On Blastocystis secreted cysteine proteases: a legumain-activated cathepsin B increases paracellular permeability of intestinal Caco-2 cell monolayers | Parasitology | Cambridge Core (original) (raw)

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.

References

Beckham, S. A., Law, R. H., Smooker, P. M., Quinsey, N. S., Caffrey, C. R., McKerrow, J. H., Pike, R. N. and Spithill, T. W. (2006). Production and processing of a recombinant Fasciola hepatica cathepsin B-like enzyme (FhcatB1) reveals potential processing mechanisms in the parasite. Biological Chemistry 387, 1053–1061.CrossRefGoogle ScholarPubMed

Chadwick, V. S., Chen, W., Shu, D., Paulus, B., Bethwaite, P., Tie, A. and Wilson, I. (2002). Activation of the mucosal immune system in irritable bowel syndrome. Gastroenterology 122, 1778–1783.CrossRefGoogle ScholarPubMed

Chen, J. M., Dando, P. M., Rawlings, N. D., Brown, M. A., Young, N. E., Stevens, R. A., Hewitt, E., Watts, C. and Barrett, A. J. (1997). Cloning, isolation, and characterization of mammalian legumain, an asparaginyl endopeptidase. Journal of Biological Chemistry 272, 8090–8098.Google Scholar

Chen, J. M., Fortunato, M. and Barrett, A. J. (2000). Activation of human prolegumain by cleavage at a C-terminal asparagine residue. Biochemical Journal 352(Pt 2), 327–334.CrossRefGoogle Scholar

Dagci, H., Ustun, S., Taner, M. S., Ersoz, G., Karacasu, F. and Budak, S. (2002). Protozoon infections and intestinal permeability. Acta Tropica 81, 1–5.Google Scholar

Dalton, J. P., Brindley, P. J., Donnelly, S. and Robinson, M. W. (2009). The enigmatic asparaginyl endopeptidase of helminth parasites. Trends in Parasitology 25, 59–61.Google Scholar

Denoeud, F., Roussel, M., Noel, B., Wawrzyniak, I., Da Silva, C., Diogon, M., Viscogliosi, E., Brochier-Armanet, C., Couloux, A., Poulain, J., Segurens, B., Anthouard, V., Texier, C., Blot, N., Poirier, P., Ng, G. C., Tan, K. S., Artiguenave, F., Jaillon, O., Aury, J. M., Delbac, F., Wincker, P., Vivares, C. P. and El Alaoui, H. (2011). Genome sequence of the stramenopile Blastocystis, a human anaerobic parasite. Genome Biology 12, R29.CrossRefGoogle ScholarPubMed

Fayer, R., Elsasser, T., Gould, R., Solano, G., Urban, J. Jr. and Santin, M. (2014). Blastocystis tropism in the pig intestine. Parasitology Research 113, 1465–1472.CrossRefGoogle ScholarPubMed

Hernandez, H. M., Figueredo, M., Garrido, N., Sanchez, L. and Sarracent, J. (2005). Intranasal immunisation with a 62 kDa proteinase combined with cholera toxin or CpG adjuvant protects against Trichomonas vaginalis genital tract infections in mice. International Journal for Parasitology 35, 1333–1337.Google Scholar

Ho, L. C., Singh, M., Suresh, G., Ng, G. C. and Yap, E. H. (1993). Axenic culture of Blastocystis hominis in Iscove's modified Dulbecco's medium. Parasitology Research 79, 614–616.Google Scholar

Mayer, E. A., Savidge, T. and Shulman, R. J. (2014). Brain-gut microbiome interactions and functional bowel disorders. Gastroenterology 146, 1500–1512.CrossRefGoogle ScholarPubMed

McKerrow, J. H., Caffrey, C., Kelly, B., Loke, P. and Sajid, M. (2006). Proteases in parasitic diseases. Annual Review of Pathology 1, 497–536.CrossRefGoogle ScholarPubMed

Mirza, H. and Tan, K. S. (2009). Blastocystis exhibits inter- and intra-subtype variation in cysteine protease activity. Parasitology Research 104, 355–361.Google Scholar

Mirza, H., Wu, Z., Teo, J. D. and Tan, K. S. (2012). Statin pleiotropy prevents rho kinase-mediated intestinal epithelial barrier compromise induced by Blastocystis cysteine proteases. Cellular Microbiology 14, 1474–1484.Google Scholar

Mottram, J. C., Coombs, G. H. and Alexander, J. (2004). Cysteine peptidases as virulence factors of Leishmania . Current Opinion in Microbiology 7, 375–381.Google Scholar

North, M. J. (1982). Comparative biochemistry of the proteinases of eucaryotic microorganisms. Microbiological Reviews 46, 308–340.CrossRefGoogle ScholarPubMed

Nourrisson, C., Scanzi, J., Pereira, B., NkoudMongo, C., Wawrzyniak, I., Cian, A., Viscogliosi, E., Livrelli, V., Delbac, F., Dapoigny, M. and Poirier, P. (2014). Blastocystis is associated with decrease of fecal microbiota protective bacteria: comparative analysis between patients with irritable bowel syndrome and control subjects. PLoS ONE 9, e111868.CrossRefGoogle ScholarPubMed

O'Hara, J. R. and Buret, A. G. (2008). Mechanisms of intestinal tight junctional disruption during infection. Frontiers in Bioscience 13, 7008–7021.Google Scholar

Ohman, L., Tornblom, H. and Simren, M. (2015). Crosstalk at the mucosal border: importance of the gut microenvironment in IBS. Nature Reviews: Gastroenterology & Hepatology 12, 36–49.Google Scholar

Poirier, P., Wawrzyniak, I., Vivares, C. P., Delbac, F. and El Alaoui, H. (2012). New insights into Blastocystis spp.: a potential link with irritable bowel syndrome. PLoS Pathogens 8, e1002545.Google Scholar

Puthia, M. K., Vaithilingam, A., Lu, J. and Tan, K. S. (2005). Degradation of human secretory immunoglobulin A by Blastocystis . Parasitology Research 97, 386–389.Google Scholar

Puthia, M. K., Sio, S. W., Lu, J. and Tan, K. S. (2006). Blastocystis ratti induces contact-independent apoptosis, F-actin rearrangement, and barrier function disruption in IEC-6 cells. Infection and Immunity 74, 4114–4123.Google Scholar

Puthia, M. K., Lu, J. and Tan, K. S. (2008). Blastocystis ratti contains cysteine proteases that mediate interleukin-8 response from human intestinal epithelial cells in an NF-kappaB-dependent manner. Eukaryotic Cell 7, 435–443.CrossRefGoogle Scholar

Que, X. and Reed, S. L. (2000). Cysteine proteinases and the pathogenesis of amebiasis. Clinical Microbiology Reviews 13, 196–206.Google Scholar

Rawlings, N. D., Waller, M., Barrett, A. J. and Bateman, A. (2014). MEROPS: the database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Research 42, D503–D509.Google Scholar

Rosenthal, P. J. (2011). Falcipains and other cysteine proteases of malaria parasites. Advances in Experimental Medicine and Biology 712, 30–48.CrossRefGoogle ScholarPubMed

Sajid, M., McKerrow, J. H., Hansell, E., Mathieu, M. A., Lucas, K. D., Hsieh, I., Greenbaum, D., Bogyo, M., Salter, J. P., Lim, K. C., Franklin, C., Kim, J. H. and Caffrey, C. R. (2003). Functional expression and characterization of Schistosoma mansoni cathepsin B and its trans-activation by an endogenous asparaginyl endopeptidase. Molecular Biochemical Parasitology 131, 65–75.CrossRefGoogle ScholarPubMed

Sambuy, Y., De Angelis, I., Ranaldi, G., Scarino, M. L., Stammati, A. and Zucco, F. (2005). The Caco-2 cell line as a model of the intestinal barrier: influence of cell and culture-related factors on Caco-2 cell functional characteristics. Cell Biology and Toxicology 21, 1–26.CrossRefGoogle Scholar

Shirahama-Noda, K., Yamamoto, A., Sugihara, K., Hashimoto, N., Asano, M., Nishimura, M. and Hara-Nishimura, I. (2003). Biosynthetic processing of cathepsins and lysosomal degradation are abolished in asparaginyl endopeptidase-deficient mice. Journal of Biological Chemistry 278, 33194–33199.CrossRefGoogle ScholarPubMed

Singh, S. M. and Panda, A. K. (2005). Solubilization and refolding of bacterial inclusion body proteins. Journal of Bioscience and Bioengineering 99, 303–310.CrossRefGoogle ScholarPubMed

Sio, S. W., Puthia, M. K., Lee, A. S., Lu, J. and Tan, K. S. (2006). Protease activity of Blastocystis hominis . Parasitology Research 99, 126–130.Google Scholar

Tan, K. S. (2008). New insights on classification, identification, and clinical relevance of Blastocystis spp. Clinical Microbiology Reviews 21, 639–665.Google Scholar

Wu, B., Yin, J., Texier, C., Roussel, M. and Tan, K. S. (2010). Blastocystis legumain is localized on the cell surface, and specific inhibition of its activity implicates a pro-survival role for the enzyme. Journal of Biological Chemistry 285, 1790–1798.CrossRefGoogle ScholarPubMed

Wawrzyniak, I., Texier, C., Poirier, P., Viscogliosi, E., Tan, K. S., Delbac, F. and El Alaoui, H. (2012). Characterization of two cysteine proteases secreted by Blastocystis ST7, a human intestinal parasite. Parasitology International 61, 437–442.Google Scholar

Wawrzyniak, I., Poirier, P., Viscogliosi, E., Dionigia, M., Texier, C., Delbac, F. and Alaoui, H. E. (2013). Blastocystis, an unrecognized parasite: an overview of pathogenesis and diagnosis. Therapeutic Advance in Infectious Diseases 1, 167–178.Google Scholar

Wawrzyniak, I., Courtine, D., Osman, M., Hubans-Pierlot, C., Cian, A., Nourrisson, C., Chabe, M., Poirier, P., Bart, A., Polonais, V., Delgado-Viscogliosi, P., El Alaoui, H., Belkorchia, A., Van Gool, T., Tan, K. S., Ferreira, S., Viscogliosi, E. and Delbac, F. (2015). Draft genome sequence of the intestinal parasite Blastocystis subtype 4-isolate WR1. Genomics Data 4, 22–23.CrossRefGoogle ScholarPubMed

Wu, Z., Mirza, H., Teo, J. D. and Tan, K. S. (2014). Strain-dependent induction of human enterocyte apoptosis by blastocystis disrupts epithelial barrier and ZO-1 organization in a caspase 3- and 9-dependent manner. Biomed Research International 2014, 209163.Google Scholar