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Cellular plasminogen (Pg) receptors (PgRs) are utilized to recruit Pg; stimulate its activation to the serine protease, plasmin (Pm); and sterically protect the surface Pm from inactivation by host inhibitors. One such PgR is the moonlighting enzyme, enolase, some of which leaves the cytoplasm and resides at the cell surface to potentially function as a PgR. Since microbes employ conscription of host Pg by PgRs as one virulence mechanism, we explored the structural basis of the ability of Streptococcus pyogenes enolase (Sen) to function in this manner. Employing single-particle cryo-electron microscopy (cryo-EM), recombinant Sen from S. pyogenes was modeled at 2.6 Å as a stable symmetrical doughnut-shaped homooctamer with point group 422 (D4) symmetry, with a monomeric subunit molecular weight of ∼49 kDa. Binding sites for hPg were reported in other studies to include an internal K 252,255 and the COOH-terminal K 434,435 residues of Sen. However, in native Sen, the latter are buried within the minor interfaces of the octamer and do not function as a Pg-binding epitope. Whereas Sen and hPg do not interact in solution, when Sen is bound to a surface, hPg interacts with Sen independently of K 252,255,434,435 . PgRs devoid of COOH-terminal lysine utilize lysine isosteres comprising a basic residue, " i ", and an anionic residue at " i + 3" around one turn of an α-helix. We highlight a number of surface-exposed potential hPg-binding lysine isosteres and further conclude that while the octameric structure of Sen is critical for hPg binding, disruption of this octamer without dissociation exposes hPg-binding epitopes.

Borrelia miyamotoi, a relapsing fever spirochete transmitted by Ixodid ticks causes B. miyamotoi disease (BMD). To evade the human host´s immune response, relapsing fever borreliae, including B. miyamotoi, produce distinct variable major proteins. Here, we investigated Vsp1, Vlp15/16, and Vlp18 all of which are currently being evaluated as antigens for the serodiagnosis of BMD. Comparative analyses identified Vlp15/16 but not Vsp1 and Vlp18 as a plasminogen-interacting protein of B. miyamotoi. Furthermore, Vlp15/16 bound plasminogen in a dose-dependent fashion with high affinity. Binding of plasminogen to Vlp15/16 was significantly inhibited by the lysine analog tranexamic acid suggesting that the protein-protein interaction is mediated by lysine residues. By contrast, ionic strength did not have an effect on binding of plasminogen to Vlp15/16. Of relevance, plasminogen bound to the borrelial protein cleaved the chromogenic substrate S-2251 upon conversion by urokinase-type plasminogen activator (uPa), demonstrating it retained its physiological activity. Interestingly, further analyses revealed a complement inhibitory activity of Vlp15/16 and Vlp18 on the alternative pathway by a Factor H-independent mechanism. More importantly, both borrelial proteins protect serum sensitive Borrelia garinii cells from complement-mediated lysis suggesting multiple roles of these two variable major proteins in immune evasion of B. miyamotoi.

Virulent strains of Streptococcus pyogenes (gram-positive group A Streptococcus pyogenes [GAS]) recruit host single-chain human plasminogen (hPg) to the cell surface-where in the case of Pattern D strains of GAS, hPg binds directly to the cells through a surface receptor, plasminogen-binding group A streptococcal M-protein (PAM). The coinherited Pattern D GAS-secreted streptokinase (SK2b) then accelerates cleavage of hPg at the R 561 -V 562 peptide bond, resulting in the disulfide-linked two-chain protease, human plasmin (hPm). hPm localizes on the bacterial surface, assisting bacterial dissemination via proteolysis of host defense proteins. Studies using isolated domains from PAM and hPg revealed that the A-domain of PAM binds to the hPg kringle-2 module (K2 hPg ), but how this relates to the function of the full-length proteins is unclear. Herein, we use intact proteins to show that the lysine-binding site of K2 hPg is a major determinant of the activation-resistant T-conformation of hPg. The binding of PAM to the lysine-binding site of K2 hPg relaxes the conformation of hPg, leading to a greatly enhanced activation rate of hPg by SK2b. Domain swapping between hPg and mouse Pg emphasizes the importance of the Pg latent heavy chain (residues 1-561) in PAM binding and shows that while SK2b binds to both hPg and mouse Pg, the activation properties of streptokinase are strictly attributed to the serine protease domain (residues 562-791) of hPg. Overall, these data show that native hPg is locked in an activation-resistant conformation that is relaxed upon its direct binding to PAM, allowing hPm to form and provide GAS cells with a proteolytic surface., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is an evolutionarily conserved essential enzyme in the glycolytic pathway. GAPDH is also involved in a wide spectrum of non-catalytic cellular 'moonlighting' functions. Bacterial surface-associated GAPDHs engage in many host interactions that aid in colonization, pathogenesis, and virulence. We have structurally and functionally characterized the recombinant GAPDH of the obligate intracellular bacteria Chlamydia trachomatis, the leading cause of sexually transmitted bacterial and ocular infections. Contrary to earlier speculations, recent data confirm the presence of glucose-catabolizing enzymes including GAPDH in both stages of the biphasic life cycle of the bacterium. The high-resolution crystal structure described here provides a close-up view of the enzyme's active site and surface topology and reveals two chemically modified cysteine residues. Moreover, we show for the first time that purified C. trachomatis GAPDH binds to human plasminogen and plasmin. Based on the versatility of GAPDH's functions, data presented here emphasize the need for investigating the Chlamydiae GAPDH's involvement in biological functions beyond energy metabolism., (© 2020 The Protein Society.)

Mycoplasma bovis is an extremely small cell wall-deficient pathogenic bacterium in the genus Mycoplasma that causes serious economic losses to the cattle industry worldwide. Fructose-1,6-bisphosphate aldolase (FBA), a key enzyme in the glycolytic pathway, is a multifunctional protein in several pathogenic bacterial species, but its role in M. bovis remains unknown. Herein, the FBA gene of the M. bovis was amplified by PCR, and subcloned into the prokaryotic expression vector pET28a (+) to generate the pET28a-FBA plasmid for recombinant expression in Escherichia coli Transetta. Expression of the 34 kDa recombinant rMbFBA protein was confirmed by electrophoresis, and enzymatic activity assays based on conversion of NADH to NAD+ revealed Km and Vmax values of 48 μM and 43.8 μmoL/L/min, respectively. Rabbit anti-rMbFBA and anti-M. bovis serum were generated by inoculation with rMbFBA and M. bovis, and antigenicity and immunofluorescence assay demonstrated that FBA is an immunogenic protein expressed on the cell membrane in M. bovis cells. Enzyme-linked immunosorbent assays revealed equal distribution of FBA in the cell membrane and cytoplasm. Complement-dependent mycoplasmacidal assays showed that rabbit anti-rMbFBA serum killed 44.1% of M. bovis cells in the presence of complement. Binding and ELISA assays demonstrated that rMbFBA binds native bovine plasminogen and in a dose-dependent manner. Fluorescent microscopy revealed that pre-treatment with antibodies against rMbFBA decreased the adhesion of M. bovis to embryonic bovine lung (EBL) cells. Furthermore, adherence inhibition assays revealed 34.4% inhibition of M. bovis infection of EBL cells following treatment with rabbit anti-rMbFBA serum, suggesting rMbFBA participates in bacterial adhesion to EBL cells., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The binding of human plasminogen (hPg) to the surface of the human pathogen group A Streptococcus pyogenes (GAS) and subsequent hPg activation to the protease plasmin generate a proteolytic surface that GAS employs to circumvent host innate immunity. Direct high-affinity binding of hPg/plasmin to pattern D GAS is fully recapitulated by the hPg kringle 2 domain (K2 hPg ) and a short internal peptide region (a1a2) of a specific subtype of bacterial surface M protein, present in all GAS pattern D strains. To better understand the nature of this binding, critical to the virulence of many GAS skin-tropic strains, we used high-resolution NMR to define the interaction of recombinant K2 hPg with recombinant a1a2 (VKK38) of the M protein from GAS isolate NS455. We found a 2:1 (m/m) binding stoichiometry of K2 hPg /VKK38, with the lysine-binding sites of two K2 hPg domains anchored to two regions of monomeric VKK38. The K2 hPg /VKK38 binding altered the VKK38 secondary structure from a helical apo-peptide with a flexible center to an end-to-end K2 hPg -bound α-helix. The K2 hPg residues occupied opposite faces of this helix, an arrangement that minimized steric clashing of K2 hPg We conclude that VKK38 provides two conformational lysine isosteres that each interact with the lysine-binding sites in K2 hPg Further, the adoption of an α-helix by VKK38 upon binding to K2 hPg sterically optimizes the side chains of VKK38 for maximal binding to K2 hPg and minimizes steric overlap between the K2 hPg domains. The mechanism for hPg/M protein binding uncovered here may facilitate targeting of GAS virulence factors for disease management., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Staphylococcus aureus is a commensal bacterium that has the ability to cause superficial and deep-seated infections. Like several other invasive pathogens, S. aureus can capture plasminogen from the human host where it can be converted to plasmin by host plasminogen activators or by endogenously expressed staphylokinase. This study demonstrates that sortase-anchored cell wall-associated proteins are responsible for capturing the bulk of bound plasminogen. Two cell wall-associated proteins, the fibrinogen- and fibronectin-binding proteins A and B, were found to bind plasminogen, and one of them, FnBPB, was studied in detail. Plasminogen captured on the surface of S. aureus- or Lactococcus lactis-expressing FnBPB could be activated to the potent serine protease plasmin by staphylokinase and tissue plasminogen activator. Plasminogen bound to recombinant FnBPB with a KD of 0.532 μm as determined by surface plasmon resonance. Plasminogen binding did not to occur by the same mechanism through which FnBPB binds to fibrinogen. Indeed, FnBPB could bind both ligands simultaneously indicating that their binding sites do not overlap. The N3 subdomain of FnBPB contains the full plasminogen-binding site, and this includes, at least in part, two conserved patches of surface-located lysine residues that were recognized by kringle 4 of the host protein., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Acinetobacter baumannii is an emerging opportunistic pathogen, responsible for up to 10% of gram-negative, nosocomial infections. The global increase of multidrug-resistant and pan-resistant Acinetobacter isolates presents clinicians with formidable challenges. To establish a persistent infection,A. baumannii must overcome the detrimental effects of complement as the first line of defense against invading microorganisms. However, the immune evasion principles underlying serum resistance inA. baumannii remain elusive. Here, we identified a novel plasminogen-binding protein, termed CipA. Bound plasminogen, upon conversion to active plasmin, degraded fibrinogen and complement C3b and contributed to serum resistance. Furthermore, CipA directly inhibited the alternative pathway of complement in vitro, irrespective of its ability to bind plasminogen. A CipA-deficient mutant was efficiently killed by human serum and showed a defect in the penetration of endothelial monolayers, demonstrating that CipA is a novel multifunctional protein that contributes to the pathogenesis ofA. baumannii., (© The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

Plasminogen (Plg) circulates in the host as two predominant glycoforms. Glycoform I Plg (GI-Plg) contains glycosylation sites at Asn289 and Thr346, whereas glycoform II Plg (GII-Plg) is exclusively glycosylated at Thr346. Surface plasmon resonance experiments demonstrated that Plg binding group A streptococcal M protein (PAM) exhibits comparative equal affinity for GI- and GII-Plg in the "closed" conformation (for GII-Plg, KD = 27.4 nM; for GI-Plg, KD = 37.0 nM). When Plg was in the "open" conformation, PAM exhibited an 11-fold increase in affinity for GII-Plg (KD = 2.8 nM) compared with that for GI-Plg (KD = 33.2 nM). The interaction of PAM with Plg is believed to be mediated by lysine binding sites within kringle (KR) 2 of Plg. PAM-GI-Plg interactions were fully inhibited with 100 mM lysine analogue ε-aminocaproic acid (εACA), whereas PAM-GII-Plg interactions were shown to be weakened but not inhibited in the presence of 400 mM εACA. In contrast, binding to the KR1-3 domains of GII-Plg (angiostatin) by PAM was completely inhibited in the presence 5 mM εACA. Along with PAM, emm pattern D GAS isolates express a phenotypically distinct SK variant (type 2b SK) that requires Plg ligands such as PAM to activate Plg. Type 2b SK was able to generate an active site and activate GII-Plg at a rate significantly higher than that of GI-Plg when bound to PAM. Taken together, these data suggest that GAS selectively recruits and activates GII-Plg. Furthermore, we propose that the interaction between PAM and Plg may be partially mediated by a secondary binding site outside of KR2, affected by glycosylation at Asn289.

Rapid kinetics demonstrate a three-step pathway of streptokinase (SK) binding to plasminogen (Pg), the zymogen of plasmin (Pm). Formation of a fluorescently silent encounter complex is followed by two conformational tightening steps reported by fluorescence quenches. Forward reactions were defined by time courses of biphasic quenching during complex formation between SK or its COOH-terminal Lys(414) deletion mutant (SKΔK414) and active site-labeled [Lys]Pg ([5-(acetamido)fluorescein]-D-Phe-Phe-Arg-[Lys]Pg ([5F]FFR-[Lys]Pg)) and by the SK dependences of the quench rates. Active site-blocked Pm rapidly displaced [5F]FFR-[Lys]Pg from the complex. The encounter and final SK ·[5F]FFR-[Lys]Pg complexes were weakened similarly by SK Lys(414) deletion and blocking of lysine-binding sites (LBSs) on Pg kringles with 6-aminohexanoic acid or benzamidine. Forward and reverse rates for both tightening steps were unaffected by 6-aminohexanoic acid, whereas benzamidine released constraints on the first conformational tightening. This indicated that binding of SK Lys(414) to Pg kringle 4 plays a role in recognition of Pg by SK. The substantially lower affinity of the final SK · Pg complex compared with SK · Pm is characterized by a ∼ 25-fold weaker encounter complex and ∼ 40-fold faster off-rates for the second conformational step. The results suggest that effective Pg encounter requires SK Lys(414) engagement and significant non-LBS interactions with the protease domain, whereas Pm binding additionally requires contributions of other lysines. This difference may be responsible for the lower affinity of the SK · Pg complex and the expression of a weaker "pro"-exosite for binding of a second Pg in the substrate mode compared with SK · Pm., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Mycoplasma synoviae is an avian pathogen that can lead to respiratory tract infections and arthritis in chickens and turkeys, resulting in serious economic losses to the poultry industry. Enolase reportedly plays important roles in several bacterial pathogens, but its role in M. synoviae has not been established. Therefore, in this study, the enolase encoding gene (eno) of M. synoviae was amplified from strain WVU1853 and expressed in E. coli BL21 cells. Then the enzymatic activity, immunogenicity and binding activity with chicken plasminogen (Plg) and human fibronectin (Fn) was evaluated., Results: We demonstrated that the recombinant M. synoviae enolase protein (rMsEno) can catalyze the conversion of 2-phosphoglycerate (2-PGA) to phosphoenolpyruvate (PEP), the Km and Vmax values of rMsEno were 1.1 × 10(-3) M and 0.739 μmol/L/min, respectively. Western blot and immuno-electron microscopy analyses confirmed that enolase was distributed on the surface and within the cytoplasm of M. synoviae cells. The binding assays demonstrated that rMsEno was able to bind to chicken Plg and human Fn proteins. A complement-dependent mycoplasmacidal assay demonstrated that rabbit anti-rMsEno serum had distinct mycoplasmacidal efficacy in the presence of complement, which also confirmed that enolase was distributed on the surface of M. synoviae. An inhibition assay showed that the adherence of M. synoviae to DF-1 cells pre-treated with Plg could be effectively inhibited by treatment with rabbit anti-rMsEno serum., Conclusion: These results reveal that M. synoviae enolase has good catalytic activity for conversion of 2-PGA to PEP, and binding activity with chicken Plg and human Fn. Rabbit anti-rMsEno serum displayed an obvious complement-dependent mycoplasmacidal effect and adherent inhibition effect. These results suggested that the M. synoviae enolase plays an important role in M. synoviae metabolism, and could potentially impact M. synoviae infection and immunity.

To examine the global function of the key surface-exposed loops of streptokinase, bearing substrate-specific exosites, namely, the 88-97 loop in the α domain, the 170 loop in the β domain, and the coiled-coil region (Leu321-Asn338) in the γ domain, mutagenic as well as peptide inhibition studies were carried out. Peptides corresponded to the primary structure of an exosite, either individual or stoichiometric mixtures of various disulfide-constrained synthetic peptide(s) inhibited plasminogen activation by streptokinase. Remarkably, pronounced inhibition of substrate plasminogen activation by the preformed streptokinase-plasmin activator complex was observed when complementary mixtures of different peptides were used compared to the same overall concentrations of individual peptides, suggesting co-operative interactions between the exosites. This observation was confirmed with streptokinase variants mutated at one, two, or three sites simultaneously. The single/double/triple exosite mutants of streptokinase showed a nonadditive, synergistic decline in kcat for substrate plasminogen activation in the order single > double > triple exosite mutant. Under the same conditions, zymogen activation by the various mutants remained essentially native- like in terms of nonproteolytic activation of partner plasminogen. Multisite mutants also retain affinity to form 1:1 stoichiometric activator complexes with plasmin when probed through sensitive equilibrium fluorescence studies. Thus, the present results strongly support a model of streptokinase action, wherein catalysis by the streptokinase-plasmin complex operates through a distributed network of substrate-interacting exosites resident across all three domains of the cofactor protein.

Our previously hypothesized mechanism for the pathway of plasminogen (Pg) activation by streptokinase (SK) was tested by the use of full time course kinetics. Three discontinuous chromogenic substrate initial rate assays were developed with different quenching conditions that enabled quantitation of the time courses of Pg depletion, plasmin (Pm) formation, transient formation of the conformationally activated SK·Pg* catalytic complex intermediate, formation of the SK·Pm catalytic complex, and the free concentrations of Pg, Pm, and SK. Analysis of full time courses of Pg activation by five concentrations of SK along with activity-based titrations of SK·Pg* and SK·Pm formation yielded rate and dissociation constants within 2-fold of those determined previously by continuous measurement of parabolic chromogenic substrate hydrolysis and fluorescence-based equilibrium binding. The results obtained with orthogonal assays provide independent support for a mechanism in which the conformationally activated SK·Pg* complex catalyzes an initial cycle of Pg proteolytic conversion to Pm that acts as a trigger. Higher affinity binding of the formed Pm to SK outcompetes Pg binding, terminating the trigger cycle and initiating the bullet catalytic cycle by the SK·Pm complex that converts the residual Pg into Pm. The new assays can be adapted to quantitate SK-Pg activation in the context of SK- or Pg-directed inhibitors, effectors, and SK allelic variants. To support this, we show for the first time with an assay specific for SK·Pg* that fibrinogen forms a ternary SK·Pg*·fibrinogen complex, which assembles with 200-fold enhanced SK·Pg* affinity, signaled by a perturbation of the SK·Pg* active site.

Aim: Study system of activation of plasminogen in Vibrio cholerae., Materials and Methods: 75 strains of V. cholerae of various origins were used in the study. Plasminogen was isolated from human plasma by using affinity chromatography on L-lysine sepharose, alpha-enolase activity was determined by a direct method assuming transformation of 2-phosphoglycerate into phopshoenolpyruvate. Vibrios were destroyed by ultrasound disintegrator to isolate membrane Omp protein, intact cells were discarded by centrifugation and cell lysate was centrifugated for 1 hour at 105000 g. The precipitate was solubilized in buffer with 1% triton X-100 and passed through a column with DE-52 cellulose., Results: Vibrio cholerae O1 and O139 strains isolated from clinical specimens and water samples from open water bodies had the ability to bind by using alpha-enolase and transform human plasminogen into plasmin under the effect of outer membrane protein OmpT A protein with molecular weight around 40 kDa had proteolytic activity with a wide specter of substrate specificity, degraded fibrin, gelatin, collagen, protamine and activated plasminogen. Computer analysis showed that OmpT protein of cholera vibrion had a low degree of relation with Enterobacteriaceae omptins., Conclusion: The study carried out showed that vibrios have a system of activation of plasminogen that includes at least alpha-enolase and OmpT membrane protein. OmpT protein is assumed to belong to a new class of porins of Vibrionaceae family and its enzymatic activity may play a significant role in pathogenesis of infection.

The Lyme disease spirochete Borrelia burgdorferi lacks endogenous, surface-exposed proteases. In order to efficiently disseminate throughout the host and penetrate tissue barriers, borreliae rely on recruitment of host proteases, such as plasmin(ogen). Here we report the identification of a novel plasminogen-binding protein, BBA70. Binding of plasminogen is dose-dependent and is affected by ionic strength. The BBA70-plasminogen interaction is mediated by lysine residues, primarily located in a putative C-terminal α-helix of BBA70. These lysine residues appear to interact with the lysine-binding sites in plasminogen kringle domain 4 because a deletion mutant of plasminogen lacking that domain was unable to bind to BBA70. Bound to BBA70, plasminogen activated by urokinase-type plasminogen activator was able to degrade both a synthetic chromogenic substrate and the natural substrate fibrinogen. Furthermore, BBA70-bound plasmin was able to degrade the central complement proteins C3b and C5 and inhibited the bacteriolytic effects of complement. Consistent with these functional activities, BBA70 is located on the borrelial outer surface. Additionally, serological evidence demonstrated that BBA70 is produced during mammalian infection. Taken together, recruitment and activation of plasminogen could play a beneficial role in dissemination of B. burgdorferi in the human host and may possibly aid the spirochete in escaping the defense mechanisms of innate immunity.

Yersinia pestis protein Pla is a plasmid-coded outer membrane protein with aspartic-protease activity. Pla exhibits a plasminogen (Plg) activator activity (PAA) that promotes the cleavage of Plg to the active serine-protease form called plasmin. Exactly how Pla activates Plg into plasmin remains unclear. To investigate this event, we performed the interactions between the predicted Plg and Pla protein structures by rigid-body docking with the HEX program and evaluated the complex stability by molecular dynamics (MD) using the GROMACS package programs. The predicted docked complex of Plg-Pla shows the same interaction site predicted by experimental site-direct mutagenesis in other studies. After a total of 8 ns of MD simulation, we observed the relaxation of the beta-barrel structure of Pla and the progressive approximation and stabilization between the cleavage site of Plg into the extracellular loops of Pla, followed by the increase in the number of H bonds. We also report here the aminoacids that participate in the active site and the sub sites of interaction. The total understanding of these interactions can be an important tool for drug design against bacterial proteases.

Phosphoglycerate kinase (PGK), present on the surface of group B streptococcus (GBS), has previously been demonstrated to bind the host proteins actin and plasminogen. The actin and plasminogen binding sites of GBS-PGK were identified using truncated GBS-PGK molecules, followed by peptide mapping. These experiments identified two actin and plasminogen binding sites located between amino acids 126-134 and 204-208 of the 398-amino acid-long GBS-PGK molecule. Substitution of the lysine residues within these regions with alanine resulted in significantly reduced binding to both actin and plasminogen. In addition, conversion of the glutamic acid residue at amino acid 133 to proline, the amino acid found at this position for the PGK protein of Streptococcus pneumoniae, also resulted in significantly reduced binding to actin and plasminogen. These results demonstrate that the lysine residues at amino acid positions 126, 127, 130, 204, and 208 along with the glutamic acid residue at amino acid position 133 are necessary for actin and plasminogen binding by GBS-PGK.

Omptins constitute a unique family of outer membrane proteases that are widespread in Enterobacteriaceae. The plasminogen activator (Pla) of Yersinia pestis is an omptin family member that is very important for development of both bubonic and pneumonic plague. The physiological function of Pla is to cleave (activate) human plasminogen to form the plasma protease plasmin. Uniquely, lipopolysaccharide (LPS) is essential for the catalytic activity of all omptins, including Pla. Why omptins require LPS for enzymatic activity is unknown. Here, we report the co-crystal structure of LPS-free Pla in complex with the activation loop peptide of human plasminogen, its natural substrate. The structure shows that in the absence of LPS, the peptide substrate binds deep within the active site groove and displaces the nucleophilic water molecule, providing an explanation for the dependence of omptins on LPS for enzymatic activity.

Bacterial plasminogen (Pg) activators generate plasmin to degrade fibrin blood clots and other proteins that modulate the pathogenesis of infection, yet despite strong homology between mammalian Pgs, the activity of bacterial Pg activators is thought to be restricted to the Pg of their host mammalian species. Thus, we found that Streptococcus uberis Pg activator (SUPA), isolated from a Streptococcus species that infects cows but not humans, robustly activated bovine but not human Pg in purified systems and in plasma. Consistent with this, SUPA formed a higher avidity complex (118-fold) with bovine Pg than with human Pg and non-proteolytically activated bovine but not human Pg. Surprisingly, however, the presence of human fibrin overrides the species-restricted action of SUPA. First, human fibrin enhanced the binding avidity of SUPA for human Pg by 4-8-fold in the presence and absence of chloride ion (a negative regulator). Second, although SUPA did not protect plasmin from inactivation by α(2)-antiplasmin, fibrin did protect human plasmin, which formed a 31-fold higher avidity complex with SUPA than Pg. Third, fibrin significantly enhanced Pg activation by reducing the K(m) (4-fold) and improving the catalytic efficiency of the SUPA complex (6-fold). Taken together, these data suggest that indirect molecular interactions may override the species-restricted activity of bacterial Pg activators; this may affect the pathogenesis of infections or may be exploited to facilitate the design of new blood clot-dissolving drugs.

In vertebrates, fibrinolysis is primarily carried out by the serine protease plasmin (Pm), which is derived from activation of the zymogen precursor, plasminogen (Pg). One of the most distinctive features of Pg/Pm is the presence of five homologous kringle (K) domains. These structural elements possess conserved Lys-binding sites (LBS) that facilitate interactions with substrates, activators, inhibitors and receptors. In human Pg (hPg), K2 displays weak Lys affinity, however the LBS of this domain has been implicated in an atypical interaction with the N-terminal region of a bacterial surface protein known as PAM (Pg-binding group A streptococcal M-like protein). A direct correlation has been established between invasiveness of group A streptococci and their ability to bind Pg. It has been previously demonstrated that a 30-residue internal peptide (VEK-30) from the N-terminal region of PAM competitively inhibits binding of the full-length parent protein to Pg. We have attempted to determine the effects of this ligand-protein interaction on the regulation of Pg zymogen activation and conformation. Our results show minimal effects on the sedimentation velocity coefficients (S degrees (20,w)) of Pg when associated to VEK-30 and a direct relationship between the concentration of VEK-30 or PAM and the activation rate of Pg. These results are in contrast with the major conformational changes elicited by small-molecule activators of Pg, and point towards a novel mechanism of Pg activation that may underlie group A streptococcal (GAS) virulence., (Copyright 2010 Elsevier B.V. All rights reserved.)

The solution structure of the complex containing the isolated kringle 2 domain of human plasminogen (K2(Pg)) and VEK-30, a 30-amino acid residue internal peptide from a streptococcal M-like plasminogen (Pg) binding protein (PAM), has been determined by multinuclear high-resolution NMR. Complete backbone and side-chain assignments were obtained from triple-resonance experiments, after which structure calculations were performed and ultimately refined by restrained molecular simulation in water. We find that, in contrast with the dimer of complexes observed in the asymmetric unit of the crystal, global correlation times and buoyant molecular weight determinations of the complex and its individual components showed the monomeric nature of all species in solution. The NMR-derived structure of K2(Pg) in complex with VEK-30 presents a folding pattern typical of other kringle domains, while bound VEK-30 forms an end-to-end alpha-helix (residues 6-27) in the complex. Most of the VEK-30/K2(Pg) interactions in solution occur between a single face of the alpha-helix of VEK-30 and the lysine binding site (LBS) of K2(Pg). The canonical LBS of K2(Pg), consisting of Asp54, Asp56, Trp60, Arg69, and Trp70 (kringle numbering), interacts with an internal pseudo-lysine of VEK-30, comprising side-chains of Arg17, His18, and Glu20. Site-specific mutagenesis analysis confirmed that the electrostatic field formed by the N-terminal anionic residues of the VEK-30 alpha-helix, viz., Asp7, and the non-conserved cationic residues of K2(Pg), viz., Lys43 and Arg55, play additional important roles in the docking of VEK-30 to K2(Pg). Structural analysis and kringle sequence alignments revealed several important features related to exosite binding that provide a structural rationale for the high specificity and affinity of VEK-30 for K2(Pg)., ((c) 2009 Elsevier Inc. All rights reserved.)

The characteristics of a streptococcal plasminogen activator (PA) displaying specificity for ruminant plasminogen (Plg) were defined using molecular approaches. The 16-kDa secreted protein PadA was found to be prevalent in Streptococcus dysgalactiae subspecies dysgalactiae isolated from cases of bovine mastitis and septic arthritis in lambs. PadA was able to activate bovine, ovine and caprine Plg, but not human Plg. Amino acid sequence analysis identified a limited level of homology to other streptococcal PAs, including streptokinase; however, PadA was found to align well with and match in size the staphylococcal PA, staphylokinase. Recombinant PadA was used to investigate interaction with bovine Plg, leading to formation of an activator complex that was capable of recruiting and converting further substrate Plg into plasmin. Individual non-overlapping peptides of PadA or bovine microplasminogen were found to block the interaction between PadA and bovine Plg, preventing the formation of the activation complex. Homology modelling based upon structures of staphylokinase complexed with human microplasminogen supported these findings by placing critical residues in close proximity to the plasmin component of the activation complex.

VEK-30, a 30-amino acid internal peptide present within a streptococcal M-like plasminogen (Pg)-binding protein (PAM) from Gram-positive group-A streptococci (GAS), represents an epitope within PAM that shows high affinity for the lysine binding site (LBS) of the kringle-2 (K2) domain of human (h)Pg. VEK-30 does not interact with this same region of mouse (m)Pg, despite the high conservation of the mK2- and hK2-LBS. To identify the molecular basis for the species specificity of this interaction, hPg and mPg variants were generated, including an hPg chimera with the mK2 sequence and an mPg chimera containing the hK2 sequence. The binding of synthetic VEK-30 to these variants was studied by surface plasmon resonance. The data revealed that, in otherwise intact Pg, the species specificity of VEK-30 binding in these two cases is entirely dictated by two K2 residues that are different between hPg and mPg, namely, Arg-220 of hPg, which is a Gly in mPg, and Leu-222 of hPg, which is a Pro in mPg, neither of which are members of the canonical K2-LBS. Neither the activation of hPg, nor the enzymatic activity of its activated product, plasmin (hPm), are compromised by replacing these two amino acids by their murine counterparts. It is also demonstrated that hPg is more susceptible to activation to hPm after complexation with VEK-30 and that this property is greatly reduced as a result of the R220G and L222P replacements in hPg. These mechanisms for accumulation of protease activity on GAS likely contribute to the virulence of PAM(+)-GAS strains and identify targets for new therapeutic interventions.

The surface subproteome of Listeria monocytogenes that includes many proteins already known to be involved in virulence and interaction with host cells has been characterized. A new method for the isolation of a defined surface proteome of low complexity has been established based on serial extraction of proteins by different salts at high concentration, and in all 55 proteins were identified by N-terminal sequencing and mass spectrometry. About 16% of these proteins are of unknown function and three proteins have no orthologue in the nonpathogenic L. innocua and might be involved in virulence mechanisms. Remarkably, a relatively high number of proteins with a function in the cytoplasmic compartment was identified in this surface proteome. These proteins had neither predicted or detectable signal peptides nor could any modification be observed except removal of the N-terminal methionine. Enolase (Lmo2455) is one of these proteins. It was shown to be present in the cell wall of the pathogen by immunoelectron microscopy and, along with heat shock factor DnaK (Lmo1473), elongation factor TU (Lmo2653), and glyceraldehyde-3-phosphate dehydrogenase (Lmo2459), it was found to be able to bind human plasminogen in overlay blots and surface plasmon resonance (SPR) experiments. The KD values of these interactions were determined by SPR measurements. The data indicate a possible role of these proteins as receptors for human plasminogen on the bacterial cell surface. The potential role of this recruitment of a host protease for extracellular invasion mechanisms is discussed.

The determinants of binding of a peptide lacking C-termini-exposed lysine residues to a kringle domain were investigated using an up-regulated lysine binding kringle (K2Pg[C4G/E56D/K72Y]) of plasminogen and a peptide (a1-PAM) with a sequence derived from a surface-exposed M-like streptococcal protein. Significant kringle-induced chemical shifts in a His side-chain of a1-PAM were revealed by two-dimensional NMR. Further studies using isothermal titration calorimetry (ITC) provided support for the involvement of His12 in the peptide/ protein complex. In an effort to screen a1-PAM-derived truncation peptides, a combinatorial mixture, a1deltaa2-PAM[H12X] (where X=Pro, Arg, His, Trp, Lys, Ala, Phe, Asp and Gly), was analyzed using the surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI) platform. The major peptide that remained bound to the surface of the K2Pg[C4G/ E56D/K72Y]-containing chip was that containing His12, corresponding to the wild-type sequence. Minor peaks, representing binding, were obtained for Lys12-, Arg12- and Trp12-containing peptides. Individual peptides containing these amino acids were then examined using ITC and the binding constants obtained correlated with the relative strengths of binding estimated from the SELDI-based screen.

A novel single polypeptide endopeptidase of 24 kDa (24k-endopeptidase) was purified with a yield of 300-400 microg/L from conditioned medium of a bacterial strain which was identified as a new species in the genus Chryseobacterium Sp. on the basis of its 16S rDNA sequence and DNA:DNA hybridizations. The NH(2)-terminal amino acid sequence (Val-Ala-Thr-Pro-Asn-Leu-Glu-.) was not found in the availabe databases. The 24k-endopeptidase specifically hydrolyzed the Ser(441)-Val(442) peptide bond in human plasmin(ogen), with additional cleavage of the Lys(78)-Val(79) and Pro(447)-Val(448) peptide bonds, and a secondary cleavage at Lys(615)-Val(616). Thereby, plasminogen is converted into an angiostatin-like fragment containing kringles 1-4 (K1-4) and miniplasminogen (kringle 5 and the serine proteinase domain). The purified K1-4 fragment showed a comparable cytotoxicity toward endothelial cells as the elastase-derived K1-3 fragment (12.7% versus 10.6% at a concentration of 10 microg/mL). Plasminogen, bound to monocytoid THP-1 cells, was also cleaved by the 24k-endopeptidase, resulting in generation of an angiostatin-like fragment and in a decreased capacity to generate cell-associated plasmin following activation by urokinase. The 24k-endopeptidase was not efficiently neutralized by specific inhibitors against the serine, cysteine, aspartic, or matrix metalloproteinase classes of enzymes. In human plasma or serum, however, it induced only very limited plasminogen degradation, apparently due to neutralization of its activity by alpha(2)-macroglobulin. Interaction of this novel 24k-endopeptidase with plasminogen thus yields an angiostatin-like fragment and affects plasmin-mediated cellular proteolytic activity.

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 x 10(7) M-1, as determined by surface plasmon resonance, a value close to that (1.6 x 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.