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Trypanosoma cruzi: differential expression and distribution of an 85-kDa polypeptide epitope by in vitro
M A Ouaissi1, J F Dubremetz, J P Kusnierz
1Centre d'Immunologie et de Biologie Parasitaire, Unité Mixte INSERM U 167-CNRS 624, Institut Pasteur, Lille, France.
Abstract:
The expression by Trypanosoma cruzi developmental stages of an 85-kDa polypeptide epitope defined by the 155D3 monoclonal antibody (mAb) has been investigated. Immunoprecipitation revealed the presence of an 85-kDa antigen in the NP-40 soluble extract of parasites freshly released from infected fibroblasts; this antigen was not found in epimastigote and Leishmania infantum promastigote. Indirect immunofluorescence revealed that the mAb 155D3 failed to react with trypomastigotes, whereas extracellular amastigotes were heavily stained. Positive organisms displayed either surface or polar fluorescence. Since the same mAb immunoprecipitated the 85-kDa antigen in both radioactive iodine- and methionine-labeled trypomastigote detergent soluble extracts, the reactive epitope is likely to be hidden in a cryptic site in trypomastigotes. An alternative explanation for the negative immunofluorescence on trypomastigotes and the positive immunoprecipitation is the presence, in the extracts, of a small population of parasites already expressing the 155D3 epitope. Immunoelectron microscopy revealed that the target epitope is heterogenously distributed among the populations of differentiating parasites. Two types of immunogold labeling were observed: (a) mAb revealed a high amount of reactive material associated with the periphery of the parasites and (b) a label was observed on the inner surface of peripheral vacuoles that might correspond to cross sections of inflated flagellar pockets and in association with vesicles which were released by the parasites. The surface expression of the epitope recognized by the 155D3 mAb was followed by fluorescence-activated cell-sorting analysis. The results showed that the epitope is increasingly accessible during trypomastigote differentiation in vitro. Taken together, these results suggest that the epitope reacting with the 155D3 mAb is heavily expressed on extracellular amastigotes after the transformation process and, thus, appears to be developmentally regulated.
Insights
The 155D3 monoclonal antibody (mAb) targets a developmentally regulated 85-kDa epitope in Trypanosoma cruzi. This epitope is primarily expressed on extracellular amastigotes after transformation, suggesting a role in parasite development.
Area of Science:
- Parasitology
- Molecular Biology
- Immunology
Background:
- Trypanosoma cruzi exhibits complex developmental stages, crucial for its transmission and pathogenesis.
- Understanding stage-specific antigen expression is vital for developing diagnostic and therapeutic strategies against Chagas disease.
Purpose of the Study:
- To investigate the expression of an 85-kDa polypeptide epitope, recognized by the 155D3 monoclonal antibody (mAb), across different developmental stages of Trypanosoma cruzi.
- To determine the localization and developmental regulation of this epitope.
Main Methods:
- Immunoprecipitation using the 155D3 mAb to identify the antigen in parasite extracts.
- Indirect immunofluorescence microscopy to visualize epitope localization on intact parasites.
- Immunoelectron microscopy for high-resolution mapping of the epitope.
- Fluorescence-activated cell sorting (FACS) to quantify surface epitope accessibility during differentiation.
Main Results:
- The 85-kDa antigen was detected in trypomastigotes but not in epimastigotes or Leishmania infantum.
- Indirect immunofluorescence showed strong staining on extracellular amastigotes, but not on trypomastigotes, suggesting a cryptic epitope in the latter.
- Immunoelectron microscopy revealed heterogeneous distribution, with labeling on the parasite periphery, peripheral vacuoles, and released vesicles.
- FACS analysis indicated increased epitope accessibility during in vitro trypomastigote differentiation.
Conclusions:
- The 155D3 mAb recognizes a Trypanosoma cruzi epitope that is developmentally regulated.
- This epitope is predominantly expressed on extracellular amastigotes following transformation, indicating its significance in the parasite's life cycle.
- The findings suggest the epitope's potential as a marker for specific developmental stages.