in the plasma of vertebrates that acts as a general proteinase inhibitor, markedly inhibits the infection of macrophages and fibroblasts by T. cruzi (Araujo-Jorge et.
Cysteine proteinase in Trypanosoma cruzi: immunocytochemical localization and involvement in parasite-host cell interaction
THAIS SOUTO-PADRON, OSCAR E. CAMPETELLA, JUAN J. CAZZULO and WANDERLEY DE SOUZA Laboratdrio de Ultra-estrutura Celular e Microscopia Eletrdnica, Departamento de Parasitologia e Bioflsica Celular, Institute de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Ilha do Fundao, 21949, Rio de Janeiro, Brasil and Instituto de Investigaciones Bioquimicas, Fundacion Campomar, Antonio Machado 151, 1405 Buenos Aires, Argentina
Summary A monospecific polyclonal antibody obtained against a cysteine proteinase isolated from epimastigotes of Trypanosoma cruzi was used for the immunocytochemical localization of the protein by electron microscopy and to analyse the role played by cysteine proteinase in the process of T. cruzi-host cell interaction. Cytoplasmic structures that correspond to elements of the endosomal-lysosomal (reservosome) system found in epimastigote, amastigote and trypomastigote forms reacted intensely with colloidal gold-labelled antibodies using on-section indirect labelling. The surface of most of the tissue culturederived trypomastigotes was not labelled. However, the flagellar pocket of this form was labelled. All epimastigotes obtained from axenic cultures and amastigote-like forms found in the supernatant of vertebrate cells heavily infected with T. cruzi had
their surface intensely labelled, indicating also the surface localization of the protein. Incubation of the parasites in the presence of a sub-agglutinating concentration of the anti-cysteine proteinase antibody led to a marked increase in their uptake by macrophages. In contrast, addition of the F(ab')2 portion of the same antibody significantly reduced the uptake of the parasites by the macrophages. The results obtained strongly suggest an important participation of cysteine proteinase in the process of T. cruzimacrophage interaction.
Introduction
zulo et al. 1989a); and (3) is a high-mannose type of glycoprotein, as shown by binding to concanavalin A Sepharose, endo-AT-acetyl-glucosaminidase H sensitivity, and determination of oligosaccharide chain composition (Cazzulo et al. 1989a, 1990a). The purified protein has been used to obtain polyclonal antibodies (Campetella et al. 1990). In the present paper we describe the results obtained using these antibodies, as well as antibodies that recognize the cytosolic enzyme NADP-linked glutamate dehydrogenase (Cazzulo et al. 19896), for the immunocytochemical localization of the proteins in thin sections of the epimastigote, amastigote and trypomastigote forms of T. cruzi. F(ab')2 portions of the antibodies are used in experiments on T. cruzi—macrophage interaction. Our observations show that: (1) cysteine proteinase is located in lysosomes of epimastigotes but is also expressed on the surface of epimastigotes and amastigote-trypomastigote transitional forms; and (2) addition of anti-proteinase antibodies to the interaction medium significantly inhibits the ingestion of parasites by macrophages.
Studies carried out in recent years have shown that Trypanosoma cruzi infects vertebrate cells by a process of endocytosis following an initial step of parasite-host cell recognition (see review by Zingales and Colli, 1985; Araujo-Jorge, 1990). In this process components located on the surface of the parasite and the host cell play an important role. There is some evidence suggesting the involvement of parasite proteinases in the process of T. cruzi-host cell interaction. Treatment of infective forms with proteinases increases their interaction with host cells (Piras et al. 1985). Addition to the interaction medium of alpha-2macroglobulin, a glycoprotein found in high concentration in the plasma of vertebrates that acts as a general proteinase inhibitor, markedly inhibits the infection of macrophages and fibroblasts by T. cruzi (Araujo-Jorge et al. 1986). We have recently isolated and characterized a lysosomal cysteine proteinase, for which we propose the trivial name 'cruzipain' (Cazzulo et al. 19906), from epimastigotes of T. cruzi. This enzyme: (1) is able to degrade bovine serum albumin, hemoglobin and azocasein at pH 5.0 (Bontempi et al. 1989); (2) contains amino acid sequences presenting considerable homology with papain and cathepsin L (CazJournal of Cell Science 96, 485-490 (1990) Printed in Great Britain © The Company of Biologists Limited 1990
Key words: Trypanosoma cruzi-macrophage interaction, cysteine proteinase, lysosomal enzyme, immunocytochemistry, electron microscopy.
Materials and methods Antigens and sera Cysteine proteinase and NADP-linked glutamate dehydrogenase 485
were purified as previously described (Cazzulo et al. 1989a,6). To obtain polyclonal antisera rabbits were immunized by intramuscular injection (hind legs) of 50 /
