Detection of matrix metallopeptidase-9-like proteins in Trypanosoma cruzi.

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.

In vitro evidence for metallopeptidase participation in hepatocyte damage induced by Leishmania chagasi-infected macrophages.

Leishmania (Leishmania) chagasi infection activates macrophages, which release several microbicidal agents, including peptidases, to eliminate the parasite. Leishmanicidal mediators released in large amounts may cause morphological and/or functional injuries to the liver. In order to investigate the involvement of peptidases in this phenomenon, an in vitro co-culture model of peritoneal macrophages infected with L. chagasi and hepatocytes was used. High levels of released hepatic transaminases were found in supernatants from infected co-cultures at the same time point in which alterations in hepatocyte morphology and maximum proteolytic activity were observed. The largest proteolytic activity being at pH 10 as well as the greatest efficiency of treatment with 1,10-phenantroline observed in supernatants from the infected co-cultures suggests the presence of metallopeptidases during the leishmanicidal activity by infected macrophages. Furthermore, TNF-alpha levels and high levels of TGF-beta were increased at this time point, and this can be related to the synthesis of metallopeptidases and the conversion of the latent form to the active form. Metallopeptidase activities were detected by gelatin SDS-PAGE in higher amounts in infected macrophages and co-culture supernatant; moreover, one metallopeptidase migrating at 85 kDa produced in excess (41% more) by infected macrophages was identified as MMP-9. This metallopeptidase may be participating in this phenomenon together with other leishmanicidal factors released by these host cells.

Insights into the role of gp63-like proteins in lower trypanosomatids.

Any actual understanding of trypanosomatids in general requires a comprehensive analysis of the less-specialized species as thorough as our knowledge of the more specialized Leishmania and Trypanosoma. In this context, we have shown by antibody cross-reactivity that purified extracellular metallopeptidases from Phytomonas françai, Crithidia deanei (cured strain) and Crithidia guilhermei share common epitopes with the leishmanial gp63. Flow cytometry and fluorescence microscopy analyses indicated the presence of gp63-like molecules on the cell surface of these lower trypanosomatids. Binding assays with explanted guts of Aedes aegypti incubated with purified gp63 and the pretreatment of trypanosomatids with anti-gp63 antibodies indicated that the gp63-like molecules are involved in the adhesive process of these trypanosomatids to the A. aegypti gut wall. In addition, our results indicate for the first time that the gp63-like molecule binds to a polypeptide of 50 kDa on the A. aegypti gut epithelium extract.

Reduced activity of matrix metalloproteinase-9 in trypanosoma cruzi-infected mouse embryo hepatocyte cell.

In this work, we are reporting differences in the proteolytic profile of Trypanosoma cruzi-infected and non-infected primary cultures of mouse embryo hepatocyte cells. In gelatin-SDS-PAGE, ours results showed the presence of a 100kDa metalloproteinase in the supernatant and in the cells of both systems and an 85kDa extracellular metalloproteinase found only in the non-infected hepatocyte cultures. An enzymatic assay using gelatin as substrate showed a decrease of 74 and 70% in metalloproteinase activity in the culture supernatant and in the cell hepatocyte system infected with T. cruzi, respectively. Western blotting analysis using anti-matrix metalloproteinase-9 (MMP-9) antibody recognized the 100 and 85kDa protein bands, indicating that hepatocyte metalloproteinases correspond to the latent and active forms of the gelatinase MMP-9, respectively. The localization of MMP-9 was established by immunocytochemistry analysis in the cytoplasm of the non-infected and infected hepatocyte cells. In normal and infected hepatocyte cells, cysteine-proteinases migrating in gelatin-SDS-PAGE at 60kDa were detected and should correspond to lysosomal cysteine-proteinases of T. cruzi (cruzipain) and hepatocytes. In T. cruzi-infected hepatocytes an increase of approximately 50% in this enzymatic activity was observed, possibly due to parasite's cruzipain.