Characterization of two cysteine proteases secreted by Blastocystis ST7, a human intestinal parasite Ivan Wawrzyniak a, b , Catherine Texier a, b , Philippe Poirier a, b , Eric Viscogliosi c, d, e, f , Kevin S.W. Tan g , Frédéric Delbac a, b , Hicham El Alaoui a, b, ⁎ a Clermont Université, Université Blaise Pascal, Laboratoire Microorganismes: Génome et Environnement, BP 10448, F-63000 Clermont-Ferrand, France b CNRS, UMR 6023, LMGE, F-63177 Aubière, France c University Lille-Nord de France, F-59000 Lille Cedex, France d Center for Infection and Immunity of Lille, Institut Pasteur de Lille, F-59019 Lille Cedex, France e Inserm U1019, F-59000 Lille Cedex, France f CNRS UMR 8402, F-59021 Lille Cedex, France g Laboratory of Molecular and Cellular Parasitology, Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 5 Science Drive 2, 117597 Singapore abstract article info Article history: Received 24 August 2011 Received in revised form 17 February 2012 Accepted 20 February 2012 Available online 25 February 2012 Keywords: Blastocystis Secreted proteases Cathepsin Legumain Pathogenicity Proteomics Blastocystis spp. are unicellular anaerobic intestinal parasites of both humans and animals and the most prevalent ones found in human stool samples. Their association with various gastrointestinal disorders raises the questions of its pathogenicity and of the molecular mechanisms involved. Since secreted proteases are well-known to be implicated in intestinal parasite virulence, we intended to determine whether Blastocystis spp. possess such pathogenic factors. In silico analysis of the Blastocystis subtype 7 (ST7) genome sequence highlighted 22 genes coding proteases which were predicted to be secreted. We characterized the proteolytic activities in the secretory products of Blastocystis ST7 using specific protease inhibitors. Two cysteine proteases, a cathepsin B and a legumain, were identified in the parasite culture supernatant by gelatin zymographic SDS-PAGE gel and MS/MS analysis. These proteases might act on intestinal cells and disturb gut function. This work provides serious molecular candidates to link Blastocystis spp. and intestinal disorders. © 2012 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Blastocystis spp. are unicellular anaerobic parasites belonging to the stramenopile group and are found in the intestinal tract of humans as well as of a wide range of animals such as mammals, birds and rodents [1]. Four major forms, called vacuolar, granular, amoeboid and cystic, are described for this polymorphic protozoan parasite. A life cycle with cysts being the infectious stages is hypoth- esized although the transitions between the different forms remain to be elucidated [1]. Blastocystis spp. prevalence in human ranges from 3% to 5% in developed countries to 60% in developing ones [1]. But it is believed to be largely underestimated [2], since data significantly vary according to the methods used for diagnosis. A recent prospective study using a real-time quantitative PCR approach demonstrated a 14.5%-prevalence of Blastocystis spp. in France, compared to a 4%-one obtained by direct light microscopy observation [3]. Thirteen different subtypes (ST1 to ST13) are defined using a partial sequence of the small ribosomal subunit rRNA gene [1,2]. Among them, subtypes ST1 to ST9 are recovered in human fecal samples with highly variable frequencies according to geographical areas [1]. The pathogenic poten- tial of Blastocystis spp. remains controversial as the parasite is described in both asymptomatic and symptomatic patients. Blastocystis spp.- associated symptomatology consists in gastrointestinal disorders such as abdominal pains, diarrhea, nausea, cramps or urticarial symptoms [1,2]. Blastocystis spp. are also suggested to be involved in the irritable bowel syndrome (IBS) [4]. Proteases are well known to play major roles in parasitic biology and in host–pathogen interactions. In enteric parasites such as Entamoeba spp. or Giardia spp., cysteine proteases are shown to be efficient virulence factors [5,6]. Entamoeba histolytica trophozoites secrete more cysteine proteases than the non invasive species Entamoeba dispar [5], supporting the role of these enzymes in pathogenesis. In particular, proteases can alter gut integrity [7] and modulate host immune system [5]. Protease activities from Blastocystis ST4 and ST7 cell lysates [8,9], and Blastocystis ST4 culture supernatants [10] were previously reported. They were shown to be able to cleave human immunoglobulins A in vitro [10], to modulate inflammatory IL-8 production [11] and to increase permeabili- ty of intestinal epithelial cells [12]. Although these data strongly suggest a role of proteases in Blastocystis spp. physiopathology, the corresponding Parasitology International 61 (2012) 437–442 ⁎ Corresponding author at: UBP, UMR CNRS 6023, LMGE, 24 Avenue des Landais, BP 80026, F-63171 Aubière, France. Tel.: +33 4 73 40 74 31; fax: +33 4 73 40 76 70. E-mail address: hicham.el_alaoui@univ-bpclermont.fr (H. El Alaoui). 1383-5769/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.parint.2012.02.007 Contents lists available at SciVerse ScienceDirect Parasitology International journal homepage: www.elsevier.com/locate/parint