On Blastocystis secreted cysteine proteases: a legumain-activated cathepsin B increases paracellular permeability of intestinal Caco-2 cell monolayers.
Blastocystis spp. pathogenic potential remains unclear as these anaerobic parasitic protozoa are frequently isolated from stools of both symptomatic and asymptomatic subjects. In silico analysis of the whole genome sequence of Blastocystis subtype 7 revealed the presence of numerous proteolytic enzymes including cysteine proteases predicted to be secreted. To assess the potential impact of proteases on intestinal cells and gut function, we focused our study on two cysteine proteases, a legumain and a cathepsin B, which were previously identified in Blastocystis subtype 7 culture supernatants. Both cysteine proteases were produced as active recombinant proteins. Activation of the recombinant legumain was shown to be autocatalytic and triggered by acidic pH, whereas proteolytic activity of the recombinant cathepsin B was only recorded after co-incubation with the legumain. We then measured the diffusion of 4-kDa FITC-labelled dextran across Caco-2 cell monolayers following exposition to either Blastocystis culture supernatants or each recombinant protease. Both Blastocystis culture supernatants and recombinant activated cathepsin B induced an increase of Caco-2 cell monolayer permeability, and this effect was significantly inhibited by E-64, a specific cysteine protease inhibitor. Our results suggest that cathepsin B might play a role in pathogenesis of Blastocystis by increasing intestinal cell permeability.
CHU Clermont-Ferrand,Centre de Biologie, Laboratoire de Parasitologie-Mycologie,Hôpital G. Montpied,F-63003 Clermont-Ferrand,France.
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Nourrisson
Clermont Université, Université Blaise Pascal, Laboratoire Microorganismes: Génome et Environnement,BP 10448, F-63000 Clermont-Ferrand,France.
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Wawrzyniak
Institut Pasteur de Lille, Centre d'Infection et d'Immunité de Lille (CIIL), Inserm U1019, CNRS UMR 8204, Université Lille Nord de France,1 rue du Professeur Calmette,BP 245, 59019 Lille cedex,France.
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Cian
CHU Clermont-Ferrand,Centre de Biologie, Laboratoire de Parasitologie-Mycologie,Hôpital G. Montpied,F-63003 Clermont-Ferrand,France.
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Livrelli
Institut Pasteur de Lille, Centre d'Infection et d'Immunité de Lille (CIIL), Inserm U1019, CNRS UMR 8204, Université Lille Nord de France,1 rue du Professeur Calmette,BP 245, 59019 Lille cedex,France.
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Viscogliosi
Clermont Université, Université Blaise Pascal, Laboratoire Microorganismes: Génome et Environnement,BP 10448, F-63000 Clermont-Ferrand,France.
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Delbac
CHU Clermont-Ferrand,Centre de Biologie, Laboratoire de Parasitologie-Mycologie,Hôpital G. Montpied,F-63003 Clermont-Ferrand,France.
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Poirier
D016844:Blastocystis / Q000201:enzymology*; D018938:Caco-2 Cells; D002401:Cathepsin B / Q000235:genetics / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D010539:Permeability / Q000187:drug effects*; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D000099240:Asparaginyl Endopeptidase
D002401:Cathepsin B; D003546:Cysteine Endopeptidases; D011994:Recombinant Proteins; D000099240:Asparaginyl Endopeptidase

               Blastocystis
            ; Caco-2; Cathepsin B; Legumain; cysteine proteases; permeability