the parabolic equation: ΔA_{405 nm}=v₂t²/2 + v₁t to obtain the initial rate of substrate hydrolysis (v₁) due to the conformationally activated SK•Pg* complex, and the rate of the increase in activity with time due to Pm formation (v₂) [23, 24, 27]. The hyperbolic SK dependence of v₁ has a K_D that reflects SK•Pg* formation and a limiting rate representing the bimolecular rate (k_cat/K_m) of D-VLK-pNA hydrolysis by SK•Pg*. The SK dependence of v₂, the rate of Pm formation, increases to a maximum and decreases to zero as SK is increased. Pg acts both as the catalyst in the SK•Pg* complex and as the substrate of this complex, and Pm generation decreases as free Pg is depleted by higher SK. The bimolecular rate constant for Pm generation is calculated from v₂. An intramolecular cleavage mechanism cannot account for this behaviour of v₂. This approach allows determining the binding and kinetic parameters of both SK•Pg* and SK•Pm formation. This analysis was validated independently by full time-courses of Pg activation, and three discontinuous chromogenic substrate initial rate assays, orthogonal to the assays described above [28]. The assays use different conditions of quenching with active site-blocked Pm (FFR-Pm), 6-AHA, and α2-AP to allow quantitation of time-courses of Pg depletion, transient formation of conformationally activated SK•Pg*, and formation of Pm and SK•Pm. Results of both kinetic approaches are in excellent agreement, supporting the unified mechanism that couples conformational and proteolytic Pg activation.

Different Interactions of SK with Pg/Pm in the Catalytic and Substrate Mode

SK consists of three tightly folded domains, α, β, and γ, linked by segments that allow flexible movement of the domains in solution. When bound to Pg or Pm, the SK domains exhibit a vastly increased level of organisation into a highly ordered structure resembling a three-sided crater that surrounds the catalytic site, and provides a binding site for substrate Pg (Figure 2A) [29, 30]. In the crystal structure of the catalytic complex of SK with μ-plasmin (the catalytic domain only), 15 N-terminal and 32 C-terminal residues are among the disordered regions, and the β-domain shows significantly less contacts with μ-plasmin than the α- and γ-domains, suggesting a possible role in binding of substrate Pg to the complex.

Studies with the deletion mutant SKΔK414 showed that the C-terminal Lys⁴¹⁴ residue of SK interacts with an LBS on a Pg/Pm kringle domain, which causes enhancement of the affinity of SK•Pg* and SK•Pm catalytic complex formation [31]. Binding of native SK and SKΔK414 to [Glu]Pg was LBS-independent, due to shielding of critical LBS in the compact α-conformation of [Glu]Pg [9, 10]. However, SKΔK414 binding to [Lys]Pg and [Lys]Pm in the catalytic mode was significantly weaker than native SK binding. SKΔK414 binding to [Lys]Pg and [Lys]Pm and conformational [Lys]Pg activation were LBS-independent, whereas [Lys]Pg substrate binding and proteolytic [Lys]Pm generation were LBS-dependent. This indicates that SK Lys⁴¹⁴ binding to [Lys]Pg and [Lys]Pm kringles enhances SK• [Lys]Pg* and SK• [Lys]Pm catalytic complex formation, and other residues are involved in substrate Pg binding. Kinetic analysis of mini-Pg activation by SKΔK414 indicated no difference in affinity between native SK and the mutant binding to this Pg form, lacking kringles 1-4, which suggests that Lys⁴¹⁴ may interact with K4 [27]. Stopped-flow fluorescence binding of SK to active-site-labelled Pg and Pm demonstrated that Lys⁴¹⁴ binding to a Pg/Pm kringle, likely K4, enhances formation of an initial rapid equilibrium encounter complex, succeeded by two sequential conformational changes [12, 26]. The weaker encounter complex affinity, and the faster off-rates for the second conformational step of SK•Pg* formation compared to SK•Pm, result in a lower affinity of the SK•Pg* complex, and expression of a weaker “pro”-exosite for binding of Pg in the substrate mode. Numerical analysis of forward and reverse rates and affinities for initial, intermediate and final complexes suggested that the Pg encounter also involves significant non-LBS interactions with the protease domain, whereas Pm binding may include additional contributions of other lysines, consistent with tight SK•Pm binding.

Previous studies reported LBS dependence of Pg substrate recognition [32], but were not in agreement on the putative SK domains involved in this interaction [33, 34]. The SK β-domain contains a protruding hairpin loop called the 250-loop (Ala²⁵¹–Ile²⁶⁴), which is ordered in the structure of the isolated β-domain [35], but disordered in the SK•μPm complex [29]. Early studies implicating Lys²⁵⁶ and Lys²⁵⁷ of the 250-loop in [Glu]Pg conformational activation and substrate recognition used constructs of recombinant SK with a methionine residue or a maltose-binding protein added onto the N-terminus, which led to diverse conclusions about Pg conformational activation and substrate binding [36, 37]. Binding of the isolated β-domain to Pg and kringle 5 (K5) was LBS-dependent [33], implicating K5 in Pg substrate recognition. We used site-directed mutagenesis of Ala substitutions on the SKΔK414 backbone to identify Arg²⁵³, Lys²⁵⁶, and Lys²⁵⁷ in the SK β-domain 250-loop, critical for recognition of Pg, and mini-Pg (lacking K1-K4), as substrates of the catalytic SK•Pg* complex. These data confirmed K5 of Pg as the site of interaction of these residues [27]. Molecular modelling suggested that the residues bound K5 in a non-canonical fashion, rather than through a pseudo-lysine motif formed by residues with cationic (Arg and His) and anionic side chains (Glu) arranged spatially on a helix. This motif is seen in binding of VEK-30, a 30-residue peptide from PAM, to K2 of Pg [38, 39], and also in α-enolase from Streptococcus pneumoniae, which has an internal Pg binding site containing two Lys residues, and acidic Asp and Glu residues located on a surface loop [40]. Lys²⁵⁶ and Lys²⁵⁷ were also critical for substrate recognition of bovine Pg (BPg), which is not activated conformationally by SK, but binds as a substrate to human SK•Pm. The complex of the double Ala mutant with fluorescently labelled Pm did not bind BPg, and the complex with active Pm did not support BPg activation. It is noteworthy that SK2a and 2b variants were found to be weak Pg activators, consistent with the non-conservation of Arg²⁵³ and Lys²⁵⁶ in their 250-loop [3, 27, 41].

Another bacterial activator, staphylokinase (SAK), with no sequence similarity to SK, but sharing a similar domain fold, forms a tight stoichiometric SAK•Pm complex that cleaves Pg bound in the substrate mode [15]. However, there is no clear functional evidence for Pg binding and conformational activation by SAK. We identified a protein, secreted by Streptococcus agalactiae, with moderate sequence identity to SK and SAK, and named it skizzle (SkzL) [42]. SkzL binds Pg, and deletion of its C-terminal Lys⁴¹⁵ reduces its binding to [Lys]Pg ~30-fold, indicative of a kringle interaction. SkzL enhances the activation of [Glu]Pg by uPA, activation of [Glu]Pg and [Lys]Pg by single chain tPA, and plasma clot lysis by these Pg activators. These results suggest a role for SkzL in S. agalactiae pathogenesis through fibrinolytic enhancement.

Specific Interactions of the SK Amino-Terminal Sequence with Pg

Equilibrium binding and kinetic studies of Pg activation with SK(2-414), an SK mutant lacking Ile¹, demonstrated that SK(2-414) bound to [Lys]Pg and [Lys]Pm with similar affinity as native SK, but induced no detectable conformational Pg activation [18]. This activity was partially rescued by the peptides SK(1-2), SK(1-5), SK(1-10), and SK(1-15). A mixture of SK(1-10) and SK(2-414) triggered formation of the active catalytic site in the Pg zymogen, allowing active site-specific fluorescence labelling of Pg, whereas sequence-scrambled SK(1-10) was inactive. Ala scanning of the SK1-10 residues (Figure 2C) showed that the rescue effect of SK(1-10) is completely lost by substituting Ile¹ and Gly³, partially by substituting Trp⁶, Leu⁷ and Arg¹⁰, and 2-fold enhanced by Asp⁹ to Ala substitution. For some residues the effect was 6-AHA-dependent: a peptide with Pro⁴ changed to Ala behaved like the wild-type peptide in the absence of 6-AHA, but lost its rescue capability in 6-AHA, which supports obligatory interactions with LBS on Pg. These data indicate the requirement of sequence-specific interactions of at least the first 10 SK residues to guide insertion of Ile¹ of SK into the Pg N-terminal binding cleft during conformational Pg activation by SKH46A. SK(2-414) binding to Pm is ~1,500-fold tighter than to Pg, and together with the N-terminal sequence specificity, may be part of a cooperative mechanism to trigger the conformational change and stabilise the active zymogen conformation.

The N-terminal Ile-Ala-Gly sequence is conserved in SKH46A and the allelic variants. SK2a and SK2b have Pro⁴→Tyr, Val²²→Met, and Thr²⁵→Ile substitutions, and a non-conserved 30 – 36 sequence cluster [3] that may affect binding to Pg and Pm. The ALAB49 SK2b variant, isolated from impetigo lesions, does not activate [Glu]Pg [3], but we observed that it causes weak conformational [Lys]Pg activation, with K_D for the catalytic SK•Pg* complex of 3 μM. Upon substitution of Tyr⁴ with Pro, Pg activation was partially restored (Figure 2D, unpublished results), with K_D ~0.6 μM, a 5-fold gain of function. However, making the corresponding Pro⁴→Tyr substitution in SKH46A had no effect on the affinity of the SK•Pg* complex, suggesting differential binding effects of the N-terminal sequences on Pg activation by the allelic SK variants. Interactions extending further into the α-domain may play a role in high affinity binding of SKH46A to Pm, as shown by a differential, <360-fold loss of affinity of the SK55–414 truncation mutant for Pm compared to the <2-fold weaker binding to [Lys]Pg [43]. The SK(1–10) peptide failed to reactivate conformational Pg activation by this mutant, suggesting additional binding interactions of the α-domain. Binding of SK N-terminal deletion mutants that lack the complete α-domain was significantly more LBS-dependent [43], and thermodynamic analysis indicated that α-domain binding interactions with Pg/Pm were mainly LBS-independent.

Surface Localisation of Pg/Pm by Bacterial Virulence Factors, and Role of Fibrin

The cell wall-linked Plasminogen-binding group A streptococcal M-like protein (PAM) of many skin-tropic GAS strains binds host Pg/Pm and promotes tissue dissemination through Pm generation mediated by SK [13, 44]. Soluble PAM exhibits a temperature-dependent equilibrium between the dimeric and monomeric state, a general property of Fbg-binding M proteins, whereas surface-bound PAM is a homodimer [45]. PAM uses a pseudo-lysine motif to capture Pg and Pm at two sites, allowing interaction with SK for inducing Pg activation to Pm that remains bound on the bacterial surface. This suggests that the Pg binding sites for PAM and SK do not overlap. Inactivation of genes encoding PAM or SK results in loss of infectivity, illustrating a mechanism of cooperation between surface-bound and secreted virulence factors [6]. PAM was also reported to bind Fbg/Fbn that may initiate an alternative pathway to Pm formation. However, the status of Fbg/Fbn binding to PAM is unclear, with some studies reporting no binding [44], and others documenting qualitative Fbg interaction [6]. One possible conformation of Fbg/Fbn binding to PAM is suggested by the crystal structure of an M1 protein fragment binding four Fbg/Fbn Fragment D (FgD) molecules, perpendicular to its two B-repeats. The triple α-helical coiled-coil of FgD interacts with the coiled-coil of the dimeric M1-fragment [45]. M1 does not bind Pm, and Fbg/Fbn binding may be an indirect mechanism to recruit SK•Pg* and SK•Pm complexes to the surface, by binding these in ternary complexes. It is likely that varying affinities for Fbg/Fbn binding to PAM variants and M1 proteins on GAS may help define their degree of infectivity. A recent study showed that Fbg binding enhances the SK•Pg* affinity by 200-fold, signalled by linkage to the SK•Pg* active site in the ternary SK•Pg*•Fbg complex [28]. SK variants may differ in how they subvert host proteins Fbg/Fbn in a dissemination mechanism by assembly of high affinity SK•Pg*•Fbg/Fbn catalytic complexes that induce a different Pg conformational/proteolytic pathway to Pm, and are resistant to inactivation by α2-AP [4, 7].

In Vivo Assessment of SK in Streptococcal Infections

Recent advances in understanding the molecular mechanisms that govern human Pg activation by SK and the resultant Pm burst triggered at the host-pathogen surface has led many to question how exactly S. pyogenes uses SK to promote dissemination. It is thought that once host Pg is activated to Pm by SK•Pg*/Pm, the SK•Pm complex coats S. pyogenes, thereby providing directed proteolysis, allowing the pathogen to cross host fibrin-barriers [46]. It is now known that SK does not activate mouse Pg [46–50], and, as a result, mice have some level of resistance to S. pyogenes infections [51]. Mouse Pg is also a substrate for SK complexes with human Pg/Pm [52], although its turnover is slow [48, 50, 51]. There is ample evidence documenting the enhanced lethality of S. pyogenes in the presence of human Pg, either provided genetically through transgenic mice [51] or via intraperitoneal human Pg bolus injections [46]. Both strategies may be aided by the prolonged blood half-life of the human zymogen [53]. The identity between human Pg and mouse Pg is high at 79% [54], indicating that modest amino acid substitution drives the preferential ability of SK to activate human Pg. Human Pg or Pm bind PAM with higher affinity than mouse Pg [48, 55], alleviating the concern that mouse Pg might competitively inhibit human Pg complex formation in vivo through saturating the PAM receptors. It is, however, unclear whether human Pg activation is a strict requirement for pathogen spreading in mice, and whether lack of this activation potential precludes disseminative infections. Plasma concentrations of human Pg in the transgenic models do not exceed ~17% of the normal level, and may not be fully representative of what occurs during infections in humans. Furthermore, it is interesting to speculate that co-inheritance of SK2b and PAM may foster GAS virulence potential [7], or that SpeB regulation may trigger pathogen dissemination [56]. GAS expressing SK1 and SK2b showed different levels of virulence in transgenic mouse models expressing human Pg [3, 51], with pathogens expressing SK2b having little effect, but SK1 producers exhibiting significant virulence. Depending on the human Pg levels in these mice, all SK variants may be virulent to some degree, and a model with physiological circulating human [Glu]Pg concentrations may be required to compare in vivo mechanisms of allelic SK infection, growth, dissemination, and virulence.

Future Prospects

SK from GAS isolates proves to be a secreted virulence factor with no known physiological inhibitors. The SK•Pg* and SK•Pm complexes by themselves, and bound to fibrin or PAM on the bacterial surface, are resistant to the physiological serpin, α2-AP, making plasmin generation essentially unstoppable [57]. Usurping host Pg activation by multiple pathways allows GAS such as S. pyogenes to disseminate through tissues and the circulation for colonisation and growth. With the advent of new small animal imaging techniques, it is becoming possible to track GAS infections over time in a single animal. Bioluminescence-expressing bacteria can be used to track GAS populations in vivo through light production driven by over-expression of either the genomically incorporated Photorhabdus luminescens lux operon [58] or by a stabilised plasmid encoding the firefly luciferase gene [59]. Tracking of GAS is also possible through administration of imaging probes that target the bacteria themselves, or reflect heightened neutrophil and monocyte function. Tracking an infection in a single animal over time (e.g. days to weeks) will ultimately allow for better assessment of GAS dissemination from initial infection sites (i.e. skin, muscle, and lung), and will facilitate evaluating adjunctive therapies to disrupt assembly of the (Pg/Pm) •SK2b•PAM or SK1•Pg*•Fbg•M1-protein complexes on the pathogen surface. Due to the ever-increasing occurrence of antibiotic resistance, there is an urgent need for mechanism-based therapeutics to combat GAS infections. Targeting biosynthesis of SK with small molecules while sparing growth is a novel way to address antibiotic resistance [60]. Mouse monoclonal antibodies that target virulence factors, e.g. critical regions of SK and PAM, may be humanised, and engineered to exhibit greater affinity. This approach may represent a first step in innovative translational research toward development of effective inhibitors of host Pg activation by bacterial pathogens.