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Protein palmitoylation is a post-translational modification that contributes to determining protein localization and function. Palmitoylation has been described in trypanosomatid protozoa, but no zDHHC palmitoyl transferase has been identified in Trypanosoma cruzi, the etiological agent of Chagas disease in Latin America. In this study we identify and show the subcellular localization of TcHIP (Tc00.1047053508199.50), a putative T. cruzi zDHHC palmitoyl transferase. Analysis of the deduced protein sequence indicates that it contains ankyrin repeats (Ank and Ank2) and the zDHHC conserved domain, typical of zDHHC palmitoyl transferases. A TcHIP polyclonal antiserum obtained from mice immunized with the purified recombinant protein was used to study the presence and subcellular localization of the native enzyme. In western blots this antiserum recognized a protein of about 95 kDa, consistent with the predicted molecular mass of TcHIP (95.4 kDa), in whole extracts of T. cruzi epimastigotes, metacyclic trypomastigotes and intracellular amastigotes. Immunolocalization by confocal microscopy showed TcHIP labeling at the Golgi complex, co-localizing with the T. cruzi Golgi marker TcRab7-GFP. Transfectant T. cruzi epimastigotes containing a construct encoding TcHIP fused to proteins A and C (TcHIP/AC) were obtained. In western blotting experiments, the TcHIP polyclonal antiserum recognized both native and TcHIP/AC proteins in extracts of the transfectants. Confocal microscopy showed co-localization of native TcHIP with TcHIP/AC. These findings demonstrate the presence of a putative zDHHC palmitoyl transferase (TcHIP) containing ankyrin and zDHHC domains in different developmental forms of T. cruzi, and its association with the Golgi complex.

Here we describe the visualization by confocal microscopy of ingested gold (15 nm)-labeled transferrin in epimastigote forms of the protozoan Trypanosoma cruzi. Intracellular gold labeling was evident at two sites, which represent the bottom of the cytopharynx and the reservosomes. The gold tracer was best observed by confocal microscopy by using the 633 nm excitation wavelength. Intracellular gold clusters larger than 60 nm could be visualized by either gold reflection (light scattering) or photoluminescence modes. The gold reflection mode, the gold photoluminescence mode and the anti-transferrin immunofluorescence image of gold-labeled transferrin showed co-localization, thus demonstrating that the gold visualization modes did not represent artifacts or mislocalization of the biomarker. Visualization of protein-gold nanoparticle complexes by confocal microscopy thus emerges as a promising imaging tool to explore the endocytic pathway in trypanosomes and other cell types, as well as to perform immunolocalization studies using gold-labeled secondary antibodies.