Enucleated L929 cells support invasion, differentiation, and multiplication of Trypanosoma cruzi parasites

Abstract

Cell infection with Trypanosoma cruzi, the agent of Chagas' disease, begins with the uptake of infective trypomastigotes within phagosomes and their release into the cytosol, where they transform into replicating amastigotes; the latter, in turn, differentiate into cytolytically released and infective trypomastigotes. We ask here if the T. cruzi infection program can develop in enucleated host cells. Monolayers of L929 cells, enucleated by centrifugation in the presence of cytochalasin B and kept at 34 degrees C to extend the survival of cytoplasts, were infected with parasites of the CL strain. Percent infection, morphology, stage-specific markers, and numbers of parasites per cell were evaluated in nucleated and enucleated cells, both of which were present in the same preparations. Parasite uptake, differentiation and multiplication of amastigotes, development of epimastigote- and trypomastigote-like forms, and initial cytolytic release of parasites were all documented for cytoplasts and nucleated cells. Although the doubling times were similar, parasite loads at 48 and 72 h were significantly lower in the cytoplasts than in nucleated cells. Similar results were obtained with the highly virulent strain Y as well as with strains CL-14 and G, which exhibit low virulence for mice. Cytoplasts could also be infected with the CL strain 24 or 48 h after enucleation. Thus, infection of cells by T. cruzi can take place in enucleated host cells, i.e., in the absence of modulation of chromosomal and nucleolar gene transcription and of RNA modification and processing in the nucleus.

FIG. 1.

Nucleated (n) and enucleated (e) L929 fibroblasts infected with T. cruzi strain CL TCTs. Cell monolayers, kept at 34°C throughout, were enucleated, chased in complete medium, infected, washed, and fixed. Parasites in cells fixed 24, 48, and 72 h after infection were labeled with 1D9, an amastigote MAb. Cultures fixed at 96 h were labeled with 3B2, a trypomastigote MAb. In both cases, the primary antibodies were followed by a fluorescein isothiocyanate (FITC)-labeled rabbit anti-mouse IgG secondary antibody. Nuclei were stained with DAPI. Arrows indicate intracellular parasites at 24 h. Bar = 10 μm for all panels.

FIG. 2.

Infection of L929 cytoplasts by T. cruzi strain CL parasites. (A to C) Two hours after enucleation, cells were infected with TCTs for 1 h, washed, chased for 15 min in complete medium, and fixed. (D to F) Similarly infected cells were washed and chased for 24 h. (A and D) DAPI staining; (B and E) anti-LAMP-1 antibody followed by FITC-labeled secondary antibody; (C and F) phase-contrast images of the same cells. Bar = 10 μm. Arrows point to a parasite with the LAMP-1 antigen, presumably localized to the phagosome membrane.

FIG. 3.

. Infection of L929 cytoplasts by TCTs of strain CL. Cells were enucleated and infected as described in Materials and Methods, fixed with methanol at different times, and stained with Giemsa stain. (A) Percentages of infection. (B) Numbers of parasites per cell. Asterisks in panel B indicate statistically significant differences (P < 0.001) in numbers of parasites in nucleated (Nuc) and enucleated (Enu) cells at 48 and 72 h compared with those for the 12-h group (one-way ANOVA with Bonferroni posttest). As indicated by the symbol “#,” the number of parasites in nucleated cells was significantly higher than that in cytoplasts (two-way ANOVA with Bonferroni posttest; P < 0.001). The figure presents the results of one of five separate concordant experiments (means ± standard errors of the means).

FIG. 4.

Frequency distributions of numbers of TCTs of strain CL in infected nucleated and enucleated L929 cells at different times after infection. Data shown are means ± standard errors of the means for the same experiment as that shown in Fig. 3. Time was a statistically significant source of variation (P < 0.001).

FIG. 5.

Intracellular multiplication of TCTs in infected cytoplasts and nucleated cells. Enucleation, infection, staining, and counts were performed as described in Materials and Methods. Estimated doubling times were 25, 46, 16, and 21 h for strains CL, CL-14, G, and Y, respectively. Differences between nucleated cells and cytoplasts were not statistically significant (P > 0.05).