scholarly journals Activation of human pro-urokinase by unrelated proteases secreted by Pseudomonas aeruginosa

2010 ◽  
Vol 428 (3) ◽  
pp. 473-482 ◽  
Author(s):  
Nathalie Beaufort ◽  
Paulina Seweryn ◽  
Sophie de Bentzmann ◽  
Aihua Tang ◽  
Josef Kellermann ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Pathogenic Bacteria ◽  
Recombinant Protein Expression ◽  
Active Form ◽  
Plasminogen Activator Inhibitor ◽  
Matrix Metalloproteinase 2 ◽  
Plasminogen Activator Inhibitor 1 ◽  
Upa Receptor ◽  
Protease Iv ◽  
Activation Cascade

Pathogenic bacteria, including Pseudomonas aeruginosa, interact with and engage the host plasminogen (Plg) activation system, which encompasses the urokinase (uPA)-type Plg activator, and is involved in extracellular proteolysis, including matrilysis and fibrinolysis. We hypothesized that secreted bacterial proteases might contribute to the activation of this major extracellular proteolytic system, thereby participating in bacterial dissemination. We report that LasB, a thermolysin-like metalloprotease secreted by Ps. aeruginosa, converts the human uPA zymogen into its active form (kcat=4.9 s−1, Km=8.9 μM). Accordingly, whereas the extracellular secretome from the LasB-expressing pseudomonal strain PAO1 efficiently activates pro-uPA, the secretome from the isogenic LasB-deficient strain PDO240 is markedly less potent in pro-uPA activation. Still, both secretomes induce some metalloprotease-independent activation of the human zymogen. The latter involves a serine protease, which we identified via both recombinant protein expression in Escherichia coli and purification from pseudomonal cultures as protease IV (PIV; kcat=0.73 s−1, Km=6.2 μM). In contrast, neither secretomes nor the pure proteases activate Plg. Along with this, LasB converts Plg into mini-Plg and angiostatin, whereas, as reported previously, it processes the uPA receptor, inactivates the plasminogen activator inhibitor 1, and activates pro-matrix metalloproteinase 2. PIV does not target these factors at all. To conclude, LasB and PIV, although belonging to different protease families and displaying quite different substrate specificities, both activate the urokinase-type precursor of the Plg activation cascade. Direct pro-uPA activation, as also reported for other bacterial proteases, might be a frequent phenomenon that contributes to bacterial virulence.

scholarly journals The human fibrinolytic system is a target for the staphylococcal metalloprotease aureolysin

2008 ◽  
Vol 410 (1) ◽  
pp. 157-165 ◽  
Author(s):  
Nathalie Beaufort ◽  
Piotr Wojciechowski ◽  
Christian P. Sommerhoff ◽  
Grzegorz Szmyd ◽  
Grzegorz Dubin ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Active Form ◽  
Catalytic Efficiency ◽  
Plasminogen Activator Inhibitor ◽  
Opportunistic Pathogen ◽  
Fibrinolytic System ◽  
Growth Media ◽  
Single Chain ◽  
Plasminogen Activator Inhibitor 1 ◽  
Proteolytic Activation

The major opportunistic pathogen Staphylococcus aureus utilizes the human fibrinolytic system for invasion and spread via plasmin(ogen) binding and non-proteolytic activation. Because S. aureus secretes several proteases recently proposed as virulence factors, we explored whether these enzymes could add to the activation of the host's fibrinolytic system. Exposure of human pro-urokinase [pro-uPA (where uPA is urokinase-type plasminogen activator)] to conditioned growth media from staphylococcal reference strains results in an EDTA-sensitive conversion of the single-chain zymogen into its two-chain active form, an activity not observed in an aureolysin-deficient strain. Using purified aureolysin, we verified the capacity of this thermolysin-like metalloprotease to activate pro-uPA, with a 2.6×103 M−1·s−1 catalytic efficiency. Moreover, activation also occurs in the presence of human plasma, as well as in conditioned growth media from clinical isolates. Finally, we establish that aureolysin (i) converts plasminogen into angiostatin and mini-plasminogen, the latter retaining its capacity to be activated by uPA and to hydrolyse fibrin, (ii) degrades the plasminogen activator inhibitor-1, and (iii) abrogates the inhibitory activity of α2-antiplasmin. Altogether, we propose that, in parallel with the staphylokinase-dependent activation of plasminogen, aureolysin may contribute significantly to the activation of the fibrinolytic system by S. aureus, and thus may promote bacterial spread and invasion.

Increased Plasminogen Activator Inhibitor-1 Concentrations in Bronchoalveolar Lavage Fluids Are Associated with Increased Mortality in a Cohort of Patients with Pseudomonas aeruginosa 

2007 ◽  
Vol 106 (2) ◽  
pp. 252-261 ◽  
Author(s):  
Yuanlin Song ◽  
Susan V. Lynch ◽  
Judith Flanagan ◽  
Hanjing Zhuo ◽  
Wynnson Tom ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Acute Respiratory Distress Syndrome ◽  
Respiratory Distress Syndrome ◽  
Bronchoalveolar Lavage ◽  
Distress Syndrome ◽  
Plasminogen Activator Inhibitor ◽  
Ventilator Associated Pneumonia ◽  
Plasminogen Activator Inhibitor 1 ◽  
Activator Inhibitor ◽  
Pai 1

Background Increased plasminogen activator inhibitor-1 (PAI-1) concentrations are found in bronchoalveolar lavage (BAL) fluids from patients with ventilator-associated pneumonia or acute respiratory distress syndrome. The authors hypothesized that PAI-1 concentrations were associated with increased mortality in patients with either Pseudomonas aeruginosa-induced ventilator-associated pneumonia or tracheobronchial colonization. Methods In a prospective cohort study, daily aspirates from intubated patients were cultured for P. aeruginosa. Positive patients had blind BAL (bBAL) that was processed for biomarker concentrations. Secretion of type III secretion cytotoxins were also analyzed from the P. aeruginosa strains. Results Thirty-three patients were enrolled. Ten of the 33 patients died. bBAL PAI-1 concentrations were significantly increased in nonsurvivors compared with survivors (31.7 vs. 3.4 ng/ml, P = 0.001 for hospital mortality; 35.9 vs. 4.7 ng/ml, P = 0.02 for 28-day mortality). Even when acute respiratory distress syndrome patients were excluded, there was a significant difference between the survivors and nonsurvivors for bBAL PAI-1 concentrations (P = 0.005). Eighty-three percent of P. aeruginosa strains isolated from patients with high concentrations of bBAL PAI-1 also had strains that secreted cytotoxins. Conclusions PAI-1 concentrations in bBALs correlated with mortality in ventilated patients with positive cultures for P. aeruginosa. Elevated bBAL PAI-1 concentrations also correlated with the secretion of type III exotoxins by P. aeruginosa.

scholarly journals Vitamin D Enhances Radiosensitivity of Colorectal Cancer by Reversing Epithelial-Mesenchymal Transition

2021 ◽  
Vol 9 ◽  
Author(s):  
Xinyue Yu ◽  
Qian Wang ◽  
Baocai Liu ◽  
Ning Zhang ◽  
Guanghui Cheng
Keyword(s):  
Colorectal Cancer ◽  
Vitamin D ◽  
Epithelial Mesenchymal Transition ◽  
Active Form ◽  
Plasminogen Activator Inhibitor ◽  
Biologically Active ◽  
Plasminogen Activator Inhibitor 1 ◽  
Mesenchymal Transition

Colorectal cancer (CRC) is often resistant to conventional therapies. Previous studies have reported the anticancer effects of vitamin D in several cancers, its role in radiotherapy (RT) remains unknown. We found that 1α, 25-dihydroxyvitamin D3 (VD3), the biologically active form of vitamin D, had antitumor effect on CRC and sensitized CRC cells to ionizing radiation (IR). VD3 demonstrated synergistic effect in combination with IR, which were detected by colony formation and cell proliferation assay. Radiosensitivity restoration induced by VD3 was associated with a series of phenotypes, including apoptosis, autophagy, and epithelial-mesenchymal transition (EMT). Using proteomics, “regulation of cell migration” and “cadherin” were found to be obviously enriched GO terms. Moreover, cystatin D and plasminogen activator inhibitor-1 (PAI-1), the differentially expressed proteins, were associated with EMT. Next, we confirmed the contributions of these two genes in enhancing IR sensitivity of CRC cells upon inhibition of EMT. As determined by proteomics, the mechanism underlying such sensitivity involved partially block of JAK/STAT3 signaling pathway. Furthermore, VD3 also elicited sensitization to RT in xenograft CRC models without additional toxicity. Our study revealed that VD3 was able to act in synergy with IR both in vitro and in vivo and could also confer radiosensitivity by regulating EMT, thereby providing a novel insight for elevating the efficacy of therapeutic regimens.

Immunoassays for the Quantitation of Porcine PAI-1 Antigen and Activity in Biological Fluid Samples

2000 ◽  
Vol 84 (12) ◽  
pp. 1082-1086 ◽  
Author(s):  
Henry M. Leng ◽  
Els Brouwers ◽  
Isabelle Knockaert ◽  
Paul Declerck
Keyword(s):  
Biological Fluid ◽  
Platelet Rich Plasma ◽  
Active Form ◽  
Plasminogen Activator Inhibitor ◽  
Experimental Models ◽  
Activity Levels ◽  
Plasminogen Activator Inhibitor 1 ◽  
Antigen Elisa ◽  
Pathophysiological Role ◽  
Pai 1

SummaryTwo monoclonal antibody-based enzyme-linked immunosorbent assays (ELISAs) for the quantitation of porcine plasminogen activator inhibitor-1 (PAI-1) antigen and activity in plasma were constructed and validated. The intra-assay, interassay, and interdilution coefficients of variation were 4.3, 13, and 8%, respectively, for the antigen ELISA and 5, 16, and 11% for the activity assay. Assay recoveries, in the antigen ELISA, of either latent or active recombinant porcine PAI-1 (10 and 50 ng/ml) added to plasma were 86 ± 9% and 92 ± 22%, respectively, for the latent form and 89 ± 9% and 87 ± 7% for the active form (mean ± SD, n = 3 to 4). In the immunofunctional assay, recoveries for the same concentrations of active PAI-1 were 108 ± 16% and 92 ± 21%, respectively. In male porcine plasma the level of PAI-1 antigen was 31 ± 11 ng/ ml and the activity, 34 ± 16 ng/ml (mean ± SD, n = 10). In female plasma PAI-1 antigen levels were 20 ± 5.2 ng/ml and the PAI-1 activity 42 ± 17 ng/ml (n = 13). A linear correlation was found between PAI-1 antigen and activity levels in male (r = 0.60) and female (r = 0.70) plasma. Immunodepletion resulted in a decrease of >95% of the original PAI-1 antigen or activity levels. Incubation of plasma samples at 37° C for 16 h resulted in a significant decrease (70 to 85%) of PAI-1 activity. Under these conditions (37° C, 16 h) PAI-1 antigen levels remained unchanged in males whereas the response of the female samples in the PAI-1 antigen assay increased two-fold.In lysed platelet-rich plasma males had 990 ± 470 ng/ml antigen and 160 ± 80 ng/ml activity and females, 920 ± 500 ng/ml antigen and 150 ± 98 ng/ml activity corresponding to 2.1 ± 0.77 fg PAI-1 antigen per platelet. Only 16% of PAI-1 released from platelets was found to be active. Linear correlations between PAI-1 antigen and activity were found for both males (r = 0.61) and females (r = 0.67).The assays are both sensitive and specific and may, therefore, aid the elucidation of the pathophysiological role of PAI-1 in swine experimental models of atherosclerosis and other thrombotic disorders.

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