Amastigotes obtained by axenic differentiation of tissue culture trypomastigotes contain a major 84-kDa iodinatable glycoprotein designated Ssp-4 that is linked to the parasite membrane through a GPI anchor (13,20). Ssp-4 and other structurally related components are the parasite's immunodominant elements since they are readily recognized by different mAbs raised against isolated amastigotes (13,23,35,40). In the present study we have described a previously uncharacterized polymorphism of T. cruzi amastigote surface components. Using mAbs directed at carbohydrate and non-carbohydrate epitopes located on the parasite surface, we could detect by ELISA both strain and clonal variation in the expression of these epitopes among amastigotes of different T. cruzi isolates. In addition, immunoblotting analysis of these parasites using the same mAbs revealed particular patterns of reaction, indicating a polymorphism in the expression of these antigens among the different strains and clones of T. cruzi. A similar behavior regarding distinct reactivities of mAbs in different immunochemical assays (e.g., immunofluorescence, immunoblotting, complement-mediated lysis and agglutination) has been extensively documented in the literature. We have previously described a similar trend in the expression of a mucin and of a major surface component of T. cruzi metacyclic trypomastigotes (10,11).
We have observed that most of the mAbs that recognize Ssp-4 react with carbohydrate epitopes (35), a fact that has facilitated the use of glutaraldehyde fixation in our ELISAs. Indeed, a survey of the literature shows that most of the mAbs raised against T. cruzi amastigotes recognize carbohydrate epitopes (13,23). These observations suggest that these developmental stages are covered with a significant proportion of sugar residues. Polymorphism of surface molecules seems to be a common feature of T. cruzi forms and has been described in epimastigotes (3), tissue culture trypomastigotes (8,9) and metacyclic trypomastigotes (10,11). If the polymorphic expression of peptide epitopes in these studies has emerged as a fairly common feature for T. cruzi (3,8,9), the variability in the carbohydrate moieties mainly attached to parasite surface glycoproteins is less well characterized (11). Other parasitic protozoans like Plasmodium and Leishmania have also been shown to display extensive antigenic polymorphism (41,42).
There are numerous strains and clones of T. cruzi being used by different laboratories. These strains have distinct biological properties. Some isolates can even contain subpopulations that range from highly lethal to mildly infective in murine models (30). Although the molecular basis for this diversity is not known, it is likely that chromosomal rearrangements also occur in T. cruzi, similar to what has been described for other protozoans like Giardia, T. brucei and Plasmodium (43).
Extracellular amastigotes of the Y strain have been shown to be infective for different cell types and are able to sustain an infective cycle in vivo (14). Comparative studies carried out with both intracellular and extracellular forms of the Y strain indicate that extracellular amastigotes are more infective and comparatively more resistant to complement-mediated lysis (Barros HC, Verbisck NV, Da-Silva S and Mortara RA, unpublished data). Although several studies have shown that amastigotes can be found in the peripheral blood of infected animals (14,44), particularly during the acute phase of the disease, the role of circulating amastigotes in the course of natural infection remains unclear (14,19). T. cruzi strains that are particularly virulent and lethal for animal models always induce high parasitemias, a fact that has been correlated with the infectivity of the circulating trypomastigotes. In preliminary studies, we have found that extracellular amastigotes from T. cruzi strains that are less virulent and only cause subpatent parasitemia, like the G and MD strains, are far more infective to HeLa cells than forms of the more virulent isolates like Tulahuen, F, Y and CL strains (Barros HC, Da-Silva S and Mortara RA, unpublished data). Whether this unexpected pattern of infectivity is significant in the natural course of infection in vivo is not known, but this phenomenon clearly deserves further investigation. Since Ssp-4 is a major surface component of T. cruzi amastigotes it could also play a role in the process of parasite adhesion and invasion.
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