Group A streptococci, a common human pathogen, secrete streptokinase, which activates the host's blood clot-dissolving protein, plasminogen. Streptokinase is highly specific for human plasminogen, exhibiting little or no activity against other mammalian species, including mouse. Here, a transgene expressing human plasminogen markedly increased mortality in mice infected with streptococci, and this susceptibility was dependent on bacterial streptokinase expression. Thus, streptokinase is a key pathogenicity factor and the primary determinant of host species specificity for group A streptococcal infection. In addition, local fibrin clot formation may be implicated in host defense against microbial pathogens.

Surface-associated plasmin(ogen) may contribute to the invasive properties of various cells. Analysis of plasmin(ogen)-binding surface proteins is therefore of interest. The N-terminal variable regions of M-like (ML) proteins from five different group A streptococcal serotypes (33, 41, 52, 53 and 56) exhibiting the plasminogen-binding phenotype were cloned and expressed in Escherichia coli. The recombinant proteins all bound plasminogen with high affinity. The binding involved the kringle domains of plasminogen and was blocked by a lysine analogue, 6-aminohexanoic acid, indicating that lysine residues in the M-like proteins participate in the interaction. Sequence analysis revealed that the proteins contain common 13-16-amino-acid tandem repeats, each with a single central lysine residue. Experiments with fusion proteins and a 30-amino-acid synthetic peptide demonstrated that these repeats harbour the major plasminogen-binding site in the ML53 protein, as well as a binding site for the tissue-type plasminogen activator. Replacement of the lysine in the first repeat with alanine reduced the plasminogen-binding capacity of the ML53 protein by 80%. The results precisely localize the binding domain in a plasminogen surface receptor, thereby providing a unique ligand for the analysis of interactions between kringles and proteins with internal kringle-binding determinants.

A common mammalian defence 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 defence mechanism allowing invasive disease initiation. To facilitate this process, S. pyogenes secrete streptokinase, a plasminogen activating protein.Streptokinase polymorphism exhibited by S. pyogenes isolates is well characterised. 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 two 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. AbstractA common mammalian defence 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 defence mechanism allowing invasive disease initiation. To facilitate this process, S. pyogenes secrete streptokinase, a plasminogen activating protein. Streptokinase polymorphism exhibited by S. pyogenes isolates is well characterised. 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 two 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 pre-bound 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...

SummaryAll virulent group A streptococcal isolates bind fibrinogen, a property that is closely linked to expression of type-specific antiphagocytic surface molecules designated M proteins. Here we show that although the M proteins from two different strains, M1 and M5, both bind fibrinogen with high affinity, they interact with different regions in the ligand. Moreover, mapping experiments demonstrated that the fibrinogen-binding regions in the M1 and M5 proteins are quite dissimilar at the amino acid sequence level and that they bind to different regions in the plasma protein. In spite of these differences, the fibrinogenbinding regions of M1 and M5 could both be shown to contribute to streptococcal survival in human blood, providing evidence for the distinct function of a plasma protein interaction in bacterial pathogenesis.

We have characterized 2 distinct mechanisms through which infectious agents may promote platelet adhesion and thrombus formation in flowing blood, thus contributing to the progression of disease. In one case, the process initiates when the integrin ␣ IIb ␤ 3 mediates platelet arrest onto immobilized bacterial constituents that have bound plasma fibrinogen. If blood contains antibodies against the bacteria, immunoglobulin (Ig) G may cluster on the same surface and activate adherent platelets through the Fc␥RIIA receptor, leading to thrombus growth. As an alternative, bacteria that cannot bind fibrinogen may attach to substrates, such as immobilized plasma proteins or components of the extracellular matrix, which also support platelet adhesion. As a result of this colocalization, IgG bound to bacteria can activate neighboring platelets and induce thrombus growth regard- IntroductionPlatelet adhesion and aggregation at sites of tissue trauma involve interactions of membrane receptors with constituents of extracellular matrices, such as collagen, and circulating macromolecules, such as von Willebrand factor (VWF) and fibrinogen, 1 which contribute to arrest bleeding during hemostasis. Bacteria, too, can induce platelet aggregation [2][3][4] or uncontrolled clotting with disseminated intravascular coagulation (DIC), 5,6 which may become disease mechanisms when the causative agent intermittently invades the bloodstream. For example, coagulation and hemostasis are activated in some localized infections, such as necrotizing fasciitis, resulting in extensive thrombosis of arterioles and veins in and around lesions. 7,8 Moreover, experimental and clinical observations have demonstrated that platelets play a key pathogenetic role when certain microorganisms establish infection in the bloodstream, as in the case of bacterial endocarditis. 4,9 In particular, platelet aggregates may allow bacteria to settle and remain at the site of infection withstanding the shear forces of flowing arterial blood. In experimental models of this disease, early vegetations grow by accretion of layers of fibrin and platelets with bacterial colonies sandwiched between them. 10 Similar mechanisms may facilitate the establishment of bacteria on artificial devices, such as arterial grafts. 11 The number of strains that can settle in the arterial circulation is limited, but an array of different species can cause septic venous thrombosis, 12,13 a condition in which platelet activation may be involved.In these studies, we have used 2 invasive species, Streptococcus pyogenes (also designated group A streptococcus) and Staphylococcus aureus, as models to examine the mechanisms involved in bacteria-induced thrombus formation under conditions mimicking the macromolecular, cellular, and hemodynamic complexity of blood circulating in different vessels. Only bacteria capable of binding a platelet-reactive factor from blood, such as fibrinogen, could initiate adhesion on a surface not intrinsically conducive to platelet deposition. This step, however, was n...

Many cells express receptors for plasminogen (Pg), although the responsible molecules in most cases are poorly defined. In contrast, the group A streptococcal surface protein PAM contains a domain with two 13-amino acid residue long repeated sequences (a1 and a2) responsible for Pg binding. Here we identify the region in Pg that interacts with PAM. A radiolabeled proteolytic plasminogen fragment containing the first three kringles (K1-K3) interacted with streptococci expressing PAM or a chimeric surface protein harboring the a1a2 sequence. In contrast, plasminogen fragments containing kringle 4 or kringle 5 and the activable serine proteinase domain failed to bind to PAM-expressing group A streptococci. A synthetic and a recombinant polypeptide containing the a1a2 sequence both bound to immobilized recombinant K2 (rK2) but not to rK1 or rK3. The interaction between the a repeat region and rK2 was reversible, and rK2 completely blocked the binding of Pg to the a1a2 region. The binding of the a repeat containing polypeptide to K2 occurred with an equilibrium association constant of 4.5 ؋ 10 M ؊1, as determined by surface plasmon resonance, a value close to that (1.6 ؋ 10 7 M ؊1 ) calculated for the a1a2-Pg interaction. Inhibition experiments suggested involvement of the lysine-binding site of K2 in the interaction. These data demonstrate that K2 contains the major Pg-binding site for PAM, providing the first well defined example of an interaction between an internal Pg-binding region in a protein and a single kringle domain.The plasma glycoprotein plasminogen (Pg) 1 is a single-chain 92-kDa precursor for the broad spectrum serine proteinase plasmin (1, 2) (see Fig. 1A). In vivo, the tissue-type and urokinase-type plasminogen activators convert the zymogen into the two-chain proteinase by cleavage of a single peptide bond (Arg 561 -Val 562 ). Activation can also be achieved by some bacterial proteins, such as streptokinase from streptococci (1, 2). Plasmin plays a key role in fibrinolysis (1-3) but also participates in several other physiological and pathophysiological processes, including wound healing, tissue penetration of cancer cells, neuronal cell death, and bacterial dissemination (4 -8).The activable serine proteinase domain is located in the COOH-terminal third of Pg. The NH 2 -terminal two-thirds of Pg contains an 8-kDa preactivation peptide and five characteristic kringle domains (K1-K5), each ϳ9 kDa. The kringles mediate interactions with multiple ligands, including fibrin, the primary target of Pg, and ␣ 2 -plasmin inhibitor, its principal regulator (1, 2). The recognition events depend upon interactions between lysine-binding sites in the kringles and exposed COOH-terminal lysines in the ligands. Lysine analogues, such as 6-aminohexanoic acid (6-AHA), mimic COOH-terminal lysines in the interaction with kringles and the structural basis of the interactions between some kringles, particularly K1 and K4, and 6-AHA has been disclosed (9 -12). The affinity of the different kringles for lysine or 6-AHA is...

Bacterial surface-associated plasmin formation is believed to contribute to invasion, although the underlying molecular mechanisms are poorly understood. To define the components necessary for plasmin generation on group A streptococci we used strain AP53 which exposes an M-like protein ("PAM") that contains a plasminogen-binding sequence with two 13-amino acid residues long tandem repeats (a1 and a2). Utilizing an Escherichia coli-streptococcal shuttle vector, we replaced a 29-residue long sequence segment of Arp4, an M-like protein that does not bind plasminogen, with a single (a1) or the combined a1a2 repeats of PAM. When expressed in E. coli, the purified chimeric Arp/PAM proteins both bound plasminogen, as well as plasmin, and when used to transform group A streptococcal strains lacking the plasminogen-binding ability, transformants with the Arp/PAM constructs efficiently bound plasminogen. Moreover, when grown in the presence of plasminogen, both Arp/PAM-and PAM-expressing streptococci acquired surface-bound plasmin. In contrast, plasminogen activation failed to occur on PAM-and Arp/PAMexpressing streptococci carrying an inactivated streptokinase gene: this block was overcome by exogenous streptokinase. Together, these results provide evidence for an unusual co-operation between a surface-bound protein, PAM, and a secreted protein, streptokinase, resulting in bacterial acquisition of a host protease that is likely to spur parasite invasion of host tissues.

Group A streptococci (GAS), a common human pathogen, secrete streptokinase (SK), which activates the host’s blood clot-dissolving protein, plasminogen (PLG). SK is highly specific for human PLG, exhibiting little or no activity against other mammalian species, including mouse. In addition to the species specificity of SK interaction with host PLG, species specificity has also been demonstrated for PLG receptors on the bacterial surface, such as the bacterial surface protein, PAM, and for interactions with fibrinogen. We generated a “humanized” transgenic mouse expressing human PLG under control of the mouse albumin gene regulatory sequences within a Bacteria Artificial Chromosome (BAC) transgene. The highest expressing transgenic founder line produced human PLG corresponding to ~17% of the PLG level in control human plasma (16.7±1.78) and largely rescued the prothromobtic morbidity otherwise observed in Plg null mice. Mice are generally highly resistant to subcutaneous infection by most human pathogenic GAS. However, introduction of human PLG expressed by the transgene markedly increased mortality to 75% from 20% in Tg− littermate control using the GAS strain 2616, exhibiting enhanced virulence due to site-directed mutation in the regulatory locus, csrRS. Similar differences in mortality were also observed with a wildtype GAS strain. The increased susceptibility of Tg+ mice to GAS was largely abrogated by deletion of the SK gene, demonstrating the major role of the PLG/SK interaction in GAS pathogenicity. Marked differences in mortality were also observed in Tg+ mice infected by the PAM expressing GAS strain AP53 and its PAM− isogenic variant, demonstrating a role for PAM in focusing PLG at the bacterial surface. We hypothesize that GAS hijack the host fibrinolytic system in order to circumvent local thrombosis and microvascular occlusion and reopen the vascular tree to systemic spread. Consistent with this model, the marked difference in mortality between Tg+ and Tg− mice was no longer observed when GAS were injected directly intravenously. In addition, a significant increase in bacterial colonies in the spleens of Tg+ mice was observed following subcutaneous GAS injection. Markedly increased mortality was also observed following GAS injection in C57BL/6J mice treated with the snake venom Ancrod, which proteolytically degrades plasma fibrinogen, consistent with a key role for fibrin deposition in host defense against GAS dissemination. In summary, activation of host plasminogen by SK leads to accelerated clearance of host fibrin and is a central mechanism for GAS invasion and spread. It is likely that similar interactions are central to the invasive program of other unrelated PA-associated pathogens that occupy diverse microenvironmental niches. The remarkable species specificity of SK for host PLG probably resulted from host and pathogen coevolution. These observations highlight the potential role of infectious disease as a critical force in the evolution of the hemostatic system and the unusual species specificity of many coagulation factor interactions.