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On Blastocystis secreted cysteine proteases: a legumain-activated cathepsin B increases paracellular permeability of intestinal Caco-2 cell monolayers

Published online by Cambridge University Press:  09 September 2016

C. NOURRISSON ,
I. WAWRZYNIAK ,
A. CIAN ,
V. LIVRELLI ,
E. VISCOGLIOSI ,
F. DELBAC  and
P. POIRIER
C. NOURRISSON
Affiliation:
CHU Clermont-Ferrand, Centre de Biologie, Laboratoire de Parasitologie-Mycologie, Hôpital G. Montpied, F-63003 Clermont-Ferrand, France Clermont Université, Université Blaise Pascal, Laboratoire Microorganismes: Génome et Environnement, BP 10448, F-63000 Clermont-Ferrand, France
I. WAWRZYNIAK
Affiliation:
Clermont Université, Université Blaise Pascal, Laboratoire Microorganismes: Génome et Environnement, BP 10448, F-63000 Clermont-Ferrand, France
A. CIAN
Affiliation:
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
V. LIVRELLI
Affiliation:
CHU Clermont-Ferrand, Centre de Biologie, Laboratoire de Parasitologie-Mycologie, Hôpital G. Montpied, F-63003 Clermont-Ferrand, France Clermont Université, Université d'Auvergne, Centre de Recherche en Nutrition Humaine Auvergne, M2iSH, ‘Microbes, Intestin, Inflammation et Susceptibilité de l'Hôte UMR INSERM/Université d'Auvergne U1071 USC-INRA 2018, BP 10448, F-63000 Clermont-Ferrand, France
E. VISCOGLIOSI
Affiliation:
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
F. DELBAC
Affiliation:
Clermont Université, Université Blaise Pascal, Laboratoire Microorganismes: Génome et Environnement, BP 10448, F-63000 Clermont-Ferrand, France
P. POIRIER*
Affiliation:
CHU Clermont-Ferrand, Centre de Biologie, Laboratoire de Parasitologie-Mycologie, Hôpital G. Montpied, F-63003 Clermont-Ferrand, France Clermont Université, Université Blaise Pascal, Laboratoire Microorganismes: Génome et Environnement, BP 10448, F-63000 Clermont-Ferrand, France
*
*Corresponding author: CHU Clermont-Ferrand, Centre de Biologie, Laboratoire de Parasitologie-Mycologie, Hôpital G. Montpied, F-63003 Clermont-Ferrand, France. E-mail: ppoirier@chu-clermontferrand.fr
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Summary

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.

Keywords

Blastocystiscysteine proteasesCathepsin BLegumainCaco-2permeability
Type
Research Article
Information
Parasitology , Volume 143 , Issue 13 , November 2016 , pp. 1713 - 1722
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Copyright
Copyright © Cambridge University Press 2016 

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