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Jason D Mcarthur - One of the best experts on this subject based on the ideXlab platform.

  • site restricted Plasminogen Activation mediated by group a streptococcal streptokinase variants
    Biochemical Journal, 2014
    Co-Authors: Simon M Cook, Amanda Skora, Mark Walker, Martina L Sandersonsmith, Jason D Mcarthur
    Abstract:

    SK (streptokinase) is a secreted Plasminogen activator and virulence factor of GAS (group A Streptococcus). Among GAS isolates, SK gene sequences are polymorphic and are grouped into two sequence clusters (cluster type-1 and cluster type-2) with cluster type-2 being further classified into subclusters (type-2a and type-2b). In the present study, we examined the role of bacterial and host-derived cofactors in SK-mediated Plasminogen Activation. All SK variants, apart from type-2b, can form an activator complex with Glu-Plg (Glu-Plasminogen). Specific ligand-binding-induced conformational changes in Glu-Plg mediated by fibrinogen, PAM (Plasminogen-binding group A streptococcal M protein), fibrinogen fragment D or fibrin, were required for type-2b SK to form a functional activator complex with Glu-Plg. In contrast with type-1 and type-2a SK, type-2b SK activator complexes were inhibited by α2-antiplasmin unless bound to fibrin or to the GAS cell-surface via PAM in combination with fibrinogen. Taken together, these data suggest that type-2b SK Plasminogen Activation may be restricted to specific microenvironments within the host such as fibrin deposits or the bacterial cell surface through the action of α2-antiplasmin. We conclude that phenotypic SK variation functionally underpins a pathogenic mechanism whereby SK variants differentially focus Plasminogen Activation, leading to specific niche adaption within the host.

  • streptokinase variants from streptococcus pyogenes isolates display altered Plasminogen Activation characteristics implications for pathogenesis
    Molecular Microbiology, 2012
    Co-Authors: Simon M Cook, Amanda Skora, Mark Walker, Christine M Gillen, Jason D Mcarthur
    Abstract:

    Streptococcus pyogenes (group A streptococcus, GAS) secretes streptokinase, a potent Plasminogen activating protein. Among GAS isolates, streptokinase gene sequences (ska) are polymorphic and can be grouped into two distinct sequence clusters (termed cluster type-1 and cluster type-2) with cluster type-2 being further divided into sub-clusters type-2a and type-2b. In this study, far-UV circular dichroism spectroscopy indicated that purified streptokinase variants of each type displayed similar secondary structure. Type-2b streptokinase variants could not generate an active site in Glu-Plasminogen through non-proteolytic mechanisms while all other variants had this capability. Furthermore, when compared with other streptokinase variants, type-2b variants displayed a 29- to 35-fold reduction in affinity for Glu-Plasminogen. All SK variants could activate Glu-Plasminogen when an activator complex was preformed with plasmin; however, type-2b and type-1 complexes were inhibited by a2-antiplasmin. Exchanging skatype-2a in the M1T1 GAS strain 5448 with skatype-2b caused a reduction in virulence while exchanging skatype-2a with skatype-1 into 5448 produced an increase in virulence when using a mouse model of invasive disease. These findings suggest that streptokinase variants produced by GAS isolates utilize distinct Plasminogen Activation pathways, which directly affects the pathogenesis of this organism.

  • allelic variants of streptokinase from streptococcus pyogenes display functional differences in Plasminogen Activation
    The FASEB Journal, 2008
    Co-Authors: Jason D Mcarthur, Martina L Sandersonsmith, Fiona C Mckay, Vidiya Ramachandran, Priya Shyam, Amanda J Cork, Jason N Cole, Ulrika Ringdahl, Ulf Sjobring, Marie Ranson
    Abstract:

    A common mammalian defense mechanism employed to prevent systemic dissemination of invasive bacteria involves occlusion of local microvasculature and encapsulation of bacteria within fibrin networks. Acquisition of plasmin activity at the bacterial cell surface circumvents this defense mechanism, allowing invasive disease initiation. To facilitate this process, S. pyogenes secretes streptokinase, a Plasminogen-activating protein. Streptokinase polymorphism exhibited by S. pyogenes isolates is well characterized. However, the functional differences displayed by these variants and the biological significance of this variation has not been elucidated. Phylogenetic analysis of ska sequences from 28 S. pyogenes isolates revealed 2 main sequence clusters (clusters 1 and 2). All strains secreted streptokinase, as determined by Western blotting, and were capable of acquiring cell surface plasmin activity after incubation in human plasma. Whereas culture supernatants from strains containing cluster 1 ska alleles also displayed soluble Plasminogen Activation activity, supernatants from strains containing cluster 2 ska alleles did not. Furthermore, Plasminogen Activation activity in culture supernatants from strains containing cluster 2 ska alleles could only be detected when Plasminogen was prebound with fibrinogen. This study indicates that variant streptokinase proteins secreted by S. pyogenes isolates display differing Plasminogen Activation characteristics and may therefore play distinct roles in disease pathogenesis.

Vincent Ellis - One of the best experts on this subject based on the ideXlab platform.

  • functional regulation of tissue Plasminogen activator on the surface of vascular smooth muscle cells by the type ii transmembrane protein p63 ckap4
    Journal of Biological Chemistry, 2003
    Co-Authors: Tahir M Razzaq, Rosemary Bass, David J Vines, Simon A. Whawell, Finn Werner, Vincent Ellis
    Abstract:

    Abstract We have demonstrated that tissue Plasminogen activator (tPA) binds specifically to human vascular smooth muscle cells (VSMC) in a functionally relevant manner, both increasing Plasminogen Activation and decreasing tPA inhibition (Ellis, V., and Whawell, S. A. (1997) Blood 90, 2312-2322; Werner, F., Razzaq, T. M., and Ellis, V. (1999) J. Biol. Chem. 274, 21555-21561). To further understand this system we have now identified and characterized the protein responsible for this binding. Rat VSMC were surface-labeled with 125I, and cell lysates were subjected to an affinity chromatography scheme based on the previously identified tPA binding characteristics. A single radiolabeled protein of 63 kDa bound specifically and was eluted at low pH. This protein was isolated from large scale preparations of VSMC and unambiguously identified as the rat homologue of the human type-II transmembrane protein p63 (CKAP4) by matrix-assisted laser desorption ionization and nano-electrospray tandem mass spectrometry of tryptic fragments. In confirmation of this, a monoclonal antibody raised against authentic human p63 recognized the isolated protein in Western blotting. Immunofluorescence microscopy demonstrated that p63 was located principally in the endoplasmic reticulum but was also detected in significant quantities on the surface of human VSMC. In support of the hypothesis that p63 is the functional tPA binding site on VSMC, an anti-p63 monoclonal antibody was found to block tPA binding. Furthermore, heterologous expression of an N-terminally truncated mutant of p63, which targets exclusively to the plasma membrane, led to an increase in tPA-catalyzed Plasminogen Activation. Therefore, p63 on the surface of VSMC may contribute to the functional regulation of the Plasminogen Activation system in the vessel wall.

  • functional regulation of tissue Plasminogen activator on the surface of vascular smooth muscle cells by the type ii
    2003
    Co-Authors: Tahir M Razzaq, Rosemary Bass, David J Vines, Simon A. Whawell, Finn Werner, Vincent Ellis
    Abstract:

    We have demonstrated that tissue Plasminogen activator (tPA) binds specifically to human vascular smooth muscle cells (VSMC) in a functionally relevant manner, both increasing Plasminogen Activation and decreasing tPA inhibition (Ellis, V., and Whawell, S. A. (1997) Blood 90, 2312–2322; Werner, F., Razzaq, T. M., and Ellis, V. (1999) J. Biol. Chem. 274, 21555–21561). To further understand this system we have now identified and characterized the protein responsible for this binding. Rat VSMC were surface-labeled with 125 I, and cell lysates were subjected to an affinity chromatography scheme based on the previously identified tPA binding characteristics. A single radiolabeled protein of 63 kDa bound specifically and was eluted at low pH. This protein was isolated from large scale preparations of VSMC and unambiguously identified as the rat homologue of the human type-II transmembrane protein p63 (CKAP4) by matrix-assisted laser desorption ionization and nano-electrospray tandem mass spectrometry of tryptic fragments. In confirmation of this, a monoclonal antibody raised against authentic human p63 recognized the isolated protein in Western blotting. Immunofluorescence microscopy demonstrated that p63 was located principally in the endoplasmic reticulum but was also detected in significant quantities on the surface of human VSMC. In support of the hypothesis that p63 is the functional tPA binding site on VSMC, an anti-p63 monoclonal antibody was found to block tPA binding. Furthermore, heterologous expression of an N-terminally truncated mutant of p63, which targets exclusively to the plasma membrane, led to an increase in tPA-catalyzed Plasminogen Activation. Therefore, p63 on the surface of VSMC may contribute to the functional regulation of the Plasminogen Activation system in the vessel wall. Vascular smooth muscle cells (VSMC) 1 play a key role in the

  • cell induced potentiation of the Plasminogen Activation system is abolished by a monoclonal antibody that recognizes the nh2 terminal domain of the urokinase receptor
    FEBS Letters, 1991
    Co-Authors: Ebbe Ronne, Vincent Ellis, Michael Ploug, Niels Behrendt, Keld Dano, Gunilla Hoyerhansen
    Abstract:

    We have raised four monoclonal antibodies recognizing different epitopes within the human cell-surface receptor for urokinase-type Plasminogen activator (u-PA). One of these antibodies completely abolishes the potentiation of plasmin generation observed upon incubation of the zymogens pro-u-PA and Plasminogen with U937 cells. This antibody, which is also the only one to completely inhibit the binding of DFP-inactivated [125I]-u-PA to U937 cells, is directed against the u-PA binding NH2-terminal domain of u-PAR, a well-defined fragment formed by limited chymotrypsin digestion of purified u-PAR, demonstrating the functional independence of the u-PA binding domain as well as the critical role of u-PAR in the assembly of the cell-surface Plasminogen Activation system.

Peter A. Andreasen - One of the best experts on this subject based on the ideXlab platform.

  • origin and diversification of the Plasminogen Activation system among chordates
    BMC Evolutionary Biology, 2019
    Co-Authors: Andres Chanamunoz, Peter A. Andreasen, Agnieszka Jendroszek, Malene Sønnichsen, Tobias Wang, Michael Ploug, Christian Bendixen, Jan K. Jensen, Frank Panitz
    Abstract:

    The Plasminogen (PLG) Activation system is composed by a series of serine proteases, inhibitors and several binding proteins, which together control the temporal and spatial generation of the active serine protease plasmin. As this proteolytic system plays a central role in human physiology and pathophysiology it has been extensively studied in mammals. The serine proteases of this system are believed to originate from an ancestral gene by gene duplications followed by domain gains and deletions. However, the identification of ancestral forms in primitive chordates supporting these theories remains elusive. In addition, evolutionary studies of the non-proteolytic members of this system are scarce. Our phylogenetic analyses place lamprey PLG at the root of the vertebrate PLG-group, while lamprey PLG-related growth factors represent the ancestral forms of the jawed-vertebrate orthologues. Furthermore, we find that the earliest putative orthologue of the PLG activator group is the hyaluronan binding protein 2 (HABP2) gene found in lampreys. The prime Plasminogen activators (tissue- and urokinase-type Plasminogen activator, tPA and uPA) first occur in cartilaginous fish and phylogenetic analyses confirm that all orthologues identified compose monophyletic groups to their mammalian counterparts. Cartilaginous fishes exhibit the most ancient vitronectin of all vertebrates, while Plasminogen activator inhibitor 1 (PAI-1) appears for the first time in cartilaginous fishes and is conserved in the rest of jawed vertebrate clades. PAI-2 appears for the first time in the common ancestor of reptiles and mammals, and represents the latest appearing Plasminogen activator inhibitor. Finally, we noted that the urokinase-type Plasminogen activator receptor (uPAR)—and three-LU domain containing genes in general—occurred later in evolution and was first detectable after coelacanths. This study identifies several primitive orthologues of the mammalian Plasminogen Activation system. These ancestral forms provide clues to the origin and diversification of this enzyme system. Further, the discovery of several members—hitherto unknown in mammals—provide new perspectives on the evolution of this important enzyme system.

  • Origin and diversification of the Plasminogen Activation system among chordates
    BMC, 2019
    Co-Authors: Andrés Chana-muñoz, Peter A. Andreasen, Agnieszka Jendroszek, Malene Sønnichsen, Tobias Wang, Michael Ploug, Christian Bendixen, Jan K. Jensen, Frank Panitz
    Abstract:

    Abstract Background The Plasminogen (PLG) Activation system is composed by a series of serine proteases, inhibitors and several binding proteins, which together control the temporal and spatial generation of the active serine protease plasmin. As this proteolytic system plays a central role in human physiology and pathophysiology it has been extensively studied in mammals. The serine proteases of this system are believed to originate from an ancestral gene by gene duplications followed by domain gains and deletions. However, the identification of ancestral forms in primitive chordates supporting these theories remains elusive. In addition, evolutionary studies of the non-proteolytic members of this system are scarce. Results Our phylogenetic analyses place lamprey PLG at the root of the vertebrate PLG-group, while lamprey PLG-related growth factors represent the ancestral forms of the jawed-vertebrate orthologues. Furthermore, we find that the earliest putative orthologue of the PLG activator group is the hyaluronan binding protein 2 (HABP2) gene found in lampreys. The prime Plasminogen activators (tissue- and urokinase-type Plasminogen activator, tPA and uPA) first occur in cartilaginous fish and phylogenetic analyses confirm that all orthologues identified compose monophyletic groups to their mammalian counterparts. Cartilaginous fishes exhibit the most ancient vitronectin of all vertebrates, while Plasminogen activator inhibitor 1 (PAI-1) appears for the first time in cartilaginous fishes and is conserved in the rest of jawed vertebrate clades. PAI-2 appears for the first time in the common ancestor of reptiles and mammals, and represents the latest appearing Plasminogen activator inhibitor. Finally, we noted that the urokinase-type Plasminogen activator receptor (uPAR)—and three-LU domain containing genes in general—occurred later in evolution and was first detectable after coelacanths. Conclusions This study identifies several primitive orthologues of the mammalian Plasminogen Activation system. These ancestral forms provide clues to the origin and diversification of this enzyme system. Further, the discovery of several members—hitherto unknown in mammals—provide new perspectives on the evolution of this important enzyme system

  • ligand binding modulates the structural dynamics and activity of urokinase type Plasminogen activator a possible mechanism of Plasminogen Activation
    PLOS ONE, 2018
    Co-Authors: Tobias Kromannhansen, Peter A. Andreasen, Gunilla Hoyerhansen, Eva Louise Lange, Ida K Lund, Elizabeth A Komives
    Abstract:

    The catalytic activity of trypsin-like serine proteases is in many cases regulated by conformational changes initiated by binding of physiological modulators to exosites located distantly from the active site. A trypsin-like serine protease of particular interest is urokinase-type Plasminogen activator (uPA), which is involved in extracellular tissue remodeling processes. Herein, we used hydrogen/deuterium exchange mass spectrometry (HDXMS) to study regulation of activity in the catalytic domain of the murine version of uPA (muPA) by two muPA specific monoclonal antibodies. Using a truncated muPA variant (muPA16-243), containing the catalytic domain only, we show that the two monoclonal antibodies, despite binding to an overlapping epitope in the 37s and 70s loops of muPA16-243, stabilize distinct muPA16-243 conformations. Whereas the inhibitory antibody, mU1 was found to increase the conformational flexibility of muPA16-243, the stimulatory antibody, mU3, decreased muPA16-243 conformational flexibility. Furthermore, the HDXMS data unveil the existence of a pathway connecting the 70s loop to the active site region. Using alanine scanning mutagenesis, we further identify the 70s loop as an important exosite for the Activation of the physiological uPA substrate Plasminogen. Thus, the data presented here reveal important information about dynamics in uPA by demonstrating how various ligands can modulate uPA activity by mediating long-range conformational changes. Moreover, the results provide a possible mechanism of Plasminogen Activation.

  • Urokinase links Plasminogen Activation and cell adhesion by cleavage of the RGD motif in vitronectin
    EMBO reports, 2016
    Co-Authors: Valentina De Lorenzi, Peter A. Andreasen, Gian Maria Sarra Ferraris, Jeppe B. Madsen, Michela Lupia, Nicolai Sidenius
    Abstract:

    Components of the Plasminogen Activation system including urokinase (uPA), its inhibitor (PAI‐1) and its cell surface receptor (uPAR) have been implicated in a wide variety of biological processes related to tissue homoeostasis. Firstly, the binding of uPA to uPAR favours extracellular proteolysis by enhancing cell surface Plasminogen Activation. Secondly, it promotes cell adhesion and signalling through binding of the provisional matrix protein vitronectin. We now report that uPA and plasmin induces a potent negative feedback on cell adhesion through specific cleavage of the RGD motif in vitronectin. Cleavage of vitronectin by uPA displays a remarkable receptor dependence and requires concomitant binding of both uPA and vitronectin to uPAR. Moreover, we show that PAI‐1 counteracts the negative feedback and behaves as a proteolysis‐triggered stabilizer of uPAR‐mediated cell adhesion to vitronectin. These findings identify a novel and highly specific function for the Plasminogen Activation system in the regulation of cell adhesion to vitronectin. The cleavage of vitronectin by uPA and plasmin results in the release of N‐terminal vitronectin fragments that can be detected in vivo , underscoring the potential physiological relevance of the process. ![][1] This study identifies a novel function for the Plasminogen Activation cascade in the regulation of cell adhesion. Urokinase and plasmin cleave the RGD motif of vitronectin in a process that is accelerated by the urokinase receptor and counteracted by the Plasminogen activator inhibitor‐1. [1]: /embed/graphic-1.gif

  • the Plasminogen Activation system in tumor growth invasion and metastasis
    Cellular and Molecular Life Sciences, 2000
    Co-Authors: Peter A. Andreasen, Rikke Egelund, Helle H Petersen
    Abstract:

    Generation of the serine proteinase plasmin from the extracellular zymogen Plasminogen can be catalyzed by either of two other serine proteinases, the urokinase- and tissue-type Plasminogen activators (uPA and tPA). The Plasminogen Activation system also includes the serpins PAI-1 and PAI-2, and the uPA receptor (uPAR). Many findings, gathered over several decades, strongly suggest an important and causal role for uPA-catalyzed plasmin generation in cancer cell invasion through the extracellular matrix. Recent evidence suggests that the uPA system is also involved in cancer cell-directed tissue remodeling. Moreover, the system also supports cell migration and invasion by plasmin-independent mechanisms, including multiple interactions between uPA, uPAR, PAI-1, extracellular matrix proteins, integrins, endocytosis receptors, and growth factors. These interactions seem to allow temporal and spatial reorganizations of the system during cell migration and a selective degradation of extracellular matrix proteins during invasion. The increased knowledge about the Plasminogen Activation system may allow utilization of its components as targets for anti-invasive therapy.

H. Roger Lijnen - One of the best experts on this subject based on the ideXlab platform.

  • Plasminogen Activation: a mediator of vascular smooth muscle cell apoptosis in atherosclerotic plaques.
    Journal of Thrombosis and Haemostasis, 2006
    Co-Authors: Patrick Rossignol, Desire Collen, Aernout Luttun, Jose Luis Martin-ventura, Florea Lupu, Peter Carmeliet, Eduardo Anglès-cano, H. Roger Lijnen
    Abstract:

    BACKGROUND: Apoptosis of vascular cells is considered to be a major determinant of atherosclerotic plaque vulnerability and potential rupture. Plasmin can be generated in atherosclerotic plaques and recent in vitro data suggest that Plasminogen Activation may trigger vascular smooth muscle cell (VSMC) apoptosis. AIM: To determine whether Plasminogen Activation may induce aortic VSMC apoptosis ex vivo and in vivo. METHODS AND RESULTS: Mice with single or combined deficiencies of apolipoprotein E (ApoE) and Plasminogen activator inhibitor-1 (PAI-1) were used. Ex vivo incubation with Plasminogen of isolated aortic tunica media from PAI-1-deficient mice induced Plasminogen Activation and VSMC apoptosis, which was inhibited by alpha2-antiplasmin. In vivo, levels of plasmin, active caspase 3 and VSMC apoptotic index were significantly higher in atherosclerotic aortas from mice with combined ApoE and PAI-1 deficiencies than in those from littermates with single ApoE deficiency. A parallel decrease in VSMC density was observed. CONCLUSIONS: These data strongly suggest that Plasminogen Activation may contribute to VSMC apoptosis in atherosclerotic plaques.

  • on the mechanism of fibrin specific Plasminogen Activation by staphylokinase
    Journal of Biological Chemistry, 1991
    Co-Authors: H. Roger Lijnen, Berthe Van Hoef, Kiyotaka Okada, Osamu Matsuo, Shigeru Ueshima, F De Cock, Desire Collen
    Abstract:

    Abstract The mechanism of Plasminogen Activation by recombinant staphylokinase was studied both in the absence and in the presence of fibrin, in purified systems, and in human plasma. Staphylokinase, like streptokinase, forms a stoichiometric complex with Plasminogen that activates Plasminogen following Michaelis-Menten kinetics with Km = 7.0 microM and k2 = 1.5 s-1. In purified systems, alpha 2-antiplasmin inhibits the Plasminogen-staphylokinase complex with k1(app) = 2.7 +/- 0.30 x 10(6) M-1 s-1 (mean +/- S.D., n = 12), but not the Plasminogen-streptokinase complex. Addition of 6-aminohexanoic acid induces a concentration-dependent reduction of k1(app) to 2.0 +/- 0.17 x 10(4) M-1 s-1 (mean +/- S.D., n = 5) at concentrations greater than or equal to 30 mM, with a 50% reduction at a 6-aminohexanoic acid concentration of 60 microM. Staphylokinase does not bind to fibrin, and fibrin stimulates the initial rate of Plasminogen Activation by staphylokinase only 4-fold. Staphylokinase induces a dose-dependent lysis of a 0.12-ml 125I-fibrin-labeled human plasma clot submersed in 0.5 ml of citrated human plasma; 50% lysis in 2 h is obtained with 17 nM staphylokinase and is associated with only 5% plasma fibrinogen degradation. Corresponding values for streptokinase are 68 nM and more than 90% fibrinogen degradation. In the absence of a fibrin clot, 50% fibrinogen degradation in human plasma in 2 h requires 790 nM staphylokinase, but only 4.4 nM streptokinase. These results suggest the following mechanism for relatively fibrin-specific clot lysis with staphylokinase in a plasma milieu. In plasma in the absence of fibrin, the Plasminogen-staphylokinase complex is rapidly neutralized by alpha 2-antiplasmin, thus preventing systemic Plasminogen Activation. In the presence of fibrin, the lysine-binding sites of the Plasminogen-staphylokinase complex are occupied and inhibition by alpha 2-antiplasmin is retarded, thus allowing preferential Plasminogen Activation at the fibrin surface.

Timo K Korhonen - One of the best experts on this subject based on the ideXlab platform.

  • lack of o antigen is essential for Plasminogen Activation by yersinia pestis and salmonella enterica
    Molecular Microbiology, 2003
    Co-Authors: Maini Kukkonen, Ilkka M. Helander, Marjo Suomalainen, Paivi Kyllonen, Kaarina Lahteenmaki, Hannu Lang, Ritva Virkola, Otto Holst, Timo K Korhonen
    Abstract:

    Summary The O-antigen of lipopolysaccharide (LPS) is a virulence factor in enterobacterial infections, and the advantage of its genetic loss in the lethal pathogen Yersinia pestis has remained unresolved. Y. pestis and Salmonella enterica express β-barrel surface proteases of the omptin family that activate human Plasminogen. Plasminogen Activation is central in pathogenesis of plague but has not, however, been found to be important in diarrhoeal disease. We observed that the presence of O-antigen repeats on wild-type or recombinant S. enterica, Yersinia pseudotuberculosis or Escherichia coli prevents Plasminogen Activation by PgtE of S. enterica and Pla of Y. pestis; the O-antigen did not affect incorporation of the omptins into the bacterial outer membrane. Purified His6-Pla was successfully reconstituted with rough LPS but remained inactive after reconstitution with smooth LPS. Expression of smooth LPS prevented Pla-mediated adhesion of recombinant E. coli to basement membrane as well as invasion into human endothelial cells. Similarly, the presence of an O-antigen prevented PgtE-mediated bacterial adhesion to basement membrane. Substitution of Arg-138 and Arg-171 of the motif for protein binding to lipid A 4′-phosphate abolished proteolytic activity but not membrane translocation of PgtE, indicating dependence of omptin activity on a specific interaction with lipid A. The results suggest that Pla and PgtE require LPS for activity and that the O-antigen sterically prevents recognition of large-molecular-weight substrates. Loss of O-antigen facilitates Pla functions and invasiveness of Y. pestis; on the other hand, smooth LPS renders Plasminogen activator cryptic in S. enterica.

  • lack of o antigen is essential for Plasminogen Activation by yersinia pestis and salmonella enterica
    Molecular Microbiology, 2003
    Co-Authors: Maini Kukkonen, Ilkka M. Helander, Marjo Suomalainen, Paivi Kyllonen, Kaarina Lahteenmaki, Hannu Lang, Ritva Virkola, Otto Holst, Timo K Korhonen
    Abstract:

    The O-antigen of lipopolysaccharide (LPS) is a virulence factor in enterobacterial infections, and the advantage of its genetic loss in the lethal pathogen Yersinia pestis has remained unresolved. Y. pestis and Salmonella enterica express beta-barrel surface proteases of the omptin family that activate human Plasminogen. Plasminogen Activation is central in pathogenesis of plague but has not, however, been found to be important in diarrhoeal disease. We observed that the presence of O-antigen repeats on wild-type or recombinant S. enterica, Yersinia pseudotuberculosis or Escherichia coli prevents Plasminogen Activation by PgtE of S. enterica and Pla of Y. pestis; the O-antigen did not affect incorporation of the omptins into the bacterial outer membrane. Purified His6-Pla was successfully reconstituted with rough LPS but remained inactive after reconstitution with smooth LPS. Expression of smooth LPS prevented Pla-mediated adhesion of recombinant E. coli to basement membrane as well as invasion into human endothelial cells. Similarly, the presence of an O-antigen prevented PgtE-mediated bacterial adhesion to basement membrane. Substitution of Arg-138 and Arg-171 of the motif for protein binding to lipid A 4'-phosphate abolished proteolytic activity but not membrane translocation of PgtE, indicating dependence of omptin activity on a specific interaction with lipid A. The results suggest that Pla and PgtE require LPS for activity and that the O-antigen sterically prevents recognition of large-molecular-weight substrates. Loss of O-antigen facilitates Pla functions and invasiveness of Y. pestis; on the other hand, smooth LPS renders Plasminogen activator cryptic in S. enterica.

  • protein regions important for Plasminogen Activation and inActivation of alpha2 antiplasmin in the surface protease pla of yersinia pestis
    Molecular Microbiology, 2001
    Co-Authors: Maini Kukkonen, Kaarina Lahteenmaki, Hannu Lang, Marjo Suomalainen, Nisse Kalkkinen, Levente Emody, Timo K Korhonen
    Abstract:

    The Plasminogen activator, surface protease Pla, of the plague bacterium Yersinia pestis is an important virulence factor that enables the spread of Y. pestis from subcutaneous sites into circulation. Pla-expressing Y. pestis and recombinant Escherichia coli formed active plasmin in the presence of the major human plasmin inhibitor, alpha2-antiplasmin, and the bacteria were found to inactivate alpha2-antiplasmin. In contrast, only poor Plasminogen Activation and no cleavage of alpha2-antiplasmin was observed with recombinant bacteria expressing the homologous gene ompT from E. coli. A beta-barrel topology model for Pla and OmpT predicted 10 transmembrane beta-strands and five surface-exposed loops L1-L5. Hybrid Pla-OmpT proteins were created by substituting each of the loops between Pla and OmpT. Analysis of the hybrid molecules suggested a critical role of L3 and L4 in the substrate specificity of Pla towards Plasminogen and alpha2-antiplasmin. Substitution analysis at 25 surface-located residues showed the importance of the conserved residues H101, H208, D84, D86, D206 and S99 for the proteolytic activity of Pla-expressing recombinant E. coli. The mature alpha-Pla of 292 amino acids was processed into beta-Pla by an autoprocessing cleavage at residue K262, and residues important for the self-recognition of Pla were identified. Prevention of autoprocessing of Pla, however, had no detectable effect on Plasminogen Activation or cleavage of alpha2-antiplasmin. Cleavage of alpha2-antiplasmin and Plasminogen Activation were influenced by residue R211 in L4 as well as by unidentified residues in L3. OmpT, which is not associated with invasive bacterial disease, was converted into a Pla-like protease by deleting residues D214 and P215, by substituting residue K217 for R217 in L4 of OmpT and also by substituting the entire L3 with that from Pla. This simple modification of the surface loops and the substrate specificity of OmpT exemplifies the evolution of a housekeeping protein into a virulence factor by subtle mutations at critical protein regions. We propose that inActivation of alpha2-antiplasmin by Pla of Y. pestis promotes uncontrolled proteolysis and contributes to the invasive character of plague.