Distribution of Epitopes of Trypanosoma cruzi Amastigotes During the Intracellular Life Cycle within Mammalian Cells

Abstract

In this study we have examined the distribution of epitopes defined by monoclonal antibodies raised against Trypanosoma cruzi amastigotes during the intracellular life cycle of the parasite. We have raised monoclonal antibodies towards amastigote forms and performed preliminary immunochemical characterization of their reactivities. MAB 1D9, 3G8, 2B7, 3B9, and 4B9 react with carbohydrate epitopes of the parasite major surface glycoprotein--Ssp-4 defined by MAB 2C2 [5]; MAB 4B5 reacts with a noncarbohydrate epitope in all developmental stages of the parasite, and MAB 3B2 also detects a noncarbohydrate epitope preferentially in T. cruzi flagellated forms. Vero cells infected with tissue culture-derived trypomastigotes of clone D11 (G strain) were fixed at different times during the intracellular proliferation of parasites, and processed for immuno-electron microscopy and confocal immunofluorescence with the different monoclonal antibodies. We observed that while the surface distribution of MAB 2C2 and 4B9 epitopes was uniform throughout the cycle, MAB 1D9, 3G8, and 2B7 reacted with cytoplasmic membrane-bound compartments of the amastigotes. MAB 3B9 displayed a unique surface dentate pattern in some amastigotes. MAB 4B5 recognized a curved-shaped structure at the flagellar pocket region in some intracellular amastigotes and localized to the membrane in dividing forms. In intracellular trypomastigotes, MAB 4B5 also displayed a punctate pattern near the flagellar pocket.

Parameters affecting cellular invasion and escape from the parasitophorous vacuole by different infective forms of Trypanosoma cruzi

“…Antibodies and immunofluorescence labeling -For invasion assay quantitations, cells fixed in 2% glutaraldehyde were incubated with monoclonal antibodies (ascitic fluids diluted 1:40 in PGN): mAb 1G7 against the 90 kDa metacyclic trypomastigote glycoprotein (Teixeira & Yoshida 1986) or mAb 1D9, reactive against the Ssp-4 glycoprotein of amastigotes (Barros et al 1997). After 1 h the coverlisps were washed 3 times with PBS and incubated 1 h with anti-mouse IgG-FITC conjugated (Sigma) diluted 1:50 in PGN and 10 µM DAPI (4',6-diamidino-2-phenylindole dihydrochloride, Molecular Probes, Eugene, Oregon, US).…”

“…Invasion index was calculated as described above. For conventional immunofluorescence infected cells were fixed with 3.5% formaldehyde, washed and then permeabilized with PGN solution containing 0.1% saponin (Barros et al 1997). All statistical calculations describe here were done with SigmaStat (Version 1.0, Jandel Scientific), using the T-test for significance and standard deviations for paired data sets.…”

Morphological comparison of axenic amastigogenesis of trypomastigotes and metacyclic forms of Trypanosoma cruzi

“…Most of the information concerning the in vitro and in vivo amastigogenesis process comes from the study of tissue-derived trypomastigote forms (Nogueira & Cohn 1976, Milder et al 1977, Ley et al 1988, Burleigh & Andrews 1995, Barros et al 1996, 1997, but little is known about the developmental process driving the metacyclic forms to amastigotes. Although, metacyclic trypomastigotes can appear similar in morphology and share some biological properties with tissue-derived trypomastigotes.…”

Features of host cell invasion by different infective forms of Trypanosoma cruzi

“…Interestingly, the infectivity to HeLa cells of extracellular amastigotes correlated with the expression of a carbohydrate epitope defined by monoclonal antibody 1D9 (Barros et al 1997, Verbisck et al 1998 (Figure).…”