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Characterization of metalloproteases and serine proteases of Toxoplasma gondii tachyzoites and their effect on epithelial cells

  • Immunology and Host-Parasite Interactions - Original Paper
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Abstract

Toxoplasma gondii can infect all nucleated cells from warm-blooded organisms. After infection, Toxoplasma spreads throughout the body and migrates across biological barriers, such as the intestinal and blood-brain barriers, as well as the placenta in pregnant women. The mechanisms for parasite dissemination are still unknown; however, proteases could play a role as a virulence factor. The aim of this study was to detect and to characterize proteases in whole-cell extracts and in excretion/secretion products from tachyzoites of the RH strain isolated from infected mice. Both fractions were analyzed by gelatin and casein zymography and by azocasein degradation. The biochemical characterization of proteases included standardization of optimal conditions for their activation, such as pH, the presence of cofactors, and a reducing agent. In both fractions, we detected at least nine gelatin-degrading metalloproteases in the range of 50 to 290 kDa. The proteases present in the excretion/secretion products were found as soluble proteins and not associated with exosome-like vesicles or other secretory vesicles. Moreover, by using casein zymography, it was possible to detect three serine proteases. Exposure of MDCK cells to excretion/secretion products modified the organization of the cell monolayer, and this effect was reverted after washing thoroughly with PBS and inhibition by metalloprotease and serine protease inhibitors. Proteomic analysis of excretion/secretion products identified 19 proteases. These findings suggest that tachyzoites of a highly virulent strain of Toxoplasma use a battery of proteases to modify the epithelium, probably as a strategy to facilitate their tissue dissemination.

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Acknowledgements

This research was supported by grants from Fundación Miguel Alemán A.C. and by Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN) to RMF and scholarships from Consejo Nacional de Ciencia y Tecnología (CONACyT, México) to CJRF and RCM (296155 and 295997, respectively). We thank R. Mondragón-González from Departamento de Genética y Biología Molecular (CINVESTAV-IPN) for his comments and A. Chagolla-López from the Departamento de Biotecnología y Bioquímica (CINVESTAV-IPN, Irapuato) for her technical support.

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Authors and Affiliations

  1. Departamento de Bioquímica, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Av. IPN No. 2508, Col. Zacatenco, C.P. 07360, Ciudad de México, Mexico

    Carlos J. Ramírez-Flores, Rosalba Cruz-Mirón, Mónica E. Mondragón-Castelán, Tais Nopal-Guerrero & Ricardo Mondragón-Flores

  2. Departamento de Infectómica y Patogénesis Molecular, CINVESTAV-IPN, Ciudad de México, Mexico

    Rossana Arroyo

  3. Unidad de Microscopía Electrónica, LaNSE, CINVESTAV-IPN, Ciudad de México, Mexico

    Sirenia González-Pozos

  4. Unidad de Genómica, Proteómica y Metabolómica, LaNSE, CINVESTAV-IPN, Ciudad de México, Mexico

    Emmanuel Ríos-Castro

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  1. Carlos J. Ramírez-Flores
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  2. Rosalba Cruz-Mirón
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  3. Rossana Arroyo
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  4. Mónica E. Mondragón-Castelán
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  8. Ricardo Mondragón-Flores
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Correspondence to Ricardo Mondragón-Flores.

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Online Resource 1

The proteases detected by zymography are not oligomers and their activity was not affected by the solvents of the inhibitors. a Effect of 4 M urea in the proteolytic pattern of WE and E/S products. U- and U+ correspond to the sample without or with urea respectively. b Zymograms of WE products incubated with the solvents used to dissolve the inhibitors. (PDF 435 kb)

Online Resource 2

Zymography of whole-cell extracts (a) and excretion/secretion products (b) are similar. Different amounts of parasites were used to prepare the WE fractions or the E/S products. (PDF 529 kb)

Online Resource 3

Viability of the parasites under the conditions used for harvesting of E/S products. Micrographs of parasites maintained in PBS for 4 h at 37 °C and then stained with SYTOX-green dye. (PDF 165 kb)

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Ramírez-Flores, C.J., Cruz-Mirón, R., Arroyo, R. et al. Characterization of metalloproteases and serine proteases of Toxoplasma gondii tachyzoites and their effect on epithelial cells. Parasitol Res 118, 289–306 (2019). https://doi.org/10.1007/s00436-018-6163-5

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