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.
Departamento 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.
Ana Cristina
AC
Nogueira de Melo
Edilma Paraguai
EP
de Souza
Camila Guarany Ramalho
CG
Elias
André Luis Souza
AL
dos Santos
Marta Helena
MH
Branquinha
Cláudia Masini
CM
d'Avila-Levy
Flávia Coelho Garcia
FC
dos Reis
Tatiana Ferreira Rocha
TF
Costa
Ana Paula Cabral de Araujo
AP
Lima
Mirian Cláudia
MC
de Souza Pereira
Maria Nazareth Leal
MN
Meirelles
Alane Beatriz
AB
Vermelho
D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D047468:Immunoprecipitation; D020780:Matrix Metalloproteinase 9 / Q000032:analysis* / Q000737:chemistry; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000254:growth & development
D020780:Matrix Metalloproteinase 9