Bacterial plasminogen activators and receptors

FEMS Microbiology Reviews 25 (2001) 531^552 www.fems-microbiology.org Bacterial plasminogen activators and receptors Kaarina La«hteenma«ki a , Pentti Kuusela b b;c , Timo K. Korhonen a; * a Division of General Microbiology, Department of Biosciences, University of Helsinki, P.O. Box 56, FIN-00014 Helsinki, Finland Department of Bacteriology and Immunology, The Haartman Institute, University of Helsinki, P.O. Box 21, FIN-00014 Helsinki, Finland c HUCH Laboratory Diagnostics, Division of Clinical Microbiology, Helsinki University Central Hospital, FIN-00029 Helsinki, Finland Received 10 May 2001; accepted 2 July 2001 First published online 26 September 2001 Abstract Invasive bacterial pathogens intervene at various stages and by various mechanisms with the mammalian plasminogen/plasmin system. A vast number of pathogens express plasmin(ogen) receptors that immobilize plasmin(ogen) on the bacterial surface, an event that enhances activation of plasminogen by mammalian plasminogen activators. Bacteria also influence secretion of plasminogen activators and their inhibitors from mammalian cells. The prokaryotic plasminogen activators streptokinase and staphylokinase form a complex with plasmin(ogen) and thus enhance plasminogen activation. The Pla surface protease of Yersinia pestis resembles mammalian activators in function and converts plasminogen to plasmin by limited proteolysis. In essence, plasminogen receptors and activators turn bacteria into proteolytic organisms using a host-derived system. In Gram-negative bacteria, the filamentous surface appendages fimbriae and flagella form a major group of plasminogen receptors. In Gram-positive bacteria, surface-bound enzyme molecules as well as M-protein-related structures have been identified as plasminogen receptors, the former receptor type also occurs on mammalian cells. Plasmin is a broadspectrum serine protease that degrades fibrin and noncollagenous proteins of extracellular matrices and activates latent procollagenases. Consequently, plasmin generated on or activated by Haemophilus influenzae, Salmonella typhimurium, Streptococcus pneumoniae, Y. pestis, and Borrelia burgdorferi has been shown to degrade mammalian extracellular matrices. In a few instances plasminogen activation has been shown to enhance bacterial metastasis in vitro through reconstituted basement membrane or epithelial cell monolayers. In vivo evidence for a role of plasminogen activation in pathogenesis is limited to Y. pestis, Borrelia, and group A streptococci. Bacterial proteases may also directly activate latent procollagenases or inactivate protease inhibitors of human plasma, and thus contribute to tissue damage and bacterial spread across tissue barriers. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Plasminogen ; Plasmin ; Plasminogen activation; Plasminogen receptor; Bacterial metastasis Contents 1. 2. 3. 4. 5. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The mammalian plasminogen system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Plasminogen and plasmin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Control of the plasminogen system: activators and inhibitors . . . . . . . . . Interaction of pathogenic bacteria with the plasminogen system . . . . . . . . . . 3.1. Bacterial e¡ects on mammalian plasminogen activators and inhibitors . . 3.2. Bacterial plasminogen activators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Bacterial plasminogen receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Biological functions of bacteria^plasminogen interactions . . . . . . . . . . . . Variations on the theme : activation of matrix metalloproteinases by bacterial bacterium-induced cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......... .......... .......... .......... .......... .......... .......... .......... .......... proteases and .......... .......... 532 532 532 533 535 535 536 539 541 544 544 * Corresponding author. Tel. : +358 (9) 19159260; Fax: +358 (9) 19159262. E-mail address : timo.korhonen@helsinki.¢ (T.K. Korhonen). 0168-6445 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 6 4 4 5 ( 0 1 ) 0 0 0 6 7 - 5 532 K. La«hteenma«ki et al. / FEMS Microbiology Reviews 25 (2001) 531^552 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 545 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 545 1. Introduction Proteolytic activity is an important factor in the pathogenesis of bacterial infections, both in the tissue damage associated with these infections and in bacterial invasiveness into secondary infection sites within the host body. Proteolysis associated with bacterial infections may serve several functions. Bacteria such as Pseudomonas, Serratia, Bacillus, Clostridium, and Porphyromonas secrete proteinases with broad substrate speci¢city and utilize these for nutritional demands by releasing amino acids or peptides from mammalian tissues, or by increasing vascular permeability, which ensures a supply of nutrients to the site of infection (for reviews on bacterial proteases, see [1,2]). Proteolysis at the infection site may also provide a suitable environment for growth and multiplication of anaerobic bacteria. Bacterial proteases may be targeted against circulating proteins that are important for our immunological defence, such as antibodies or complement proteins, or for the control of proteolytic activity in our body, i.e. protease inhibitors. Finally, the bacterial protease can activate a precursor of mammalian protease, such as procollagenase. The latter two cases can lead to uncontrolled proteolytic activity at the infection site and to massive tissue damage, and degradation of tissue structures by bacterial proteases may potentiate bacterial spread through tissue barriers. Within the mammalian body, tissue barriers are mainly formed by extracellular matrices (ECM) and basement membranes (BM), which invasive bacteria must penetrate in order to reach the circulation. ECMs and BMs contain collagens, laminins, ¢bronectins, proteoglycans and elastin as major constituents. These molecules interact with each other and other tissue and cell components (reviewed in [3]). In general, ECM collagens constitute ca. one-third of the total protein in mammalian organisms. Consequently, degradation of and penetration through the insoluble collagen ¢bers or networks in the tissues represents a major problem in migration of bacterial or eukaryotic cells. Production of ECM-degrading proteases and of collagenases in particular is, however, limited to a restricted number of bacterial pathogens and infectious diseases (for a recent review on bacterial collagenases, see [4]). Degradation of collagens, elastin, and ¢bronectin by secreted bacterial proteases leads to massive tissue destruction seen in diseases like corneal keratitis (caused by Serratia marcescens and Pseudomonas aeruginosa), periodontitis (Porphyromonas gingivalis), cystic ¢brosis (P. aeruginosa), and gangrene (Clostridium perfringens) [1]. A number of severe bacterial pathogens produce low levels of proteinases and, in particular, have not been reported to produce collagenases. Such bacteria include most enteric bacteria as well as major agents causing bacterial meningitis : Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus in£uenzae, and Escherichia coli K1. Some of these bacteria are intracellular pathogens, and their penetration through cellular layers apparently involves an intracellular phase. However, a number of invasive bacteria are extracellular pathogens that obviously must rely on other mechanisms for invasiveness. Pathogenic bacteria are known to interact with proteinase-dependent cascade systems of their hosts, including coagulation, ¢brinolysis, complement activation, phagocytosis, and the kallikrein^kinin cascade [1,5,6]. These pathways are tightly regulated by host proteinase activators or inhibitors. Bacteria may activate or inactivate these cascades directly through their proteases or other surface components, or indirectly by causing release of e¡ector molecules from epithelial or endothelial cells or of proteolytic enzymes or their precursors from phagocytic cells. Some of these protease-dependent pathways can be utilized by bacteria to ensure growth or spread within the host. Due to the high concentration of the serine proteinase precursor plasminogen (Plg) in plasma and the broad proteolytic activity of the enzymatic form plasmin, the mammalian Plg system o¡ers a highly potential proteolytic system that could be utilized by pathogenic bacteria. Plasmin has been proposed to play a role in several physiological processes in mammals (Fig. 1): it is a key enzyme in ¢brinolysis, degrades various ECM components, and is involved in activation of certain prohormones and growth factors as well as in tumor cell metastasis (reviewed in [7^ 9]). Bacteria interact with the Plg system in various ways: they have been found to produce Plg activators (PA) and Plg receptors (PlgR), in£uence the production of host PAs and their inhibitors, and have an e¡ect on the host plasmin inhibitors. In recent years, understanding of the mechanisms and the role of the Plg system in bacterial infections has increased signi¢cantly, and this review summarizes our current knowledge on the molecular mechanisms and pathogenetic functions of bacteria^Plg interactions. 2. The mammalian plasminogen system 2.1. Plasminogen and plasmin Plg is a 90-kDa plasma proenzyme that is converted to the active serine protease plasmin by proteolytic activation K. La«hteenma«ki et al. / FEMS Microbiology Reviews 25 (2001) 531^552 Fig. 1. The mammalian Plg system. The proenzyme plasminogen is converted to the active protease plasmin by tissue-type Plg activator (tPA) or urokinase (uPA). Plasmin degrades ¢brin and various ECM components, such as laminin and ¢bronectin, and also activates procollagenases to active collagen-degrading enzymes. Dashed arrows indicate inhibition of the enzymes: Plg activator inhibitors (PAIs) inhibit Plg activation, and K2 -antiplasmin is the main inhibitor of plasmin. [8,9]. The mammalian PAs cleave the peptide bond between Arg560 and Val561 in the Plg molecule, leaving the heavy and light chains of the formed plasmin molecule joined via two disul¢de bonds (Fig. 2). The 65-kDa heavy chain contains ¢ve disul¢de-bonded triple-loop kringle structures, and the 25-kDa light chain harbors the active site with His602 , Asp645 and Ser740 forming the characteristic catalytic triad of serine proteases. Plasmin is a broadspectrum protease that preferably cleaves peptide bonds next to lysine or arginine residues [10,11]. The native form of Plg contains 791 amino acids and is called GluPlg because it has an amino-terminal glutamic acid residue. Glu-Plg can be modi¢ed by digestion with plasmin between the residues Arg67 -Met68 , Lys76 -Lys77 , or Lys77 Val78 , thereby releasing an 8-kDa activation peptide (see Fig. 2). The truncated forms of the zymogen are desig- 533 nated Lys-Plg. The conformation of Lys-Plg enables stronger interaction with receptor and target molecules of Plg and also facilitates conversion to plasmin [12]. The threedimensional structure of the full-length Plg has not been reported yet, but the crystal structures of the kringles as well as of microplasminogen and microplasmin, truncated forms that consist of a 20 amino acid long fragment of the heavy chain connected to the light chain by two disul¢de bridges, have been resolved ([13^18], reviewed in [11]; see Fig. 2). The catalytic domain of plasmin is a compact module that can recruit adapter molecules, or cofactors, such as the bacterial PAs streptokinase and staphylokinase, which modify the substrate presentation to the enzyme and modulate its speci¢city [11]. The structural changes in the catalytic domain that are associated with the activation of Plg have recently been resolved by crystallography [18]. 2.2. Control of the plasminogen system : activators and inhibitors Plg circulates in the body in large amounts: in adult human plasma the concentration is 180^200 Wg ml31 (ca. 2 WM) [8]. It is therefore understandable that Plg activation must be tightly regulated. This is achieved by speci¢c PAs, by inhibitors that control the Plg system both at the level of Plg activation and plasmin (Fig. 1), and by immobilization of Plg to cellular receptors or to target molecules. Binding of Plg to PlgRs is usually inhibited by lysine or lysine analogs, such as O-aminocaproic acid and tranexamic acid, indicating that the binding is mediated by lysine-binding sites in Plg [19]. Binding to a lysine analog leads to a dramatic conformational change in the Plg molecule: the closed molecule opens to an extended £exible conformation, which renders it more susceptible to cleavage by PAs [20]. Mammalians have two PAs, tissue-type plasminogen activator (tPA) and urokinase (uPA), which both are serine proteases that are secreted in a single-chain form (sctPA ; scuPA or pro-uPA) and processed proteolytically Fig. 2. Structure of human Plg. The kringle domains containing lysine-binding sites are marked as K1^K5. The amino acids forming the active site are indicated (Ser740 , Asp645 , His602 ), and the cleavage sites for plasminogen activators (PAs) and plasmin are shown with arrows. Disul¢de bonds are indicated by dashed lines. Numbering of amino acids according to [8]. 534 K. La«hteenma«ki et al. / FEMS Microbiology Reviews 25 (2001) 531^552 Table 1 Plasminogen activators Activator Molecular mass Characteristics Mammalian tPA 70 kDa uPA 50 kDa Produced mainly by endothelial cells. Concentration in plasma 5^10 ng ml31 . Activates Plg by limited proteolysis at the Arg560 -Val561 bond. Major circulating activator; main activator in ¢brinolysis. Produced by various normal and malignant cells. Concentration in plasma ca. 3.5 ng ml31 and in urine 200^300 ng ml31 . Activates Plg by limited proteolysis at the Arg560 -Val561 bond. Major cell-bound activator (binds to cell surface uPAR). Functions in cell-mediated degradation of extracellular matrix during in£ammation, wound healing, tissue remodeling, and tumor metastasis. Bacterial Streptokinase Staphylokinase 47 kDa 16 kDa Pla 35 kDa PauA Dimer of 29-kDa subunits Secreted by group A, C and G streptococci. Forms a 1:1 complex with Plg, which acts as a PA. Secreted by lysogenic Staphylococcus aureus. Forms a 1:1 complex with plasmin(ogen). Only the complex with plasmin acts as a PA. Activates preferably ¢brin-bound Plg. Surface-bound protease of Yersinia pestis. Activates Plg by cleaving the same Arg560 -Val561 bond in Plg as do tPA and uPA. Secreted by Streptococcus uberis. Mechanism for Plg activation not known yet. into a fully active two-chain form (tctPA ; tcuPA) (Table 1; for reviews, see [21^23]). The processed molecules consist of an A-chain and a B-chain held together by disul¢de bonds. The His, Asp, and Ser residues forming the active site are located in the B-chain in both PAs. Crystal structures of the catalytic domains of tPA and uPA have been resolved [24,25]; their overall structures exhibit the typical serine proteinase fold, with insertion loops around the active site cleft determining their speci¢city for Plg. In uPA, the A-chain consists of one kringle structure and a growth factor-like domain, which contains the receptor-binding amino acid sequence [26]. tPA contains two kringle structures, a growth factor-like domain, and an amino-terminal domain that is homologous to the ¢brin-binding ¢nger domain in ¢bronectin. The ¢nger-like domain, together with the second kringle domain, provides tPA with high a¤nity for ¢brin [27,28]. For uPA a speci¢c, high-a¤nity 55-kDa glycoprotein receptor (uPAR) has been identi¢ed in various cell types [29,30]. The main inhibitors of the Plg system belong to the serine protease inhibitor (serpin) group of antiproteases that speci¢cally inhibit catalytic activity of serine proteinases (Table 2; for a review, see [31]). The mode of action of serpins relies on formation of a stable complex between the substrate-like region of the serpin and the active site substrate-binding region of the target enzyme. Consequently, the serpin reactive center loop is cleaved by the protease, resulting in formation of a covalent acyl-enzyme intermediate and distortion of the active site of the enzyme in a way that prevents deacylation but rather traps the inhibitor^enzyme complex [32,33]. The primary PA inhibitors are PAI-1 and PAI-2 (Table 2), and other proteins able to inhibit PAs include protease nexin, a broad-spectrum serpin found in tissues, and PAI3, which has been isolated from urine. PAI-3 inhibits both tPA and uPA, but with a slower rate than PAI-1 and PAI2 [23,34]. The main physiological inhibitor of plasmin is the serpin K2 -antiplasmin, which forms a complex with plasmin by binding to the lysine-binding kringles 1^3 of plasmin in a rapid, reversible reaction. This is followed by a slower, irreversible reaction in which a covalent bond is formed between the Ser residue in the active site of plasmin and the reactive site of K2 -antiplasmin [9,31,35,36]. As the binding of Plg to receptor structures is mediated via the same lysine-binding sites as the binding to K2 -antiplasmin, receptor-bound plasmin(ogen) is protected from inhibition by K2 -antiplasmin. The most abundant plasmin inhibitor in human plasma is the broad-spectrum proteinase inhibitor K2 -macroglobulin (Table 2). However, inhibition Table 2 Main inhibitors of the Plg system Inhibitor Molecular mass Principal target Characteristics PAI-1 52 kDa tPA, uPA PAI-2 60 kDa uPA K2 -Antiplasmin 70 kDa K2 -Macroglobulin 725 kDa Soluble plasmin (receptor-bound plasmin is protected from inhibition). Plasmin and various other serine proteinases, various thiol, carboxyl, and metalloproteinases. Produced by endothelial and various other cells. Normal plasma concentration 10^30 ng ml31 . Found in monocytes and macrophages, detected in plasma only during pregnancy. Concentration in plasma 60^70 Wg ml31 . Concentration in plasma 2.5 mg ml31 . K. La«hteenma«ki et al. / FEMS Microbiology Reviews 25 (2001) 531^552 of plasmin by K2 -macroglobulin begins only if the local or systemic concentration of K2 -antiplasmin is signi¢cantly decreased [7,31]. 2.2.1. Mammalian plasminogen receptors and plasmin target molecules Activators and inhibitors precisely control plasmin formation in the body, and via PlgRs plasmin activity is directed to locations where proteolytic activity is required. Various mammalian cells have receptors for Plg on their surface (reviewed in [19,37]). The best-characterized cellular PlgRs are the glycolytic enzyme K-enolase [38,39], the calcium- and phospholipid-binding protein annexin II [40,41], and amphoterin, which was originally characterized by its a¤nity to heparin [42,43]. Amphoterin is found in large amounts in malignant cells, and it contributes e¡ectively to their migration [44]. The lysine-binding sites of Plg, which reside in the kringle structures, recognize particularly well carboxy-terminal lysine residues [19]. In K-enolase and annexin II, carboxy-terminal lysine residues mediate the interaction with Plg [38,41], whereas amphoterin seems to represent a PlgR without a carboxy-terminal lysine residue [42,43]. Fibrin is a target for binding of both Plg and tPA, as well as for subsequent plasmin-mediated degradation [9]. Upon binding of Plg and tPA to ¢brin, Plg activation is accelerated roughly 1000-fold [45]. Likewise, binding of Plg to ECM enhances tPA-mediated Plg activation [46]. Various ECM proteins, including laminin [47], ¢bronectin [48], vitronectin [49,50], and heparan sulfate proteoglycan [51], bind Plg and are targets for plasmin-mediated degradation. ECM degradation is required during cellular migration in various physiological and pathological processes. Plasmin-mediated degradation of matrix components has been proposed to play a role in migration of in£ammatory cells to the site of in£ammation [52], in several processes connected to reproduction (reviewed in [7,21,34]), and in neuronal cell death [53]. Production of PAs, as well as expression of PlgRs and uPAR, is characteristic for malignant cells, and abundant evidence suggests that the Plg system is important in tumor cell invasion (for a review, see [54]). Several studies suggest that uPA, in particular, is associated with metastasis [55^57], although certain malignant cell lines have been reported to secrete tPA [58,59]. Development of knock-out mice de¢cient in Plg, tPA, uPA, uPAR, or PAI-1 [60^64] has provided new tools to analyze the function of each component of the Plg system. In studies of in£ammatory responses, wound healing, and tumor invasion, Plg-de¢cient mice have exhibited reduced cell migration compared to normal mice (reviewed in [65]). Degradation of the complex ECM structure also requires collagenolytic activity. Type IV collagen is known to immobilize Plg and thus enhance tPA-mediated Plg activation [66], but plasmin is relatively ine¤cient in the breakdown of collagens [67]. In addition to PAs, invasive 535 cells produce proteinases that can directly degrade collagens and other ECM components [54]. Matrix metalloproteinases (MMPs) are a large group of enzymes, of which interstitial collagenases and type IV collagenases degrade di¡erent types of collagens, and stromelysins degrade ¢bronectin, laminin, elastin, and proteoglycans [54]. The collagenases are secreted in a proenzyme form and require proteolytic cleavage for activation. Plasmin can activate procollagenases [68^70] as well as a latent macrophage elastase [71], and thus acts in concert with other proteinase systems in ECM breakdown. 3. Interaction of pathogenic bacteria with the plasminogen system 3.1. Bacterial e¡ects on mammalian plasminogen activators and inhibitors The concentrations of PAs in plasma are normally small, and the amount of tPA varies considerably in response to various physiological and pathological conditions [23]. Several mammalian cell types secrete tPA or uPA (Table 1). There is limited evidence that their production is a¡ected by bacterial components, and that Plg activation may take place at an early phase of bacterial infection. Production of uPA has been found to be enhanced in human monocytes infected with Borrelia burgdorferi [72], and in bovine mammary epithelial cells infected with Staphylococcus aureus [73]. A 35-kDa protease of Bacteroides gingivalis stimulates PA activity in gingival ¢broblasts [74]. In meningococcal septicemia, formation of plasmin is detected in patient plasma at an early phase, but at a later phase the amount of PAI-1 rises suggesting that Plg activation is inhibited [75]. In healthy human volunteers, injection of endotoxin resulted in a rapid rise of plasma tPA level and in formation of plasmin^K2 -antiplasmin complexes [76]. This was followed by a slower increase in plasma PAI-1 level with a concomitant decline in tPA activity [76]. Endotoxin also upregulates uPAR : lipopolysaccharide administration in mice increased uPAR mRNA levels in various tissues [77], and in experimental human endotoxemia the expression of uPAR in monocytes is increased [78]. Monocyte uPAR has also been found to be upregulated by intact B. burgdorferi cells, by puri¢ed Borrelia outer surface protein A, and by group A streptococcal lipoteichoic acid [79]. It is not yet known whether the observed changes in PA and PAI levels are directly induced by contact of bacterial components with host cells or whether they are mediated by other host factors that are produced during in£ammatory reactions. Bacteria are known to induce secretion of cytokines from epithelial and endothelial cells [80,81]. Some cytokines, such as interleukin-1 (IL-1), increase the permeability of cellular layers, which enables accumulation of £uid and migration of in£ammatory cells to the infec- 536 K. La«hteenma«ki et al. / FEMS Microbiology Reviews 25 (2001) 531^552 tion site. It is possible that these in£ammatory processes act together with the proteolytic systems. The ¢mbrial ¢lament curli of E. coli functions as an ECM-speci¢c adhesin as well as a PlgR (discussed in Section 3.3.3 below) and was found to enhance secretion of IL-6, IL-8 and tumor necrosis factor-K (TNF-K) from human macrophages [82] as well as to bind proin£ammatory contact phase proteins and ¢brinogen onto the bacterial surface [6]. Plasmin has been reported to release IL-1 from endotoxin-induced monocytes [83] ; on the other hand, IL-1 has been proposed to upregulate the Plg system by increasing tPA synthesis and decreasing PAI-1 synthesis in human mesangial cells [84]. IL-1 has also been reported to activate MMPs and thereby promote cartilage degradation in a rabbit model of Haemophilus arthritis [85]. These ¢ndings indicate a possible interplay between various host responses associated with invasive bacterial infections. 3.2. Bacterial plasminogen activators Several invasive human pathogens have evolved PAs that are either secreted or surface-bound proteins (Table 1). Functionally, the bacterial PAs fall into two groups. Streptokinase (SK) and staphylokinase (SAK) are not enzymes themselves but form 1:1 complexes with Plg and plasmin, leading to changes in conformation and speci¢city of plasmin(ogen). In contrast to plasmin alone, the SK^plasmin and SAK^plasmin complexes acquire a remarkable e¤ciency to activate Plg (for a review, see [11]). SK and SAK share little sequence homology but their crystal structures reveal that they have adopted a similar fold, known as the L-grasp, where the SAK molecule and the three SK domains (SKK, SKL, and SKQ) each consist of ¢ve- or four-stranded L-sheets and a central Khelix or a coiled coil [17,86,87]. The mechanism of Plg activation by SAK and SK is basically similar but di¡ers in some essential aspects, such as the ¢brin dependence of the activation in human plasma. The Pla surface protease of Yersinia pestis resembles the mammalian PAs in function and activates Plg by limited proteolysis at the same Arg560 -Val561 bond as do tPA and uPA. 3.2.1. Streptokinase SK is a single-chain 414-amino acid protein secreted by L-hemolytic group A, C, and G streptococci. It is an e¤cient PA which has been associated with the pathogenesis of post-streptococcal glomerulonephritis (PSGN). The overall amino acid sequence identity between the SKs produced by human group A, C and G streptococci is 80^ 98%. The variable amino acid residues are clustered in two regions designated V1 and V2 (residues 147^218 and 244^ 264 of the mature protein). Sequence analysis of the V1 coding region in a number of streptococcal isolates has enabled division of the V1 region into seven classes, V1IÿVII , of which classes V1I , V1II and V1VI are found in SKs of PSGN-associated isolates and the others in non-nephritogenic ones [88]. The structure^function relationships in SK have been reviewed recently [88, 89]. The current view of how the SK-mediated (as well as SAK-mediated) Plg activation takes place is largely based on recent ¢ndings from crystallography and complexing of microplasmin(ogen) and kringle domains with SK fragments [11,87,90]. Plg activation is initiated by formation of a binary Plg^SK complex involving interactions between the COOH-terminal domain of SK and the catalytic domain of Plg as well as between another site in SK and the kringle domains of Plg. In the binary complex, the active center of Plg is exposed and functional without hydrolysis of the Arg560 -Val561 peptide bond. Formation of the binary Plg^SK complex is not inhibited by lysine, and the proteolytic activity is not regulated by K2 -antiplasmin [91,92]. In the next step, another, the so-called substrate Plg molecule binds to the SKK domain in the binary complex to form a ternary Plg^SK^Plg complex and is then converted to plasmin. Formation of the ternary complex is inhibited by lysine analogs, indicating that the kringles are involved in the binding of the substrate Plg molecule [90^ 92]. An 8-kDa fragment is cleaved from the NH2 -terminus of SK and the altered SK remains associated with the complex [93]. The activation model [90] depicts that the formed plasmin is released from the ternary complex and does not directly explain how SK activates Plg bound to streptococcal PlgRs via its kringle domains. There is evidence that SK also activates receptor-bound Plg [94,95] and that SK-producing streptococci can acquire surface-bound plasmin after cultivation in human plasma [96,97]. The regions in SK involved in formation of the binary SK^Plg and the ternary Plg^SK^Plg complex have been identi¢ed. Deletion analysis revealed that the peptide Ser60 -Lys333 in SK is the minimal structure required for formation of the binary complex and that the COOH-terminal portion Ala334 -Lys387 is also required for high-a¤nity binding of SK to Plg [90,98]. Random mutagenesis of SK indicated that the NH2 -terminal residues Asp41 -His48 as well as Asp220 provide interaction sites for the substrate Plg molecule in formation of the ternary complex [99], which is in agreement with the crystallographic data suggesting that the SK residues Arg45 -Gly50 interact with the kringle 5 of the substrate Plg [87]. SKs produced by strains of human and non-human origin di¡er structurally from each other and activate Plg in a species-speci¢c manner. SKs of equine and porcine group C streptococci (S. equisimilis) are 380 and 374 amino acids long, respectively, and display 25% and 35% sequence identity with SK from S. equisimilis of human origin [100]. These SKs form the binary Plg^SK complex with Plg from any host studied, and the species speci¢city in Plg activation seems to depend on the formation of the ternary Plg^SK^Plg complex [100]. The cleavage site in both K. La«hteenma«ki et al. / FEMS Microbiology Reviews 25 (2001) 531^552 human and porcine Plg is the Arg560 -Val561 bond ; in equine Plg cleavage takes place at the corresponding Arg-Ile bond [100]. In accordance with the species-speci¢c function of the SKs, the non-human streptococci are able to generate surface-associated plasmin activity after cultivation in plasma originating from the homologous host only [94]. 3.2.2. Staphylokinase SAK is a 136-amino acid protein produced by strains of S. aureus carrying one of the three prophages which contain the sak gene and mediate lysogenic conversion of staphylococci (for a review, see [101]). SAK belongs to the group of staphylococcal proteins whose synthesis takes place during the late exponential growth phase and is positively regulated by agr, the accessory gene regulator [102]. Four SAK variants di¡ering in sequence at four amino acid positions have been identi¢ed [103^107]. A few coagulase-negative staphylococci, such as S. lentus and S. lugdunensis, alternatively express either L-hemolysin or SAK. In these strains the sak gene is carried by a converting phage which inactivates the L-hemolysin gene during lysogeny [108]. Plg activation mediated by SAK is complex and di¡ers from SK-mediated Plg activation on some major aspects. (i) The SAK^Plg complex is enzymatically inactive and requires conversion of Plg to plasmin. (ii) Active SAK^ plasmin complex is e¤ciently inactivated by K2 -antiplasmin. (iii) Binding of K2 -antiplasmin to the SAK^plasmin complex releases SAK from the complex and makes it ready to interact with other plasmin(ogen) molecules again [109^111]. (iv) SAK primarily activates ¢brin-bound Plg [101]. The initial step in the activation involves association of SAK with the trace amounts of plasmin formed as a result of weak spontaneous Plg activation. SAK^plasmin complex formation is favored by the 160-fold higher a¤nity of SAK to plasmin than to Plg [112]. The primary SAK^plasmin modi¢es SAK in the complex by cleaving it at the Lys10 -Lys11 peptide bond and subsequently converts SAK^Plg to SAK^plasmin, which in turn converts free Plg to plasmin. The kringle domains of Plg are not involved in the interaction with SAK, and there is evidence that Arg719 in Plg and Met26 in SAK are important for the binding [107,109]. The NH2 -terminal region of truncated SAK is important for the recon¢guration of the active site of the plasmin molecule in the binary complex [110]. The NH2 -terminal residue Lys11 seems to be especially important, since its deletion or substitution with Cys abolishes the activator function. The SAK molecule shows an asymmetric distribution of hydrophobic and hydrophilic residues, which is important for its activation capacity [113]. Substitution of the NH2 -terminal charged residues Lys11 , Asp13 , and Asp14 with Ala results in a mutated SAK that binds plasmin(ogen) but does not convert soluble Plg to plasmin. Similar substitutions of Glu46 and Lys50 as well as Glu65 and Asp69 yield mutant 537 proteins with reduced a¤nity to plasmin(ogen) and, subsequently, impaired conversion of Plg to plasmin [114]. In the absence of ¢brin, the primary formation of the SAK^plasmin complex in plasma and other physiological £uids is e¤ciently inhibited by K2 -antiplasmin, preventing subsequent modi¢cations as well as Plg activation. However, inhibition does not take place if the lysine-binding sites of plasmin in the complex are occupied by binding to ¢brin, ¢brinogen fragments [111] or to PlgRs on eukaryotic or bacterial cells. SAK also binds much more e¤ciently to substrate-bound Plg than to soluble Plg [112]. The observation that SAK primarily activates Plg bound to ¢brin without causing systemic Plg activation has raised great enthusiasm to use SAK as a thrombolytic agent to dissolve the ¢brin of blood clots (reviewed in [101]). SAK-producing S. aureus is able to generate surfaceassociated, inhibitor-protected plasmin activity after cultivation in plasma containing Plg and K2 -antiplasmin [115]. However, no evidence of SAK as a staphylococcal virulence factor exists so far. No di¡erences were found in the capacity to produce SAK or in the amount of secreted SAK among S. aureus isolates from nasal carriers and from patients su¡ering from invasive infections such as multiple abscesses or bacteremia (Mo«lka«nen, T. and Kuusela, P., unpublished data). 3.2.3. Y. pestis Pla and the omptin family of surface proteases Y. pestis Pla is a surface protease responsible for the invasive character of plague [116]. Pla is an outer membrane protein encoded by the 9.5-kb virulence plasmid termed pPCP1, which also contains genes encoding the bacteriocin pesticin as well as the pesticin immunity protein [117]. A pla homolog gene is lacking in Yersinia enterocolitica and Yersinia pseudotuberculosis, whereas E. coli and Salmonella typhimurium carry chromosomally homologs termed ompT and pgtE (also called gene E) [118,119]. Whereas the PA function of Pla is well established, a similar activity in OmpT and PgtE has remained less clear. Originally, E. coli outer membrane preparations were found to express PA activity [120] associated with expression of OmpT. However, at least under laboratory conditions, PA activity is rarely detected in E. coli cells. The majority of the 282 clinical E. coli isolates screened by Lundrigan and Webb [121] harbored the ompT gene, but only one isolate expressed PA activity in a ¢brin lysis assay. Similarly, other groups have failed to detect PA activity in E. coli or S. typhimurium cells [117,122,123]. A recombinant E. coli expressing S. typhimurium pgtE in a multicopy plasmid exhibited 1000-fold lower PA activity than did a Pla-positive Y. pestis strain [118], and we detected only very weak PA activity in E. coli cells expressing cloned ompT [124]. Taken together, these results indicate that Pla has much higher PA activity than OmpT. On the other hand, OmpT is likely to have other functions. OmpT 538 K. La«hteenma«ki et al. / FEMS Microbiology Reviews 25 (2001) 531^552 has been shown to be active under extreme denaturing conditions and to have a¤nity for denatured substrates, and it may have a role in turnover or degradation of membrane proteins in E. coli [125]. OmpT has also been proposed to have a role in complicated urinary tract infections [126] and to degrade antimicrobial peptides in the urine [127]. Recently, Salmonella was reported to gain resistance against antimicrobial peptides by PgtE-mediated cleavage [119]. The predicted amino acid sequences of Pla, OmpT and PgtE are highly similar. They share a total of 41% conserved residues and together with SopA, a homologous protein in Shigella £exneri [128], have been proposed to form a family of closely related serine proteases termed omptins [129]. The sizes of the mature omptins vary from 280 to 300 amino acid residues. A L-barrel topology model with 10 transmembrane L-chains and ¢ve surface loops was recently proposed for OmpT [130,131]. Kramer and co-workers [131] identi¢ed by substitution mutagenesis the surface-exposed residues Ser99 and His212 as active site residues in OmpT. We have found that the corresponding residues in Pla are needed for PA activity, and that the speci¢city of Pla and OmpT towards di¡erent peptide or polypeptide substrates is in£uenced by residues in the last three predicted surface loops that are somewhat di¡erent in Pla and OmpT [124]. Mature Pla (KPla with an apparent molecular mass of 37 kDa) is slowly processed into smaller membrane-bound forms termed LPla (35 kDa) and QPla (31 kDa) [117,132]. LPla is formed by an autoprocessing event at the COOH-terminal surface loop of the Pla molecule [124]. Somewhat surprisingly, prevention of the formation of the L form by mutagenesis does not have a detectable e¡ect on the PA activity of Pla, and the biological signi¢cance of the processing remains unknown [124]. 3.2.3.1. Biological functions of Pla. Plague is transmitted by £eas that infect both rodents and humans. Y. pestis exhibits remarkably e¤cient organ invasion during plague infection. It spreads from the subcutaneous site of the £ea bite to lymph nodes, where it multiplies to large numbers and causes extensive swelling of the lymph nodes (bubonic plague). Bacteria then invade the circulation, travel to the liver and the spleen, and ¢nally colonize the lungs, causing the most severe form of the disease, pneumonic plague. During the pneumonic phase, direct transmission from human to human may also occur. For a recent review of the multiple virulence factors of Y. pestis, the reader is referred to [133]. The importance of Pla for Y. pestis virulence has been convincingly demonstrated in vivo [116]. When bacteria were injected subcutaneously into mice, the LD50 value of Pla-negative mutant strains was close to 107 bacteria, as compared to an LD50 value of less than 50 bacteria for the isogenic Pla-positive strain. In contrast, when bacteria were injected intravenously, there was no di¡erence be- tween the virulence of the strains, suggesting that Pla speci¢cally enables the dissemination of Y. pestis from the peripheral infection site into the circulation [116]. Although the virulence function of Pla in plague is well established, the mechanisms by which it contributes to the infection have remained elusive. Pla may have multiple functions during infection ; the following hypotheses about the role of Pla have been proposed. 1. Kienle and co-workers proposed that Pla mediates adhesion to eukaryotic cells, to glycolipid extracts, and to murine type IV collagen [134]. Later studies showed that Pla binds strongly to laminin and with a weak a¤nity to heparan sulfate proteoglycan [135]. These adhesive characteristics may serve to localize Y. pestis to ECMs and BMs. 2. Pla-mediated adhesion to ECM and Plg activation may be of great importance in potentiating spread of Y. pestis through tissue barriers. Pla-mediated plasmin formation results in degradation of ECM produced by lung epithelial cells as well as radiolabeled laminin in vitro [135]. These structures are not degraded by Pla directly, indicating that ECM degradation is dependent on the PA activity of Pla [135]. Plasmin generated by Pla may also clear ¢brin deposits that could hinder bacterial migration in the circulation [136]. Goguen and co-workers have found in experimental Y. pestis infection that Plg-de¢cient mice have an approximately 100-fold increase in LD50 compared to normal mice [137], indicating that Plg activation is indeed involved in the pathogenesis of plague. 3. Pla may interfere with the complement system. Sodeinde and co-workers [116] found that Pla cleaves the complement component C3. They also found that infectious lesions in mice infected with a wild-type Y. pestis contained fewer in£ammatory cells than the lesions in mice infected with an isogenic Pla-negative mutant. They suggested that Pla-mediated cleavage of C3 or another component of the complement system may reduce the production of the chemoattractant C5a, and thereby suppress the migration of in£ammatory cells to the infection site [116]. 4. Pla possesses a weak coagulase activity [117,136]. This activity is hardly detected with human plasma and is normally tested with rabbit plasma [136,138]; it is not known whether it is important in human infection. However, it has been suggested that the coagulase activity may have an e¡ect in the invertebrate host [139]. 5. Pla has been reported to degrade Yops [140], Yersinia outer proteins that are essential virulence factors in Yersinia species. The Pla-mediated degradation seems to be speci¢cally addressed to Yops, as other Yersinia proteins are not signi¢cantly degraded [140] and E. coli outer membrane proteins are not degraded by expression of Pla [117,135]. However, the function of the degradation is not yet known. 539 K. La«hteenma«ki et al. / FEMS Microbiology Reviews 25 (2001) 531^552 6. We have found that Pla, but not OmpT, inactivates K2 antiplasmin [124]. Inactivation apparently results from cleavage of a single peptide bond in the inhibitor, and is critically a¡ected by the residue Arg211 in Pla. Such inactivation of the major plasmin inhibitor is likely to enhance uncontrolled proteolysis during infection. 3.2.4. Plasminogen activators in other bacteria Streptococcus uberis is an important etiological agent of bovine mastitis, infection of the udder. The bacterium activates bovine, ovine and equine Plg, but does not activate human Plg [141^143]. Plg activation is mediated by PauA, a 251-amino acid protein which shows only low sequence homology to SK [144]. Bovine milk contains Plg, and peptides released from bovine casein by plasmin have been shown to satisfy amino acid requirements of S. uberis [145], which suggests a mechanism of how PauA may enhance colonization of the lactating gland. Mycobacterium tuberculosis was recently demonstrated to express several PlgRs as well as a yet unidenti¢ed PA [146]. PAs have also been detected in bacteria associated with periodontal disease, which involves destruction of tooth-supporting tissues and reduction of gingival collagen ¢bers. An 80-kDa trypsin-like protease of P. gingivalis has been demonstrated to activate Plg [147]. Interestingly, the protease was also found to degrade several antiproteases, which, together with the plasmin formation, may result in an uncontrolled degradation of periodontal tissue and progression of the disease. Fig. 3. Formation of plasmin activity on a Salmonella PlgR. A: Formation of cell-bound plasmin on S. typhimurium cells. Bacteria were incubated with Plg and tPA in the absence (bars a and b) or presence (bars c and d) of the lysine analog O-aminocaproic acid, washed thoroughly, and then incubated with the chromogenic plasmin substrate S-2251, the degradation of which was detected by measuring OD405 . Plg was used in two concentrations, 20 Wg ml31 (bars a and c) and 40 Wg ml31 (bars b and d). Control tests were performed without tPA (bar e) or without Plg (bar f). B: Enhancement of tPA-mediated Plg activation in the presence of puri¢ed type 1 ¢mbriae from S. typhimurium. Plasmin formation was measured with S-2251 at a ¢mbria concentration of 10 Wg ml31 in the absence (b) or presence (a) of O-aminocaproic acid. For comparison, plasmin formation in the presence of bovine serum albumin (100 Wg ml31 ) (8), in bu¡er alone (F) and when Plg or tPA was omitted from the assay suspension (O) is shown. Note that the lysine analog inhibits plasmin formation in both systems. Reprinted from [123,148] with permission of the American Society for Microbiology. 3.3. Bacterial plasminogen receptors 3.3.1. What does a bacterial plasminogen receptor do? Bacterial PlgRs immobilize Plg on the bacterial surface and thus enhance Plg activation. In essence, bacterial PlgRs function to turn bacteria into proteolytic organisms. An example of Plg activation by tPA on the surface of S. typhimurium is shown in Fig. 3. It can be seen that formation of active plasmin (Fig. 3A) takes place on the cell surface but not in solution. Type 1 ¢mbria is one of the PlgR molecules on the Salmonella surface [148], and incu- Table 3 Bacterial species reported to express plasmin(ogen) receptors Bacterial species Reference Group A, C, and G streptococci Streptococcus pneumoniae Staphylococcus aureus Escherichia coli Salmonella enteritidis Salmonella typhimurium Haemophilus in£uenzae Branhamella catarrhalis Proteus mirabilis Pseudomonas aeruginosa Neisseria meningitidis, N. gonorrhoeae Borrelia burgdorferi Borrelia coriacae, B. garinii, B. parkerii, B. anserina, B. turicatae, B. hermsii Treponema denticola Helicobacter pylori Mycoplasma fermentans Fusobacterium nucleatum Mycobacterium tuberculosis [154^161] [158] [162,163] [148,164^167] [167] [123,148] [168] [168] [168] [168] [151] [149,169^171] [149,171] [172] [173^175] [176,177] [178] [146] 540 K. La«hteenma«ki et al. / FEMS Microbiology Reviews 25 (2001) 531^552 Table 4 Bacterial Plasmin(ogen) receptors Receptor Bacterial species Other functions Reference Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, SDH, Plr) M-like protein (PAM) Group A and C streptococci Glycolytic enzyme. Adhesion to ¢bronectin, lysozyme, myosin and actin. [179^184] Group A, C, and G streptococci Group A streptococci Escherichia coli, Salmonella Adhesion to ¢brinogen and actin. Mediators resistance to phagocytosis. Glycolytic enzyme. [153,161,185^189] K-Enolase (SEN) Fimbriae Type 1 ¢mbriae, S ¢mbriae, G ¢mbriae Curli Flagella Escherichia coli, Salmonella Aspartase Outer surface protein A (OspA) 70-kDa surface protein (OppA) Haemophilus in£uenzae Borrelia burgdorferi Borrelia burgdorferi, Treponema denticola Adhesion to cellular receptors and ECM components (laminin, ¢bronectin, collagens). Binding to ¢bronectin. Binding of contact phase proteins in human plasma. Induction of proin£ammatory cytokines in human macrophages. Motility organelle. Catabolic enzyme. Homology to periplasmic oligopeptide-binding proteins. bation of puri¢ed ¢mbriae, Plg and tPA together results in plasmin formation (Fig. 3B). PlgR activity has been detected in a number of bacterial pathogens (Table 3), and several PlgR molecules have been characterized (Table 4). It is noteworthy that most of the identi¢ed bacterial PlgRs have other important functions as well, such as adhesion, movement, enzymatic activity, nutrient uptake, or interaction with the immunological system. Furthermore, it is obvious that a single bacterial species can express multiple PlgR types. These facts complicate the analysis of the functions and possible virulence association of PlgRs by mutant construction or epidemiological analysis, and may in part explain the observed lack of correlation of virulence and PlgR function in isolates of Borrelia, group A streptococci, and meningococci [149^151]. A positive correlation between pathogenicity and PlgR activity has been found for E. coli strains from patients with ulcerative colitis compared to those from healthy subjects [152]. The patient isolates also had a higher a¤nity to various solubilized ECM proteins [152]. In addition, the group A streptococci isolated from invasive skin infections express the M53 protein with PlgR activity more often that do other group A streptococcal isolates [153]. 3.3.2. Identi¢ed plasminogen receptors on Gram-positive bacteria The best-characterized PlgRs on Gram-positive bacteria are those identi¢ed on group A and C streptococci isolated from humans. These PlgRs include the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH; also called SDH and Plr) and K-enolase (called SEN), as well as the streptococcal M-like protein PAM [161, 179,190]. [190] [148,164,165,191^194] [6,82,167,195] [166], La«hteenma«ki, K. and Korhonen, T.K., unpublished data [196] [197] [172,198] GAPDH was ¢rst identi¢ed as a streptococcal receptor for plasmin and found to bind Glu^Plg very weakly [179^ 181]. The localization of GAPDH on the cell surface of group A streptococci has been demonstrated by immunological and enzymatic methods, hence the name SDH (streptococcal surface dehydrogenase) [184]. The COOHterminal lysine residue in SDH seems to be important for the interaction with plasmin, as substitution of this residue with leucine abolishes binding [182]. SDH also displays auto-ADP-ribosylating and phosphorylation activities in vitro, which suggests a role for the enzyme in signal transduction between streptococci and pharyngeal cells [199,200]. Replacement of SDH with a surface GAPDH variant lacking plasmin-binding capacity had no e¡ect on plasmin(ogen) binding by the recombinant bacteria, which indicates that group A streptococci probably express multiple PlgRs [182]. The surface localization of SEN, another glycolytic enzyme, has been demonstrated for most streptococcal groups and serotypes [190]. Removal of the COOH-terminal lysine of SEN by carboxypeptidase B treatment diminished binding of Plg only partially, suggesting that multiple lysine residues in the molecule are involved in the interaction with Plg [190]. Comparison of the a¤nities of SDH and SEN to Plg suggests that SEN is the principal PlgR in streptococci [190]. It is noteworthy that K-enolase has been identi¢ed as a Plg-binding molecule also on the surface of several eukaryotic cells [38,201,202]. Antibodies to K-enolase can be detected in sera of patients su¡ering from PSGN, suggesting that the protein is expressed in vivo [203]. Human and streptococcal K-enolases share immunological cross-reactivity ; these ¢ndings suggest that streptococcal K-enolase is involved in consequences of the diseases caused by group A streptococci [204]. Taken K. La«hteenma«ki et al. / FEMS Microbiology Reviews 25 (2001) 531^552 together, SDH and SEN form a novel group of enzymes that are found on both eukaryotic and prokaryotic cells and known to be located both in the cytoplasm and on the cell surface. The mechanism of their surface expression remains to be resolved. M-proteins are antiphagocytic ¢brillar, helical surface proteins that contain abundant repeat regions (reviewed in [188]). Within the PAM (plasminogen-binding group A streptococcal M protein) molecule, plasmin(ogen) binding has been localized to the NH2 -terminal portion that contains two 13-amino acid repeats termed a1 and a2. Both repeats contain a single central lysine residue (for a recent review, see [95]). The a1 repeat contains the sequence DAELQRLKNERHE, where substitution of the central Lys69 residue by Ala speci¢cally abolished Plg binding, indicating that this residue in PAM is involved in the interaction with Plg [153]. The role of the a1 and a2 regions in Plg binding was further demonstrated by expressing the a1 and the a1a2 regions as a fusion to Arp4, an M-like protein lacking Plg binding. Expression of the fusion constructs conferred Plg binding on the streptococcal host strain [185]. The a1^Arp4 fusion in an SK-producing strain led to surface-associated plasmin activity in the presence of Plg, whereas a host strain with an inactivated SK gene required exogenous PA [185]. The kringle 2 in Plg has been found to be involved in the interaction with PAM [186]. PAM is associated with streptococcal isolates from impetigo, a rapidly spreading super¢cial skin infection [205], which strongly supports the idea that PAM and SK production have a role in the pathogenesis of streptococcal skin infections. M-like proteins seem to function as PlgRs also on group C and G streptococci and mediate acquisition of surface-bound plasmin in the presence of SK [187]. There seems to be a clear di¡erence between PAM and other group A streptococcal PlgR molecules in their ability to generate surface-associated plasmin in plasma in the presence of either secreted or exogenous SK. Streptococci expressing PAM generate surface-associated plasmin directly bound to PAM, whereas streptococci devoid of PAM use an indirect mechanism employing a poorly characterized complex formed of ¢brinogen, ¢brinogen-binding structures such as the Mrp protein [206], and SK. The complex activates soluble Plg to plasmin and subsequently incorporates it into the complex (reviewed in [207]). 3.3.3. Plasminogen receptors on Gram-negative bacteria In Gram-negative bacteria, the ¢lamentous surface appendages ¢mbriae and £agella seem to form a major class of PlgR molecules. Fimbriae function as adherence organelles and are composed of thousands of copies of a major subunit, ¢mbrillin, responsible for the immunogenic properties of the ¢lament. The adhesive functions are located on minor subunits which are one to three in number, depending on the ¢mbrial type, and occur in a few copies per ¢lament (for recent reviews on ¢mbria structure and 541 biogenesis, see [208,209]). A major function identi¢ed for ¢mbriae is adhesion to epithelial receptors which promotes bacterial colonization and diminishes mechanical removal of bacteria from tissue sites. We have found that many ¢mbrial types of pathogenic E. coli also bind to ECM receptors [210]. Fimbrial binding to ECM or BM proteins seems to be a true tissue adhesion mechanism as it is seen both in vitro on frozen tissue sections or with extracted ECM preparations and in vivo in rats injected with puri¢ed type IV collagen-binding ¢mbriae [211,212]. Overall, a surprisingly large number of intracellular and extracellular human pathogens have been found to adhere to the mammalian ECM (reviewed in [210,213]), and the pathogenetic importance of this adhesion property is not yet fully understood. Puri¢ed type 1, S, and G ¢mbriae, and curli of E. coli express PlgR activity [148,164,167]. tPA has also been found to bind to ¢mbriae and curli in a lysine-sensitive manner. The ¢mbrial subunit that is recognized by Plg has not been identi¢ed for any of the identi¢ed ¢mbrial PlgRs. Fimbrial ¢laments which have been genetically deleted for the lectin subunit or made de¢cient in carbohydrate binding are not a¡ected in PlgR function [148,165], which indicates that the interaction is not dependent on the lectin activity of the ¢mbriae. Other remaining questions on ¢mbrial PlgRs include the signi¢cance of structural variability within a ¢mbrial type [214] on PlgR function, as well as whether ¢mbrial types other than the ones described in Table 4 also exhibit PlgR activity. The £agellar ¢laments of E. coli and S. typhimurium bind Plg and enhance its activation by tPA ([166]; La«hteenma«ki, K., and Korhonen, T.K., unpublished data), and the PlgR activity does not seem to be related to a particular H serotype. Also, in the case of the £agellar ¢laments, the subunit(s) interacting with Plg has not been identi¢ed. The function of type 1 ¢mbriae (which are expressed by ca. 80% or more of E. coli isolates), curli and £agella as PlgRs indicates that the vast majority of E. coli strains have the capacity to immobilize Plg. Treponema denticola and B. burgdorferi express 70-kDa surface proteins that bind Plg and show homology to the oligopeptide-binding components of ABC transporter proteins [172,198]. The permease functions of these proteins have not been characterized, but the homologies suggest an interesting interplay of plasmin proteolysis and bacterial peptide transport. 3.4. Biological functions of bacteria^plasminogen interactions That Plg activation is involved in systemic infection was inferred from the observations that meningococcal meningitis is associated with enhanced ¢brinolytic activity [75]. The ¢nding of PlgR or PA function in several invasive bacterial pathogens has led to suggestions that plasmin formation has a role in tissue damage associated with or 542 K. La«hteenma«ki et al. / FEMS Microbiology Reviews 25 (2001) 531^552 needed for invasive infections, including meningitis [215^ 218]. Although the number of bacterial species found to express PlgRs is appreciable (see Table 3), the pathogenetic function of Plg activation has been addressed with a limited group of invasive bacteria. These include Y. pestis [116] and other enteric bacteria [123,215], Borrelia [171,219^221], H. in£uenzae [222], staphylococci [73], group A streptococci [223], and Fusobacterium nucleatum [178]. The studies have mainly focused on testing whether bacterium-associated plasmin activity degrades proteins of the ECM or BM and whether plasmin formation potentiates bacterial penetration through these tissue barriers. Other proposed functions for plasmin in invasive bacterial infections include ¢brin degradation [136,224], degradation of protease inhibitors in tissues [178], as well as release of peptides for nutrition of bacteria [145]. Demonstration of SK- and PlgR-related structures of group A streptococcus in kidney lesions suggests that localized Plg activation plays a role in the pathogenesis of PSGN [225]. The evidence for a pathogenetic role of plasmin in bacterial infections has largely resulted from in vitro studies. In the cases of Y. pestis, Borrelia, and group A streptococci, in vivo challenge tests have also been reported [116,137,219,221,223]. 3.4.1. Involvement of plasminogen activation in bacterial metastasis through tissue barriers ^ in vitro studies Many of the bacterial species causing systemic infections and expressing PlgR or PA activity (see Tables 1, 3, and 4) are known to adhere to BM [210,213], which could target bacteria into an environment favorable for Plg activation and plasmin activity. Adherence to BM and Plg activation are involved in tumor cell metastasis (reviewed in [226]). In order to emphasize the similar behavior of tumor cells and invasive bacteria and to distinguish the phenomenon from bacterial invasion into epithelial or phagocytic cells, we have proposed the term bacterial metastasis for the process of bacterial penetration through BM [215]. The term bacterial metastasis was actually used decades earlier to describe bacterial translocation into secondary infection sites [227]. The methodology used to demonstrate ECM degradation by bacterium-bound plasmin, i.e. degradation of radiolabeled ECM proteins or metabolically labeled ECM from cultured epithelial cells, as well as penetration of bacteria into or through BMs, has been adapted from the technology used in tumor cell metastasis studies (see [54] for a critical review of the methods and [228] for applications in bacterial infection studies). It appears that under in vitro conditions, plasmin-coated bacteria have been found to display all characteristics that probably are su¤cient for bacterial penetration through the BM or ECM: degradation of individual plasmin-sensitive proteins of BM/ECM, degradation of BM/ECM, and activation of procollagenases. The current evidence for ECM degradation by bacterium-bound plasmin can be summarized as follows. 1. Plasmin formed on the bacterial surface degrades radiolabeled noncollagenous ECM proteins laminin, ¢bronectin, and vitronectin. Fig. 4 shows degradation of ¢bronectin and laminin immobilized on glass by plasmin-coated H. in£uenzae cells [222]. Degradation is seen with plasmin-coated cells but not with plain bacterial cells, and aprotinin (an inhibitor of plasmin) prevents degradation. Similar ¢ndings have been reported for plasmin-coated cells of S. typhimurium [123], B. burgdorferi [220], and F. nucleatum [178], as well as for Pla-expressing cells of recombinant E. coli incubated with Plg [135]. 2. Plasmin generated on the surface of bacteria degrades a more complex ECM structure, such as ECM secreted by human endothelial or epithelial cells (Fig. 4). Again, in the case of S. typhimurium [123], H. in£uenzae [222], and B. burgdorferi [220] it has been shown that ECM degradation is exhibited by bacterium-bound plasmin. 3. Surface-bound plasmin enhances penetration of S. typhimurium, E. coli, H. in£uenzae and S. pneumoniae [123,215,222,229] through the reconstituted basement membrane Matrigel. Matrigel is secreted by mouse tumor cells and contains type IV collagen and laminin as major components [230,231], and at room temperature it polymerizes into a BM-like gel resembling in structure the laminin and type IV collagen networks visualized in tissues [232]. Matrigel has been frequently used in eukaryotic cell migration research, in particular tumor metastasis studies [233,234] and, more recently, also in bacterial adherence and penetration studies [123,191,222,235]. Matrigel also contains PAs, as well as procollagenases and Plg that can be activated and remain active, and, in general, BMs are considered reservoirs of Plg system components [236,237]. 4. Plasmin-coated bacteria have an enhanced potential to penetrate through cultured cell monolayers. Plasmincoated cells of B. burgdorferi penetrated human endothelial monolayers grown on connective tissue substrate, and the penetration was inhibited by aprotinin but not by K2 -antiplasmin [171]. Transcytosis of S. aureus through a bovine mammary epithelial cell monolayer was enhanced in the presence of Plg and inhibited by addition of aprotinin [73]. Interestingly, the staphylococci preferentially penetrated the cell layer in the basal-to-apical direction, i.e. from the side where the BM proteins are expected to be accessible for bacterial adhesins. 5. Bacterium-bound plasmin activates procollagenases which are needed for breakdown of the collagen network. Plasmin bound on the S. aureus surface cleaves the interstitial procollagenase pro-MMP-1 at the same peptide bond as does soluble plasmin, and the MMP-1 thus formed is proteolytically active [238]. Recently, borreliae were shown to upregulate the release of pro- K. La«hteenma«ki et al. / FEMS Microbiology Reviews 25 (2001) 531^552 543 MMPs in human monocytes and, when coated with plasmin, to convert pro-MMP-9 to active MMP-9 [239]. Human monocytes were also found to enhance penetration of B. burgdorferi across layers of ECM proteins. The penetration was partially inhibited by an MMP inhibitor, indicating that it was at least partly mediated by MMPs produced by the monocytes [239]. Fig. 4. Degradation of 35 S-labeled human endothelial cell extracellular matrix and 125 I-labeled laminin and ¢bronectin by plasmin-coated H. in£uenzae. Bacteria were incubated with Plg, unbound Plg was washed away, and tPA was added. The matrix was prepared from metabolically labeled human EA.hy926 cells and the iodinated matrix proteins were immobilized on glass. The substrates were incubated with bacterial suspensions, and the release of radioactivity was measured at timed intervals. The degradation is seen with plasmin-coated cells and inhibited by the plasmin inhibitor aprotinin. Reprinted from [222] with permission of the University of Chicago Press. 3.4.2. In vivo evidence for the role of the plasminogen system in bacterial infections In vivo data about the role of the Plg system in bacterial infections are limited so far and have resulted mainly from studies using Plg-de¢cient mice as infectious disease models. Coleman and co-workers [219] compared the dissemination of B. burgdorferi in Plg-de¢cient and normal mice after intradermal inoculation and found that the frequency of positive blood cultures was reduced in the Plg-de¢cient mice. However, similar amounts of bacteria were found in the spleen, heart, and bladder of both normal and Plgde¢cient mice. As borreliosis is transmitted via ticks, it is of interest that acquisition of Plg and PA from murine blood was found to enhance the subsequent spread of B. burgdorferi also in the tick [219]. In another study, a Borrelia sp. causing relapsing fever was detected in similar amounts in the blood of Plg-positive and Plg-negative animals, but bacterial invasion into the brain and heart of normal mice was three to ¢ve times more e¤cient than invasion into the organs of the Plg-de¢cient mice [221]. Thus, Plg seems to enhance migration of borreliae in the host. Acquisition of a SK^Plg^¢brinogen complex on the bacterial surface increases the virulence of group A streptococci in normal mice, but has no such e¡ect in Plg-de¢cient mice [223]. Plg-de¢cient mice have also been found to be more resistant to Y. pestis infection than normal mice, although the di¡erence in survival is less evident than between normal mice infected with either Pla-negative or Pla-positive bacteria [116,137]. This underscores the role of Pla-mediated Plg activation, but also indicates that other functions of Pla are likely to be active as well. Antibodies recognizing PA may give protection against invasive infection. Immunization of dairy cows with bacterial culture £uid containing PauA of S. uberis raised protection against streptococcal mastitis [240]. An immune response to PauA was detected and the protection was concluded to result from inhibition of PauA activity [240]. 4. Variations on the theme : activation of matrix metalloproteinases by bacterial proteases and bacterium-induced cytokines Degradation or loss of integrity of collagen ¢bers or network has signi¢cant consequences for the tissue dam- 544 K. La«hteenma«ki et al. / FEMS Microbiology Reviews 25 (2001) 531^552 Fig. 5. Hypothesis on bacterial migration through the extracellular matrix. The models are based on the current knowledge of bacterial interaction with the plasminogen system and the extracellular matrix. Thick arrows indicate the direction of bacterial migration which is here depicted to take place via pericellular route. Red arrows indicate conversion of plasminogen (Plg) to plasmin. Green arrows indicate inhibition by the plasmin inhibitor K2 -antiplasmin (K2AP): plain arrow indicates susceptibility to inhibition by K2AP, whereas the arrow with a bar indicates that K2AP cannot inhibit the target molecule. A. Migration of bacteria through an epithelial layer by using host-produced Plg and plasminogen activator (PA). Bacterial contact with epithelial cells stimulates secretion of PA from the cells. Bacteria bind Plg and PA, and this enhances the PA-mediated conversion of Plg to active plasmin on bacterial surface. In contrast to free plasmin, bacterium-bound plasmin is protected from inhibition by K2AP. After passing through the cell layer, bacteria adhere to the extracellular matrix (ECM), and, in order to penetrate into underlying tissues, degrade ECM by using the surface-bound plasmin. B. Streptokinase (SK) produced by group A, C, and G streptococci and staphylokinase (SAK) produced by Staphylococcus aureus form a complex with Plg. This complex activates other Plg molecules to plasmin (see text for details). The free SAK-plasmin complex is inhibited by K2AP, whereas the SKPlg complex is not inhibited. Streptococci also express various mechanisms for binding Plg, the SK-Plg-complex or plasmin, and the plasmin thus generated on bacterial surface enhances bacterial migration through the ECM. C. Migration of Yersinia pestis from the subcutaneous infection site into lymphatic vessels and circulation. The Pla surface protease mediates bacterial adherence to laminin on the ECM and also activates Plg. The formed plasmin then degrades ECM and thereby enhances bacterial invasion. Plasmin is here illustrated as cell-bound, however, no Plg receptor in Y. pestis has yet been reported. Pla also proteolytically inactivates K2AP (black arrow). This renders K2AP unable to inhibit plasmin, and thus may potentiate systemic proteolysis. age and outcome of bacterial infections, and the pathogenetic role of plasmin formation described above most likely results partly from activation of MMPs. Pathogenic bacteria also appear to have alternative mechanisms to activate metalloproteases. Direct activation of MMPs by bacterial proteases has been studied in relation to periodontal disease and P. gingivalis ([74,241,242], see also the review [4]). P. gingivalis and other bacterial species commonly associated with periodontitis were found to activate latent human ¢broblast-type and neutrophil interstitial procollagenases. Bacterial proteases were found to cleave collagen fragments generated by the activated host collagenases, and it was concluded that the host and bacterial proteases act in concert to cause periodontal tissue destruction. It is interesting to note that P. gingivalis also expresses protease activity that degrades various protease inhibitors [147,243]. Increased expression of type IV collagenases on endothelial or monocytic cells is noted in vitro after treatment with pathogenic bacteria [239,244,245]. Collagenases have recently been shown to contribute to brain damage and collapse of the blood^brain barrier in experimental bacterial meningitis [246^248]. It appears that in this disease the upregulation of MMP-9 involves the cytokine TNF-K [248]. Concentrations of MMP-9 and TNF-K in cerebrospinal £uid were increased in rats with bacterial meningitis compared to uninfected animals, and treatment with a hydroxamic acid-type MMP inhibitor reduced the MMP9 and TNF-K levels as well as neuronal damage. It seems logical that bacterial adherence to endothelial and epithe- lial cells in choroid plexus and meninges contributes to the observed release of TNF-K. 5. Conclusions The experimental evidence discussed above clearly demonstrates that a vast number of severe bacterial pathogens interact with the Plg system by expressing PlgRs or PAs, or both, or by interacting with the system in other ways. That these interactions enhance bacterial virulence is suggested by in vitro evidence showing enhanced proteolytic activity, tissue damage as well as spread through reconstituted tissue barriers by bacteria with surface-bound plasmin. These studies have led to the hypothesis (Fig. 5) that metastatic bacteria utilize similar principles for migration through tissue barriers as do metastatic tumor cells. Given the large number of bacteria observed to interact with the Plg system in vitro, it is somewhat disturbing that the in vivo evidence from experimental infections is not abundant. This may result from di¤culties encountered in the experimental approaches that are available. It is di¤cult to interfere experimentally with Plg activation in vivo; Plg is very abundant in the circulation and inhibitors or anticatalytic antibodies should be present in large amounts and be targeted against the correct conformation of Plg or PAs. The conventional approach in bacterial pathogenesis ^ site-speci¢c mutagenesis ^ is also problematic, particularly in the case of bacterial PlgRs. The identi¢ed bacterial PlgRs seem as a rule to be multifunctional surface pro- K. La«hteenma«ki et al. / FEMS Microbiology Reviews 25 (2001) 531^552 teins, such as metabolically important enzymes, adhesins, motility organelles, or proteins a¡ecting immunological reactivity against the invading pathogen. These functions in£uence the survival or infectivity of the bacterium in the host. Very detailed information on the structure^function relationships in bacterial PlgRs/PAs will be needed to construct mutants speci¢cally impaired in the PlgR/PA function only. Such structural information exists at present for the activators SK and SAK (discussed in [18]), but our information on the structural details of bacterial PlgRs, in particular, is still limited. The multifunctional nature as well as multiplicity of PlgRs on pathogenic bacteria probably re£ects a¤nity of Plg for a range of terminal and internal Lys residues. It is noteworthy, however, that a class of bacterial PlgRs is composed of ¢brillar surface proteins (PAM, ¢mbriae, £agella) that have morphological similarity to ¢brin. The other major class of bacterial PlgRs is surface-bound enzyme molecules, a theme also seen in eukaryotic organisms. Plg-negative as well as PA-negative knock-out mice have been constructed [60^65] and tested in experimental infections (reviewed in [137]). While it is obvious that these animal models will give valuable and de¢nite answers on the signi¢cance of bacteria^Plg interactions, they also have certain limitations in this respect. Some bacterial PAs exhibit marked host speci¢city in their interaction with Plg [94,224], which makes them unsuited to a murine infection model. It also appears that Plg-negative animals demonstrate increased thrombus formation and problems in growth and in£ammatory responses, which may increase their sensitivity to certain bacterial pathogens. It is encouraging that these animals have been successfully used in functional studies of Yersinia, Borrelia, and streptococcal PAs or PlgRs [137,219,221,223]. Such in vivo studies will be needed to analyze which of the numerous bacterial species found to interact with Plg (Table 3) indeed take advantage of plasmin proteolysis in their pathogenesis. Various more speci¢c questions concerning the bacteria^Plg interaction remain to be answered: what is the role of MMP activation in bacterial metastasis; which of the host PAs, tPA or uPA, is more important for bacterial migration; what is the role of bacterial adherence to ECM and endothelial/epithelial cells in localizing the surfacebound plasmin at sensitive targets and, on the other hand, in modulating the production of host PAs, PAIs, and cytokines ? It remains to be analyzed how common the inactivation of the inhibitors of the Plg system is among pathogenic bacteria and whether it a¡ects other proteolytic cascades of the human body as well. Altogether, proteinase inhibitors represent nearly 10% of the total protein in plasma and control a variety of critical events associated with connective tissue turnover, coagulation, apoptosis, angiogenesis, ¢brinolysis, complement activation, and in£ammatory reactions (for a review, see [31]). 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