Expression, localization and function of stage-specific TcTASV-B protein by means of transgenic Trypanosoma cruzi TcI parasites

RIZZI, M; CAEIRO, L; RODRIGUEZ, ME; TEKIEL, V; MASIP, Y

Caxambu

Congreso; XXXV Reunião Anual da Sociedade Brasileira de Protozoologia; XLVI Reunião Anual da Pesquisa Básica em Doença de Chagas.; 2019

Sociedad Brasileira de Protozoología

TcTASV is a surface protein family of T. cruzi comprised of about 40 genes. The amino and carboxy terminal portions are conserved, and a variable central core allows dividing TcTASV in subfamilies A, B, C and W. All TcTASV are expressed in trypomastigotes and have no orthologs in other organisms, hence being potential candidates for vaccines or drug targets. Recently, we have been studying TcTASV-B, which has the peculiarity that it seems to be absent in T. cruzi strains belonging to DTU TcI. By cloning, sequencing and/or detailed in silico inspection we detected TcTASV-B in genomes of TcII, TcV and TcVI strains (no in TcI). By using polyclonal antisera we detected TcTASV-B expression in both trypomastigotes and amastigotes, localized at the surface as discrete points, although it is not secreted. As the function of TcTASV-B is unknown and taking advantage of the absence of TcTASV-B in TcI strains, we expressed a tagged TcTASV-B gene in TcI parasites (Dm28 strain), using a tetracycline inducible vector. Our final goal is to obtain transgenic trypomastigotes to interrogate TcTASV-B function both in vitro and in vivo. Expression of TcTASV-B was detectable in transfected epimastigotes after 4hs of induction and did not affect parasite growth rate. Indirect immunofluorescence with monoclonal anti HA antibody revealed that TcTASV-B localizes all along on the membrane surface. Metacyclic trypomastigotes were used to infect Vero cells to obtain transgenic Dm28 amastigotes and trypomastigotes. Although TcTASV-B expression was detected in transgenic amastigotes, cell-released trypomastigotes were scarce and tended to round up quickly. TcTASV-B was localized at the membrane surface with two characteristic labeling patterns (discret points or polarized), which were different to the observed in transgenic epimastigotes. We are currently optimizing TcTASV-B expression in transgenic trypomastigotes, to evaluate its infection and transmigration ability in 3D culture systems.