Vol. 61, No. 9 INFECrION AND IMMUNITY, Sept. 1993, p. 3636-3641 0019-9567/93/093636-06$02.00/0 Copyright X) 1993, American Society for Microbiology Involvement of the Stage-Specific 82-Kilodalton Adhesion Molecule of Trypanosoma cruzi Metacyclic Trypomastigotes in Host Cell Invasion MARCEL IVAN RAMIREZ, RITA DE CASSIA RUIZ, JORGE ENRIQUE ARAYA, JOSE FRANCO DA SILVEIRA, AND NOBUKO YOSHIDA* Department of Microbiology, Immunology and Parasitology, Escola Paulista de Medicina, 04023-062 Sdo Paulo, S.P., Brazil Received 9 February 1993/Returned for modification 12 May 1993/Accepted 21 June 1993 This study provides several pieces of evidence indicating that 3F6-Ag, identified by monoclonal antibody (MAb) 3F6 as a stage-specific glycoprotein of approximately 82 kDa on the surface of metacyclic trypomasti- gotes of different Trypanosoma cruzi strains, promotes the entry of parasites into host cells through a ligand-receptor type interaction. First, invasion of Vero cells by metacyclic trypomastigotes of both CL and Tulahuen strains was significantly inhibited by MAb 3F6 or its Fab fragments. Second, purified 3F6-Ag bound to Vero cells in a dose-dependent and saturable fashion. Third, soluble 3F6-Ag reduced the infection of Vero cells by metacyclic forms of CL and Tulahuen strains by 90 to 97 and 50%Yo, respectively. Unrelated proteins, as well as extracellular matrix components, such as heparan sulfate and collagen, had no effect. Our studies also show that in the Tulahuen strain, 10D8-Ag, a 35/50-kDa glycoprotein identified by MAb 10D8, participates in target cell invasion, confirming previous observations, but the variant form of 10D8-Ag expressed by highly invasive CL strain metacyclic trypomastigotes appears to be irrelevant. Overall, our results indicate that the surface components of T. cruzi metacyclic trypomastigotes involved in the process of host cell penetration are developmentally regulated molecules, such as 3F6-Ag and 10D8-Ag, that have no counterpart in blood- or tissue culture-derived trypomastigotes. For many microorganisms that replicate intracellularly in the mammalian host, the crucial step for successful estab- lishment of infection is the penetration into host cells, a process thought to be receptor mediated (6, 7, 10, 17). Trypanosoma cruzi, a protozoan parasite that causes Cha- gas' disease, can be included among such microbial agents. Interaction of T. cruzi with host cells is initiated by metacyclic trypomastigotes, the developmental stages from insect vectors. Once inside the cells, the metacyclic forms escape from endocytic vacuoles to the cytoplasm and trans- form into amastigotes, which after several rounds of multi- plication differentiate into trypomastigotes. Upon rupture of host cells, trypomastigotes circulate in the blood until they encounter appropriate target cells and then go through another intracellular cycle. In the past 10 years, many laboratories have attempted to identify the surface components of T. cruzi implicated in host cell penetration. Several molecules of tissue culture- derived trypomastigotes, ranging from 80 to 250 kDa, have emerged as prospective ligands for target cell receptors, on the basis of either their cell binding capacity or the inhibitory effects of monoclonal antibodies (MAbs) (1, 3, 8, 21). Re- cently, Ortega-Barria and Pereira (16) have reported that penetrin, a novel T. cruzi heparin-binding protein of 60 kDa, promotes adhesion and penetration of trypomastigotes into mammalian cells. On the other hand, metacyclic trypo- mastigotes from insects or axenic cultures express a set of cell surface components that have no counterpart in blood or tissue culture trypomastigotes, such as the molecules of 90, 82, 75, 50, and 35 kDa (2, 24, 26), which may play a role in T. cruzi-host cell interaction. The doublet 35/50-kDa antigen * Corresponding author. defined by MAb 10D8, for instance, appears to be enlisted for invasion of mammalian cells (20). All of these findings point to the complexity of the cell invasion process by T. cruzi. Not only are metacyclic and blood trypomastigotes apparently equipped with distinct sets of molecules for target cell recognition and/or invasion, but it is also possible that different T. cruzi strains do not use the same repertoire of surface components to enter host cells. Furthermore, expression of variant forms of surface antigens has been observed among T. cruzi strains (14, 15), adding another variable to an already complicated picture. In this study, we used metacyclic trypomastigotes of two different strains of T. cruzi to further investigate the surface molecules that promote parasite entry into mammalian cells. We examined in particular the role of 3F6-Ag, a stage- specific surface glycoprotein of approximately 82 kDa de- tectable by MAb 3F6 in different T. cruzi strains (24). MATERIALS AND METHODS Parasites. T. cruzi CL (4), Tulahuen (18), and G (25) were used. Parasites were maintained cyclically in mice and in liver infusion tryptose medium (5). To accelerate differenti- ation into trypomastigotes, T. cruzi was also grown in Grace's medium (GIBCO Laboratories, Grand Island, N.Y.) for 7 to 10 days before the parasites were harvested. Meta- cyclic trypomastigotes from Grace's medium or liver infu- sion tryptose cultures were purified through passage in a DEAE-cellulose column as previously described (24). In some experiments, T. cruzi cultures at the stationary growth phase, rich in metacyclic forms, were maintained for 24 h in the presence of tunicamycin (2 ,ug/ml), an inhibitor of N glycosylation of proteins. Reagents. Type IV collagen was from Sigma Chemical Co. 3636 on July 23, 2020 by guest http://iai.asm.org/ Downloaded from