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Detection of matrix metallopeptidase-9-like proteins in Trypanosoma cruzi
Ana Cristina Nogueira de Melo1, Edilma Paraguai de Souza, Camila Guarany Ramalho Elias
1Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Góes (IMPPG), Centro de Ciências da Saúde (CCS), Bloco I, Universidade Federal do Rio de Janeiro (UFRJ), Ilha do Fundão, Rio de Janeiro, RJ, Brazil.
Abstract:
In this study, the cell-associated and extracellular peptidases of Trypanosoma cruzi grown in modified Roitman's complex (MRC) medium were analyzed by measuring peptidase activity in gelatin-containing zymograms. Our results showed that the cell-associated peptidases as well as peptidases extracellularly released by T. cruzi displayed two distinct proteolytic classes: cysteine and metallopeptidase activities. The major cysteine peptidase, cruzipain, synthesized by T. cruzi cells was detected in cellular parasite content, as a 50kDa reactive polypeptide, after probing with anti-cruzipain antibody. In addition, metallo-type peptidases belonging to the matrix metallopeptidase-9 (MMP-9) family were revealed, after Western blotting, as a 97kDa protein band in cellular extract and an 85kDa polypeptide in both cellular and secreted parasite extracts. The MMP-9-like activity present in cells and spent culture medium was immunoprecipitated by an anti-MMP-9 polyclonal antibody. The surface location of MMP-9-like proteins in T. cruzi was also evidenced by means of flow cytometry analysis. Furthermore, doxycycline that has direct MMP-9 inhibiting properties in vitro, inhibited MMP-9-like activities in gel zymography, immunoprecipitation and flow cytometry analyses. This is the first report of the presence of MMP-9-like molecules in T. cruzi. The presence of a matrix extracellular-degrading enzyme may play a role in the T. cruzi-host cell interaction, making this enzyme a potential target for future drug development against this pathogenic trypanosomatid.
Insights
This study identifies cysteine and metallopeptidase activities in Trypanosoma cruzi, including matrix metallopeptidase-9 (MMP-9)-like molecules. These findings suggest MMP-9 may be a drug target for Chagas disease.
Area of Science:
- Parasitology
- Biochemistry
- Molecular Biology
Background:
- Trypanosoma cruzi causes Chagas disease.
- Understanding parasite enzymes is crucial for drug development.
Purpose of the Study:
- To analyze cell-associated and extracellular peptidases of Trypanosoma cruzi.
- To investigate the presence and function of matrix metallopeptidase-9 (MMP-9)-like molecules.
Main Methods:
- Gelatin zymography to detect peptidase activity.
- Western blotting and immunoprecipitation using specific antibodies.
- Flow cytometry to determine surface protein localization.
- In vitro inhibition assays with doxycycline.
Main Results:
- T. cruzi secretes cysteine and metallopeptidase activities.
- Matrix metallopeptidase-9 (MMP-9)-like proteins were detected in cellular and secreted extracts.
- MMP-9-like molecules are present on the parasite surface.
- Doxycycline inhibited MMP-9-like activities.
Conclusions:
- This is the first report of MMP-9-like molecules in T. cruzi.
- MMP-9-like enzymes may facilitate T. cruzi-host cell interactions.
- MMP-9-like molecules represent a potential therapeutic target for Chagas disease.
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