Plasminogen activator inhibitor 1 (PAI-1) is a serine protease inhibitor (serpin) and a key molecule that regulates fibrinolysis by inactivating human plasminogen activators. Here we show that two important human pathogens, the plague bacterium Yersinia pestis and the enteropathogen Salmonella enterica serovar Typhimurium, inactivate PAI-1 by cleaving the R346-M347 bait peptide bond in the reactive center loop. No cleavage of PAI-1 was detected with Yersinia pseudotuberculosis, an oral/fecal pathogen from which Y. pestis has evolved, or with Escherichia coli. The cleavage and inactivation of PAI-1 were mediated by the outer membrane proteases plasminogen activator Pla of Y. pestis and PgtE protease of S. enterica, which belong to the omptin family of transmembrane endopeptidases identified in Gram-negative bacteria. Cleavage of PAI-1 was also detected with the omptins Epo of Erwinia pyrifoliae and Kop of Klebsiella pneumoniae, which both belong to the same omptin subfamily as Pla and PgtE, whereas no cleavage of PAI-1 was detected with omptins of Shigella flexneri or E. coli or the Yersinia chromosomal omptins, which belong to other omptin subfamilies. The results reveal a novel serpinolytic mechanism by which enterobacterial species expressing omptins of the Pla subfamily bypass normal control of host proteolysis.Plasminogen activator inhibitor 1 (PAI-1) is a key regulator of the mammalian fibrinolytic/plasminogen system (29, 37). The fibrinolytic system comprises the serine protease zymogen plasminogen, urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), PAI-1, and plasmin inhibitor ␣ 2 -antiplasmin (␣ 2 AP) (for a review, see reference 52). Plasminogen is converted to plasmin, which is a broad-spectrum serine protease that dissolves fibrin in blood clots, degrades laminin of basement membranes, and activates matrix metalloproteinases that degrade collagens and gelatins in tissue barriers. Herewith, plasmin controls physiological processes such as fibrinolysis/coagulation, cell migration and invasion, and tumor metastasis (29, 37). PAI-1 maintains normal hemostasis by inhibiting the function of the plasminogen activators tPA and uPA, which are serine proteases and highly specific for cleavage of the plasminogen molecule. tPA binds to fibrin and is associated with plasmin-mediated breakdown of fibrin clots, whereas uPA has low affinity for fibrin and associates with cell surface proteolysis, cellular migration, and damage of tissue barriers (52).The mammalian fibrinolytic and coagulation systems are targeted by invasive bacterial pathogens during infection (reviewed in references 6, 11, 34, and 61). In bacterial sepsis, increased production of fibrin clots at a damaged endothelium results from enhanced thrombin-catalyzed fibrin generation and from an increased serum level of PAI-1. Coagulation can protect the host by activating immune systems or by physically restraining the bacteria (6,15,25,41). On the other hand, several invasive bacterial pathogens enhance fibrinolysis eithe...

Pls, the surface protein of methicillin-resistant Staphylococcus aureus (MRSA), prevents adhesion of clinical strain 1061 to immobilized fibronectin (Fn) and immunoglobulin G (IgG). Invasion of mammalian cells by S. aureus depends on Fn-mediated binding of staphylococcal Fn-binding proteins to host cell beta (1)-integrins. In the present study, we show that, for 10 clinical Pls-positive (Pls(+)) MRSA strains, adhesion to immobilized Fn, fibrinogen (Fg), IgG, and laminin, as well as binding to soluble Fn and Fg, was less efficient than adhesion and binding associated with 4 Pls-negative (Pls(-)) MRSA strains. However, binding to soluble IgG was comparable among both types of strains. For 293 cells, Pls(+) strains were less invasive than were Pls(-) strains (median [range], 35% [22%-70%] and 110% [89%-141%], respectively, compared with strain Cowan 1). Disruption of the pls gene of strain 1061 increased invasiveness, but it did not affect binding of soluble Fn, Fg, and IgG. Complementation restored the low level of invasiveness, but it did not restore the low level of adhesion to immobilized Fn. In conclusion, the reduced adhesiveness and invasiveness of MRSA appear to generally correlate with expression of Pls.

We have shown previously that pls, which codes for the surface protein Pls of methicillin-resistant Staphylococcus aureus (MRSA), reduces adhesion to immobilized fibronectin, fibrinogen, laminin, and immunoglobulin G as well as invasion of host cells. Here, we tested a collection of 66 clinical MRSA isolates--48 negative for pls/Pls (pls(-)/Pls(-)), 15 positive for pls/Pls (pls(+)/Pls(+)), and 3 harboring the pls gene but not expressing Pls (pls(+)/Pls(-))--for cellular invasiveness. Invasion of 293 cells by pls(+)/Pls(+) strains was lower than that by the pls(-)/Pls(-) strains (median [range], 36% [22%-70%] vs. 93% [25%-162%]). The 3 pls(+)/Pls(-) strains (median [range], 95% [63%-103%]) were as invasive as the pls(-)/Pls(-) strains. In addition, we identified a pls(+)/Pls(+) staphylococcal chromosomal cassette mec (SCCmec) IV strain. In conclusion, 3 properties--pls/Pls, SCCmec type, and spa type--strongly predicted the cellular invasiveness of MRSA strains, as indicated by good clustering. In contrast, the spa type-deduced multilocus sequence typing clonal complex (MLST-CC) was not able to predict the invasiveness of MRSA strains equally well. The underlying mechanism remains to be elucidated.

Expression of cell wall-anchored Pls reduces adherence and invasiveness independently of the MRSA/SCCmec background. This occurs despite early up-regulation of fnbA transcription and FnBP surface expression. Thus, Pls acts by steric hindrance rather than another mechanism.

Aims: The focus of the research was to identify yeasts from barley kernels in order to study their folate production capability while maintaining high viscosity caused by soluble fibres in oat bran fermentation. Methods and Results: The 65 isolated yeasts were characterized by API carbohydrate utilization tests, and assays for extracellular enzyme activities were the following: amylase, beta-glucanase, cellulase or CMCase, lipase, protease and xylanase. Yeasts were identified by partial DNA sequencing of the 25S D1/D2 and ITS1-5.8S-ITS2 regions. They belonged to the genera Aureobasidium, Cryptococcus, Pseudozyma and Rhodotorula. Folate production was determined from supernatant and cells grown in a rich laboratory medium or directly from oat bran solution inoculated with the appropriate yeast. Food yeasts, Saccharomyces cerevisiae, Candida milleri, Kluyveromyces marxianus and Galactomyces geotrichum, were used for comparison. Most of the yeasts isolated from barley destroyed the solid, viscous structure of the oat bran solution, indicating that they degraded the viscosity-generating soluble fibres, considered to be nutritionally advantageous. The best folate producers were S. cerevisiae, followed by Pseudozyma sp., Rhodotorula glutinis and K. marxianus. The yeasts maintaining high viscosity were used together with lactic acid bacteria (LAB) Streptococcus thermophilus or Lactobacillus rhamnosus to ferment oat bran solution. None of the yeasts isolated from barley, contrary to S. cerevisiae and C. milleri, produced together with LAB significant amounts of folate. Conclusions: Fermentative yeasts together with LAB are potential for use in developing novel high folate content healthy foods and snacks from oat bran. Significance and Impact of the Study: High soluble fibre content and high natural folate content but low energy content food and snack products with pleasant fermentation aroma provide possibilities for new developments in the food industry.