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Timo K Korhonen - One of the best experts on this subject based on the ideXlab platform.
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Fibrinolytic and procoagulant activities of Yersinia pestis and Salmonella enterica.
Journal of Thrombosis and Haemostasis, 2015Co-Authors: Timo K KorhonenAbstract:Summary Pla of the plague bacterium Yersinia pestis and PgtE of the enteropathogen Salmonella enterica are surface-exposed, transmembrane β-barrel proteases of the Omptin family that exhibit a complex array of interactions with the hemostatic systems in vitro, and both proteases are established virulence factors. Pla favors fibrinolysis by direct activation of plasminogen, inactivation of the serpins plasminogen activator inhibitor-1 and α2-antiplasmin, inactivation of the thrombin-activable fibrinolysis inhibitor, and activation of single-chain urokinase. PgtE is structurally very similar but exhibits partially different functions and differ in expression control. PgtE proteolysis targets control aspects of fibrinolysis, and mimicry of matrix metalloproteinases enhances cell migration that should favor the intracellular spread of the bacterium. Enzymatic activity of both proteases is strongly influenced by the environment-induced variations in lipopolysaccharide that binds to the β-barrel. Both proteases cleave the tissue factor pathway inhibitor and thus also express procoagulant activity.
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The outer membrane protease PgtE of Salmonella enterica interferes with the alternative complement pathway by cleaving factors B and H
Frontiers in microbiology, 2015Co-Authors: Rauna Riva, Timo K Korhonen, Seppo MeriAbstract:The virulence factor PgtE is an outer membrane protease (Omptin) of the zoonotic pathogen Salmonella enterica that causes diseases ranging from gastroenteritis to severe enteric fever. It is surface exposed in bacteria that have a short-chain, i.e. rough LPS, as observed e.g. in bacteria residing inside macrophages or just emerging from them. We investigated whether PgtE cleaves the complement factors B (B) and H (H), key proteins controlling formation and inactivation of the complement protein C3b and thereby the activity of the complement system. S. enterica serovar Typhimurium or Omptin-expressing recombinant E. coli bacteria were incubated with purified human complement proteins or recombinant H fragments. PgtE cleaved both B and H, whereas its close homolog Pla of Yersinia pestis cleaved only H. H was cleaved at both N- and C-termini, while the central region resisted proteolysis. Because of multiple effects of PgtE on complement components (cleavage of C3, C3b, B and H) we assessed its effect on the opsonophagocytosis of Salmonella. In human serum, C3 cleavage was dependent on proteolytically active PgtE. Human neutrophils interacted less with serum-opsonized FITC-stained S. enterica 14028R than with the isogenic ΔpgtE strain, as analyzed by flow cytometry. In conclusion, cleavage of B and H by PgtE, together with C3 cleavage, affects the C3-mediated recognition of S. enterica by human neutrophils, thus thwarting the immune protection against Salmonella.
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International Endotoxin & Innate Immunity Society
2014Co-Authors: Marjo Suomalainen, Kaarina Lahteenmaki, Timo K Korhonen, Johanna Haiko, Teija OjalaAbstract:enterobacterial pathogens Invited review: Breaking barriers attack on innate immune defences by Omptin surface proteases o
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Human single-chain urokinase is activated by the Omptins PgtE of Salmonella enterica and Pla of Yersinia pestis despite mutations of active site residues.
Molecular Microbiology, 2013Co-Authors: Hanna M. Järvinen, Marjo Suomalainen, Johanna Haiko, Liisa Laakkonen, Nisse Kalkkinen, Tiira Johansson, Carmen Buchrieser, Katri Juuti, Timo K KorhonenAbstract:Fibrinolysis is important in cell migration and tightly regulated by specific inhibitors and activators; of the latter, urokinase (uPA) associates with enhancement of cell migration. Active uPA is formed through cleavage of the single-chain uPA (scuPA). The Salmonella enterica strain 14028R cleaved human scuPA at the peptide bond Lys158-Ile159, the site cleaved also by the physiological activator human plasmin. The cleavage led to activation of scuPA, while no cleavage or activation were detected with the mutant strain 14028R lacking the Omptin protease PgtE. Complementation and expression studies confirmed the role of PgtE in scuPA activation. Similar cleavage and activation of scuPA were detected with recombinant Escherichia coli expressing the Omptin genes pla from Yersinia pestis, ompT and ompP from E. coli, sopA from Shigella flexneri, and leo from Legionella pneumophila. For these Omptins the activation of scuPA is the only shared function so far detected. Only poor cleavage and activation of scuPA were seen with YcoA of Y. pestis and YcoB of Yersinia pseudotuberculosis that are considered to be proteolytically inactive Omptin variants. Point mutations of active site residues in Pla and PgtE had different effects on the proteolysis of plasminogen and of scuPA, indicating versatility in Omptin proteolysis. © 2013 John Wiley & Sons Ltd.
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Fibrinolytic and coagulative activities of Yersinia pestis
Frontiers in cellular and infection microbiology, 2013Co-Authors: Timo K Korhonen, Johanna Haiko, Liisa Laakkonen, Hanna M. Järvinen, Benita Westerlund-wikströmAbstract:The outer membrane protease Pla belongs to the Omptin protease family spread by horizontal gene transfer into Gram-negative bacteria that infect animals or plants. Pla has adapted to support the life style of the plague bacterium Yersinia pestis. Pla has a -barrel fold with 10 membrane-spanning strands and five surface loops, and the barrel surface contains bound lipopolysaccharide (LPS) that is critical for the conformation and the activity of Pla. The biological activity of Pla is influenced by the structure of the surface loops around the active site groove and by temperature-induced LPS modifications. Several of the putative virulence-related functions documented for Pla in vitro address control of the human hemostatic system, i.e. coagulation and fibrinolysis. Pla activates human plasminogen to the serine protease plasmin and activates the physiological plasminogen activator urokinase. Pla also inactivates the protease inhibitors alpha-2-antiplasmin and plasminogen activator inhibitor 1 and prevents the activation of thrombin-activatable fibrinolysis inhibitor. These functions enhance uncontrolled fibrinolysis which is thought to improve Y. pestis dissemination and survival in the mammalian host, and lowered fibrin(ogen) deposition has indeed been observed in mice infected with Pla-positive Y. pestis. However, Pla also inactivates an anticoagulant, the tissue factor pathway inhibitor, which should increase fibrin formation and clotting. Thus Pla and Y. pestis have complex interactions with the hemostatic system. Y. pestis modifies its LPS upon transfer to the mammalian host and we hypothesize that the contrasting biological activities of Pla in coagulation and fibrinolysis are influenced by LPS changes during infection.
Johanna Haiko - One of the best experts on this subject based on the ideXlab platform.
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International Endotoxin & Innate Immunity Society
2014Co-Authors: Marjo Suomalainen, Kaarina Lahteenmaki, Timo K Korhonen, Johanna Haiko, Teija OjalaAbstract:enterobacterial pathogens Invited review: Breaking barriers attack on innate immune defences by Omptin surface proteases o
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Human single-chain urokinase is activated by the Omptins PgtE of Salmonella enterica and Pla of Yersinia pestis despite mutations of active site residues.
Molecular Microbiology, 2013Co-Authors: Hanna M. Järvinen, Marjo Suomalainen, Johanna Haiko, Liisa Laakkonen, Nisse Kalkkinen, Tiira Johansson, Carmen Buchrieser, Katri Juuti, Timo K KorhonenAbstract:Fibrinolysis is important in cell migration and tightly regulated by specific inhibitors and activators; of the latter, urokinase (uPA) associates with enhancement of cell migration. Active uPA is formed through cleavage of the single-chain uPA (scuPA). The Salmonella enterica strain 14028R cleaved human scuPA at the peptide bond Lys158-Ile159, the site cleaved also by the physiological activator human plasmin. The cleavage led to activation of scuPA, while no cleavage or activation were detected with the mutant strain 14028R lacking the Omptin protease PgtE. Complementation and expression studies confirmed the role of PgtE in scuPA activation. Similar cleavage and activation of scuPA were detected with recombinant Escherichia coli expressing the Omptin genes pla from Yersinia pestis, ompT and ompP from E. coli, sopA from Shigella flexneri, and leo from Legionella pneumophila. For these Omptins the activation of scuPA is the only shared function so far detected. Only poor cleavage and activation of scuPA were seen with YcoA of Y. pestis and YcoB of Yersinia pseudotuberculosis that are considered to be proteolytically inactive Omptin variants. Point mutations of active site residues in Pla and PgtE had different effects on the proteolysis of plasminogen and of scuPA, indicating versatility in Omptin proteolysis. © 2013 John Wiley & Sons Ltd.
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Fibrinolytic and coagulative activities of Yersinia pestis
Frontiers in cellular and infection microbiology, 2013Co-Authors: Timo K Korhonen, Johanna Haiko, Liisa Laakkonen, Hanna M. Järvinen, Benita Westerlund-wikströmAbstract:The outer membrane protease Pla belongs to the Omptin protease family spread by horizontal gene transfer into Gram-negative bacteria that infect animals or plants. Pla has adapted to support the life style of the plague bacterium Yersinia pestis. Pla has a -barrel fold with 10 membrane-spanning strands and five surface loops, and the barrel surface contains bound lipopolysaccharide (LPS) that is critical for the conformation and the activity of Pla. The biological activity of Pla is influenced by the structure of the surface loops around the active site groove and by temperature-induced LPS modifications. Several of the putative virulence-related functions documented for Pla in vitro address control of the human hemostatic system, i.e. coagulation and fibrinolysis. Pla activates human plasminogen to the serine protease plasmin and activates the physiological plasminogen activator urokinase. Pla also inactivates the protease inhibitors alpha-2-antiplasmin and plasminogen activator inhibitor 1 and prevents the activation of thrombin-activatable fibrinolysis inhibitor. These functions enhance uncontrolled fibrinolysis which is thought to improve Y. pestis dissemination and survival in the mammalian host, and lowered fibrin(ogen) deposition has indeed been observed in mice infected with Pla-positive Y. pestis. However, Pla also inactivates an anticoagulant, the tissue factor pathway inhibitor, which should increase fibrin formation and clotting. Thus Pla and Y. pestis have complex interactions with the hemostatic system. Y. pestis modifies its LPS upon transfer to the mammalian host and we hypothesize that the contrasting biological activities of Pla in coagulation and fibrinolysis are influenced by LPS changes during infection.
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Human single‐chain urokinase is activated by the Omptins PgtE of Salmonella enterica and Pla of Yersinia pestis despite mutations of active site residues
Molecular microbiology, 2013Co-Authors: Hanna M. Järvinen, Marjo Suomalainen, Johanna Haiko, Liisa Laakkonen, Katri M. Juuti, Nisse Kalkkinen, Tiira Johansson, Carmen Buchrieser, Timo K KorhonenAbstract:Fibrinolysis is important in cell migration and tightly regulated by specific inhibitors and activators; of the latter, urokinase (uPA) associates with enhancement of cell migration. Active uPA is formed through cleavage of the single-chain uPA (scuPA). The Salmonella enterica strain 14028R cleaved human scuPA at the peptide bond Lys158-Ile159, the site cleaved also by the physiological activator human plasmin. The cleavage led to activation of scuPA, while no cleavage or activation were detected with the mutant strain 14028R lacking the Omptin protease PgtE. Complementation and expression studies confirmed the role of PgtE in scuPA activation. Similar cleavage and activation of scuPA were detected with recombinant Escherichia coli expressing the Omptin genes pla from Yersinia pestis, ompT and ompP from E. coli, sopA from Shigella flexneri, and leo from Legionella pneumophila. For these Omptins the activation of scuPA is the only shared function so far detected. Only poor cleavage and activation of scuPA were seen with YcoA of Y. pestis and YcoB of Yersinia pseudotuberculosis that are considered to be proteolytically inactive Omptin variants. Point mutations of active site residues in Pla and PgtE had different effects on the proteolysis of plasminogen and of scuPA, indicating versatility in Omptin proteolysis. © 2013 John Wiley & Sons Ltd.
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Reviewed by:
2013Co-Authors: Johanna Haiko, Liisa Laakkonen, Hanna M. Järvinen, Benita Westerlund-wikström, Matthew S. Francis, Wyndham W. Lathem, Timo K KorhonenAbstract:The outer membrane protease Pla belongs to the Omptin protease family spread by horizontal gene transfer into Gram-negative bacteria that infect animals or plants. Pla has adapted to support the life style of the plague bacterium Yersinia pestis. Pla has a β-barrel fold with 10 membrane-spanning β strands and five surface loops, and the barrel surface contains bound lipopolysaccharide (LPS) that is critical for the conformation and the activity of Pla. The biological activity of Pla is influenced by the structure of the surface loops around the active site groove and by temperature-induced LPS modifications. Several of the putative virulence-related functions documented for Pla in vitro address control of the human hemostatic system, i.e., coagulation and fibrinolysis. Pla activates human plasminogen to the serine protease plasmin and activates the physiological plasminogen activator urokinase. Pla also inactivates the protease inhibitors alpha-2-antiplasmin and plasminogen activator inhibitor 1 (PAI-1) and prevents the activation of thrombin-activatable fibrinolysis inhibitor (TAFI). These functions enhance uncontrolled fibrinolysis which is thought to improve Y. pestis dissemination and survival in the mammalian host, and lowered fibrin(ogen) deposition has indeed been observed in mice infected wit
Marjo Suomalainen - One of the best experts on this subject based on the ideXlab platform.
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International Endotoxin & Innate Immunity Society
2014Co-Authors: Marjo Suomalainen, Kaarina Lahteenmaki, Timo K Korhonen, Johanna Haiko, Teija OjalaAbstract:enterobacterial pathogens Invited review: Breaking barriers attack on innate immune defences by Omptin surface proteases o
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Human single-chain urokinase is activated by the Omptins PgtE of Salmonella enterica and Pla of Yersinia pestis despite mutations of active site residues.
Molecular Microbiology, 2013Co-Authors: Hanna M. Järvinen, Marjo Suomalainen, Johanna Haiko, Liisa Laakkonen, Nisse Kalkkinen, Tiira Johansson, Carmen Buchrieser, Katri Juuti, Timo K KorhonenAbstract:Fibrinolysis is important in cell migration and tightly regulated by specific inhibitors and activators; of the latter, urokinase (uPA) associates with enhancement of cell migration. Active uPA is formed through cleavage of the single-chain uPA (scuPA). The Salmonella enterica strain 14028R cleaved human scuPA at the peptide bond Lys158-Ile159, the site cleaved also by the physiological activator human plasmin. The cleavage led to activation of scuPA, while no cleavage or activation were detected with the mutant strain 14028R lacking the Omptin protease PgtE. Complementation and expression studies confirmed the role of PgtE in scuPA activation. Similar cleavage and activation of scuPA were detected with recombinant Escherichia coli expressing the Omptin genes pla from Yersinia pestis, ompT and ompP from E. coli, sopA from Shigella flexneri, and leo from Legionella pneumophila. For these Omptins the activation of scuPA is the only shared function so far detected. Only poor cleavage and activation of scuPA were seen with YcoA of Y. pestis and YcoB of Yersinia pseudotuberculosis that are considered to be proteolytically inactive Omptin variants. Point mutations of active site residues in Pla and PgtE had different effects on the proteolysis of plasminogen and of scuPA, indicating versatility in Omptin proteolysis. © 2013 John Wiley & Sons Ltd.
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Human single‐chain urokinase is activated by the Omptins PgtE of Salmonella enterica and Pla of Yersinia pestis despite mutations of active site residues
Molecular microbiology, 2013Co-Authors: Hanna M. Järvinen, Marjo Suomalainen, Johanna Haiko, Liisa Laakkonen, Katri M. Juuti, Nisse Kalkkinen, Tiira Johansson, Carmen Buchrieser, Timo K KorhonenAbstract:Fibrinolysis is important in cell migration and tightly regulated by specific inhibitors and activators; of the latter, urokinase (uPA) associates with enhancement of cell migration. Active uPA is formed through cleavage of the single-chain uPA (scuPA). The Salmonella enterica strain 14028R cleaved human scuPA at the peptide bond Lys158-Ile159, the site cleaved also by the physiological activator human plasmin. The cleavage led to activation of scuPA, while no cleavage or activation were detected with the mutant strain 14028R lacking the Omptin protease PgtE. Complementation and expression studies confirmed the role of PgtE in scuPA activation. Similar cleavage and activation of scuPA were detected with recombinant Escherichia coli expressing the Omptin genes pla from Yersinia pestis, ompT and ompP from E. coli, sopA from Shigella flexneri, and leo from Legionella pneumophila. For these Omptins the activation of scuPA is the only shared function so far detected. Only poor cleavage and activation of scuPA were seen with YcoA of Y. pestis and YcoB of Yersinia pseudotuberculosis that are considered to be proteolytically inactive Omptin variants. Point mutations of active site residues in Pla and PgtE had different effects on the proteolysis of plasminogen and of scuPA, indicating versatility in Omptin proteolysis. © 2013 John Wiley & Sons Ltd.
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Temperature-Induced Changes in the Lipopolysaccharide of Yersinia pestis Affect Plasminogen Activation by the Pla Surface Protease
Infection and immunity, 2010Co-Authors: Marjo Suomalainen, Ritva Virkola, Otto Holst, Leandro Araujo Lobo, Klaus Brandenburg, Buko Lindner, Yuriy A. Knirel, Andrey P. Anisimov, Timo K KorhonenAbstract:The Pla surface protease of Yersinia pestis activates human plasminogen and is a central virulence factor in bubonic and pneumonic plague. Pla is a transmembrane -barrel protein and member of the Omptin family of outer membrane proteases which require bound lipopolysaccharide (LPS) to be proteolytically active. Plasminogen activation and autoprocessing of Pla were dramatically higher in Y. pestis cells grown at 37°C than in cells grown at 20°C; the difference in enzymatic activity by far exceeded the increase in the cellular content of the Pla protein. Y. pestis modifies its LPS structure in response to growth temperature. We purified His6-Pla under denaturing conditions and compared various LPS types for their capacity to enhance plasmin formation by His6-Pla solubilized in detergent. Reactivation of His6-Pla was higher with Y. pestis LPSs isolated from bacteria grown at 37°C than with LPSs from cells grown at 25°C. Lack of O antigens and the presence of the outer core region as well as a lowered level of acylation in LPS were found to enhance the Pla-LPS interaction. Genetic substitution of arginine 138, which is part of a three-dimensional protein motif for binding to lipid A phosphates, decreased both the enzymatic activity of His6-Pla and the amount of Pla in Y. pestis cells, suggesting the importance of the Pla-lipid A phosphate interaction. The temperature-induced changes in LPS are known to help Y. pestis to avoid innate immune responses, and our results strongly suggest that they also potentiate Pla-mediated proteolysis. Plague is a zoonosis that primarily affects rodents but also has caused three waves of plague pandemics in the human population, leading to tens of millions of deaths (51). The etiological agent of plague, Yersinia pestis, is transmitted to humans primarily by the bite of infected fleas. The common form of the disease is bubonic plague, where the bacteria spread from the intradermal fleabite site into the lymphatic system, multiply in lymph nodes, and cause formation of bubos (51, 71). Bubonic plague may progress to systemic infection and reach the lungs, causing pneumonic plague. A high level of bacteremia is essential for the blood feeding by fleas and the efficient transmission of Y. pestis from host to host (51).
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Invited review: Breaking barriers--attack on innate immune defences by Omptin surface proteases of enterobacterial pathogens.
Innate immunity, 2009Co-Authors: Johanna Haiko, Marjo Suomalainen, Kaarina Lahteenmaki, Teija Ojala, Timo K KorhonenAbstract:The Omptin family of Gram-negative bacterial transmembrane aspartic proteases comprises surface proteins with a highly conserved β-barrel fold but differing biological functions. The Omptins OmpT of Escherichia coli, PgtE of Salmonella enterica, and Pla of Yersinia pestis differ in their substrate specificity as well as in control of their expression. Their functional differences are in accordance with the differing pathogenesis of the infections caused by E. coli, Salmonella, and Y. pestis, which suggests that the Omptins have adapted to the life-styles of their host species. The Omptins Pla and PgtE attack on innate immunity by affecting the plasminogen/plasmin, complement, coagulation, fibrinolysis, and matrix metalloproteinase systems, by inactivating antimicrobial peptides, and by enhancing bacterial adhesiveness and invasiveness. Although the mechanistic details of the functions of Pla and PgtE differ, the outcome is the same: enhanced spread and multiplication of Y. pestis and S. enterica in the ho...
Maini Kukkonen - One of the best experts on this subject based on the ideXlab platform.
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Opponent
2013Co-Authors: Maini KukkonenAbstract:Cover illustration: A cladogram presention of Omptin protein sequence alignment
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The Single Substitution I259T, Conserved in the Plasminogen Activator Pla of Pandemic Yersinia pestis Branches, Enhances Fibrinolytic Activity
Journal of bacteriology, 2009Co-Authors: Johanna Haiko, Maini Kukkonen, Benita Westerlund-wikström, Janne J. Ravantti, Timo K KorhonenAbstract:The outer membrane plasminogen activator Pla of Yersinia pestis is a central virulence factor in plague. The primary structure of the Pla beta-barrel is conserved in Y. pestis biovars Antiqua, Medievalis, and Orientalis, which are associated with pandemics of plague. The Pla molecule of the ancestral Y. pestis lineages Microtus and Angola carries the single amino acid change T259I located in surface loop 5 of the beta-barrel. Recombinant Y. pestis KIM D34 or Escherichia coli XL1 expressing Pla T259I was impaired in fibrinolysis and in plasminogen activation. Lack of detectable generation of the catalytic light chain of plasmin and inactivation of plasmin enzymatic activity by the Pla T259I construct indicated that Microtus Pla cleaved the plasminogen molecule more unspecifically than did common Pla. The isoform pattern of the Pla T259I molecule was different from that of the common Pla molecule. Microtus Pla was more efficient than wild-type Pla in alpha(2)-antiplasmin inactivation. Pla of Y. pestis and PgtE of Salmonella enterica have evolved from the same Omptin ancestor, and their comparison showed that PgtE was poor in plasminogen activation but exhibited efficient antiprotease inactivation. The substitution (259)IIDKT/TIDKN in PgtE, constructed to mimic the L5 region in Pla, altered proteolysis in favor of plasmin formation, whereas the reverse substitution (259)TIDKN/IIDKT in Pla altered proteolysis in favor of alpha(2)-antiplasmin inactivation. The results suggest that Microtus Pla represents an ancestral form of Pla that has evolved into a more efficient plasminogen activator in the pandemic Y. pestis lineages.
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Activation of pro-matrix metalloproteinase-9 and degradation of gelatin by the surface protease PgtE of Salmonella enterica serovar Typhimurium.
International journal of medical microbiology : IJMM, 2007Co-Authors: Päivi Ramu, Maini Kukkonen, Marjo Suomalainen, Otto Holst, Leandro Araujo Lobo, Eva Bjur, Hanna Raukola, Minja Miettinen, Ilkka Julkunen, Mikael RhenAbstract:Mammalian matrix metalloproteinases (MMPs) degrade collagen networks in extracellular matrices by cleaving collagen and its denatured form gelatin, and thus enhance migration of mammalian cells. The gastrointestinal pathogen Salmonella enterica survives and grows within host macrophages and dendritic cells, and can disseminate in the host by travelling within infected host cells. Here, we report that S. enterica serovar Typhimurium activates proMMP-9 (gelatinase B) secreted by human primary macrophages, and degrades gelatin after growth within J774A.1 murine macrophage-like cells. Both proMMP-9 activation and gelatin degradation were due to expression of the Salmonella surface protease PgtE. Following intraperitoneal infection in BALB/c mice, the amount of a pgtE deletion derivative was nearly ten-fold lower in the livers and spleens of mice than the amount of wild-type S. enterica, suggesting that PgtE contributes to dissemination of Salmonella in the host. PgtE belongs to the Omptin family of bacterial beta-barrel transmembrane proteases. The ortholog of PgtE in Yersinia pestis, Pla, which is central for bacterial virulence in plague, was poor in proMMP-9 activation and in gelatin degradation. To model the evolution of these activities in the Omptin barrel, we performed a substitution analysis in Pla and genetically modified it into a PgtE-like gelatinase. Our results indicate that PgtE and Pla have diverged in substrate specificity, and suggest that Salmonella PgtE has evolved to functionally mimic mammalian MMPs.
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Activation of pro-matrix metalloproteinase-9 and degradation of gelatin by the surface protease PgtE of Salmonella enterica serovar Typhimurium.
International Journal of Medical Microbiology, 2007Co-Authors: Päivi Ramu, Maini Kukkonen, Marjo Suomalainen, Otto Holst, Leandro Araujo Lobo, Eva Bjur, Hanna Raukola, Minja Miettinen, Ilkka Julkunen, Mikael RhenAbstract:Mammalian matrix metalloproteinases (MMPs) degrade collagen networks in extracellular matrices by cleaving collagen and its denatured form gelatin, and thus enhance migration of mammalian cells. The gastrointestinal pathogen Salmonella enterica survives and grows within host macrophages and dendritic cells, and can disseminate in the host by travelling within infected host cells. Here, we report that S. enterica serovar Typhimurium activates proMMP-9 (gelatinase B) secreted by human primary macrophages, and degrades gelatin after growth within J774A.1 murine macrophage-like cells. Both proMMP-9 activation and gelatin degradation were due to expression of the Salmonella surface protease PgtE. Following intraperitoneal infection in BALB/c mice, the amount of a pgtE deletion derivative was nearly ten-fold lower in the livers and spleens of mice than the amount of wild-type S. enterica, suggesting that PgtE contributes to dissemination of Salmonella in the host. PgtE belongs to the Omptin family of bacterial β-barrel transmembrane proteases. The ortholog of PgtE in Yersinia pestis, Pla, which is central for bacterial virulence in plague, was poor in proMMP-9 activation and in gelatin degradation. To model the evolution of these activities in the Omptin barrel, we performed a substitution analysis in Pla and genetically modified it into a PgtE-like gelatinase. Our results indicate that PgtE and Pla have diverged in substrate specificity, and suggest that Salmonella PgtE has evolved to functionally mimic mammalian MMPs.
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Using every trick in the book: the Pla surface protease of Yersinia pestis.
Advances in experimental medicine and biology, 2007Co-Authors: Marjo Suomalainen, Maini Kukkonen, Kaarina Lahteenmaki, Ritva Virkola, Johanna Haiko, Benita Westerlund-wikström, Päivi Ramu, Leandro Araujo Lobo, Timo K KorhonenAbstract:The Pla surface protease of Yersinia pestis, encoded by the Y. pestis-specific plasmid pPCP1, is a versatile virulence factor. In vivo studies have shown that Pla is essential in the establishment of bubonic plague, and in vitro studies have demonstrated various putative virulence functions for the Pla molecule. Pla is a surface protease of the Omptin family, and its proteolytic targets include the abundant, circulating human zymogen plasminogen, which is activated by Pla to the serine protease plasmin. Plasmin is important in cell migration, and Pla also proteolytically inactivates the main circulating inhibitor of plasmin, alpha2-antiplasmin. Pla also is an adhesin with affinity for laminin, a major glycoprotein of mammalian basement membranes, which is degraded by plasmin but not by Pla. Together, these functions create uncontrolled plasmin proteolysis targeted at tissue barriers. Other proteolytic targets for Pla include complement proteins. Pla also mediates bacterial invasion into human endothelial cell lines; the adhesive and invasive charateristics of Pla can be genetically dissected from its proteolytic activity. Pla is a 10-stranded antiparallel beta-barrel with five surface-exposed short loops, where the catalytic residues are oriented inwards at the top of the beta-barrel. The sequence of Pla contains a three-dimensional motif for protein binding to lipid A of the lipopolysaccharide. Indeed, the proteolytic activity of Pla requires rough lipopolysaccharide but is sterically inhibited by the O antigen in smooth LPS, which may be the selective advantage of the loss of O antigen in Y. pestis. Members of the Omptin family are highly similar in structure but differ in functions and virulence association. The catalytic residues of Omptins are conserved, but the variable substrate specificities in proteolysis by Pla and other Omptins are dictated by the amino acid sequences near or at the surface loops, and hence reflect differences in substrate binding. The closest orthologs of Pla are PgtE of Salmonella and Epo of Erwinia, which functionally differ from Pla. Pla gives a model of how a horizontally transferred protein fold can diverge into a powerful virulence factor through adaptive mutations.
Liisa Laakkonen - One of the best experts on this subject based on the ideXlab platform.
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Human single-chain urokinase is activated by the Omptins PgtE of Salmonella enterica and Pla of Yersinia pestis despite mutations of active site residues.
Molecular Microbiology, 2013Co-Authors: Hanna M. Järvinen, Marjo Suomalainen, Johanna Haiko, Liisa Laakkonen, Nisse Kalkkinen, Tiira Johansson, Carmen Buchrieser, Katri Juuti, Timo K KorhonenAbstract:Fibrinolysis is important in cell migration and tightly regulated by specific inhibitors and activators; of the latter, urokinase (uPA) associates with enhancement of cell migration. Active uPA is formed through cleavage of the single-chain uPA (scuPA). The Salmonella enterica strain 14028R cleaved human scuPA at the peptide bond Lys158-Ile159, the site cleaved also by the physiological activator human plasmin. The cleavage led to activation of scuPA, while no cleavage or activation were detected with the mutant strain 14028R lacking the Omptin protease PgtE. Complementation and expression studies confirmed the role of PgtE in scuPA activation. Similar cleavage and activation of scuPA were detected with recombinant Escherichia coli expressing the Omptin genes pla from Yersinia pestis, ompT and ompP from E. coli, sopA from Shigella flexneri, and leo from Legionella pneumophila. For these Omptins the activation of scuPA is the only shared function so far detected. Only poor cleavage and activation of scuPA were seen with YcoA of Y. pestis and YcoB of Yersinia pseudotuberculosis that are considered to be proteolytically inactive Omptin variants. Point mutations of active site residues in Pla and PgtE had different effects on the proteolysis of plasminogen and of scuPA, indicating versatility in Omptin proteolysis. © 2013 John Wiley & Sons Ltd.
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Fibrinolytic and coagulative activities of Yersinia pestis
Frontiers in cellular and infection microbiology, 2013Co-Authors: Timo K Korhonen, Johanna Haiko, Liisa Laakkonen, Hanna M. Järvinen, Benita Westerlund-wikströmAbstract:The outer membrane protease Pla belongs to the Omptin protease family spread by horizontal gene transfer into Gram-negative bacteria that infect animals or plants. Pla has adapted to support the life style of the plague bacterium Yersinia pestis. Pla has a -barrel fold with 10 membrane-spanning strands and five surface loops, and the barrel surface contains bound lipopolysaccharide (LPS) that is critical for the conformation and the activity of Pla. The biological activity of Pla is influenced by the structure of the surface loops around the active site groove and by temperature-induced LPS modifications. Several of the putative virulence-related functions documented for Pla in vitro address control of the human hemostatic system, i.e. coagulation and fibrinolysis. Pla activates human plasminogen to the serine protease plasmin and activates the physiological plasminogen activator urokinase. Pla also inactivates the protease inhibitors alpha-2-antiplasmin and plasminogen activator inhibitor 1 and prevents the activation of thrombin-activatable fibrinolysis inhibitor. These functions enhance uncontrolled fibrinolysis which is thought to improve Y. pestis dissemination and survival in the mammalian host, and lowered fibrin(ogen) deposition has indeed been observed in mice infected with Pla-positive Y. pestis. However, Pla also inactivates an anticoagulant, the tissue factor pathway inhibitor, which should increase fibrin formation and clotting. Thus Pla and Y. pestis have complex interactions with the hemostatic system. Y. pestis modifies its LPS upon transfer to the mammalian host and we hypothesize that the contrasting biological activities of Pla in coagulation and fibrinolysis are influenced by LPS changes during infection.
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Human single‐chain urokinase is activated by the Omptins PgtE of Salmonella enterica and Pla of Yersinia pestis despite mutations of active site residues
Molecular microbiology, 2013Co-Authors: Hanna M. Järvinen, Marjo Suomalainen, Johanna Haiko, Liisa Laakkonen, Katri M. Juuti, Nisse Kalkkinen, Tiira Johansson, Carmen Buchrieser, Timo K KorhonenAbstract:Fibrinolysis is important in cell migration and tightly regulated by specific inhibitors and activators; of the latter, urokinase (uPA) associates with enhancement of cell migration. Active uPA is formed through cleavage of the single-chain uPA (scuPA). The Salmonella enterica strain 14028R cleaved human scuPA at the peptide bond Lys158-Ile159, the site cleaved also by the physiological activator human plasmin. The cleavage led to activation of scuPA, while no cleavage or activation were detected with the mutant strain 14028R lacking the Omptin protease PgtE. Complementation and expression studies confirmed the role of PgtE in scuPA activation. Similar cleavage and activation of scuPA were detected with recombinant Escherichia coli expressing the Omptin genes pla from Yersinia pestis, ompT and ompP from E. coli, sopA from Shigella flexneri, and leo from Legionella pneumophila. For these Omptins the activation of scuPA is the only shared function so far detected. Only poor cleavage and activation of scuPA were seen with YcoA of Y. pestis and YcoB of Yersinia pseudotuberculosis that are considered to be proteolytically inactive Omptin variants. Point mutations of active site residues in Pla and PgtE had different effects on the proteolysis of plasminogen and of scuPA, indicating versatility in Omptin proteolysis. © 2013 John Wiley & Sons Ltd.
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2013Co-Authors: Johanna Haiko, Liisa Laakkonen, Hanna M. Järvinen, Benita Westerlund-wikström, Matthew S. Francis, Wyndham W. Lathem, Timo K KorhonenAbstract:The outer membrane protease Pla belongs to the Omptin protease family spread by horizontal gene transfer into Gram-negative bacteria that infect animals or plants. Pla has adapted to support the life style of the plague bacterium Yersinia pestis. Pla has a β-barrel fold with 10 membrane-spanning β strands and five surface loops, and the barrel surface contains bound lipopolysaccharide (LPS) that is critical for the conformation and the activity of Pla. The biological activity of Pla is influenced by the structure of the surface loops around the active site groove and by temperature-induced LPS modifications. Several of the putative virulence-related functions documented for Pla in vitro address control of the human hemostatic system, i.e., coagulation and fibrinolysis. Pla activates human plasminogen to the serine protease plasmin and activates the physiological plasminogen activator urokinase. Pla also inactivates the protease inhibitors alpha-2-antiplasmin and plasminogen activator inhibitor 1 (PAI-1) and prevents the activation of thrombin-activatable fibrinolysis inhibitor (TAFI). These functions enhance uncontrolled fibrinolysis which is thought to improve Y. pestis dissemination and survival in the mammalian host, and lowered fibrin(ogen) deposition has indeed been observed in mice infected wit
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Molecular adaptation of a plant-bacterium outer membrane protease towards plague virulence factor Pla
BMC Evolutionary Biology, 2011Co-Authors: Johanna Haiko, Liisa Laakkonen, Benita Westerlund-wikström, Timo K KorhonenAbstract:Background Omptins are a family of outer membrane proteases that have spread by horizontal gene transfer in Gram-negative bacteria that infect vertebrates or plants. Despite structural similarity, the molecular functions of Omptins differ in a manner that reflects the life style of their host bacteria. To simulate the molecular adaptation of Omptins, we applied site-specific mutagenesis to make Epo of the plant pathogenic Erwinia pyrifoliae exhibit virulence-associated functions of its close homolog, the plasminogen activator Pla of Yersinia pestis . We addressed three virulence-associated functions exhibited by Pla, i.e., proteolytic activation of plasminogen, proteolytic degradation of serine protease inhibitors, and invasion into human cells. Results Pla and Epo expressed in Escherichia coli are both functional endopeptidases and cleave human serine protease inhibitors, but Epo failed to activate plasminogen and to mediate invasion into a human endothelial-like cell line. Swapping of ten amino acid residues at two surface loops of Pla and Epo introduced plasminogen activation capacity in Epo and inactivated the function in Pla. We also compared the structure of Pla and the modeled structure of Epo to analyze the structural variations that could rationalize the different proteolytic activities. Epo-expressing bacteria managed to invade human cells only after all extramembranous residues that differ between Pla and Epo and the first transmembrane β-strand had been changed. Conclusions We describe molecular adaptation of a protease from an environmental setting towards a virulence factor detrimental for humans. Our results stress the evolvability of bacterial β-barrel surface structures and the environment as a source of progenitor virulence molecules of human pathogens.
