TRYPOMASTIGOTE SMALL SURFACE ANTIGEN ABLATION CAUSES INFECTION IMPAIRMENT IN TRYPANOSOMA CRUZI

BALOUZ, VIRGINIA; CÁMARA, MARÍA DE LOS MILAGROS; CRUZ BUSTOS, TERESA; DOCAMPO, ROBERTO; BUSCAGLIA, CARLOS A.

Resistencia

Congreso; XXX Reunión de la Sociedad Argentina de Protozoología; 2018

Sociedad Argentina de Protozoología

Trypanosoma cruzi is the etiological agent of Chagas disease. tssa (Trypomastigote Small Surface Antigen) is a mucin-like gene coding for a glycoprotein displayed on the surface of infective trypomastigote forms that shows amino acids polymorphisms among parasite isolates. These polymorphisms correlate with differential antibody responses in T. cruzi-infected humans and differential adhesion towards non-macrophagic cell monolayers. The TSSAII variant (present in TcII, TcV and TcVI DTUs) has been characterized as a parasite adhesin, engaging surface receptor(s) and inducing signaling pathways on the host cell as a prerequisite for trypomastigote internalization. Most interestingly, trypomastigotes over-expressing TSSAII displayed improved adhesion and infectivity towards non-macrophagic cell lines in vitro. Moreover, TSSAII overexpression leads to an enhanced trypomastigote-to-amastigote conversion, indicating a possible role of TSSA also in parasite differentiation. To get further insights into the functional significance of TSSA, we applied CRISPR/Cas9 technique in the RA strain (TcVI) to obtain TSSAII-KO parasites. After antibiotic selection, epimastigotes were cloned and genotypified by PCR and sequencing. tssa genes were successfully ablated. Clones of interest were cycled in vitro to obtain infective forms. We confirmed the lack of expression of TSSAII protein in trypomastigotes by Western blot and IFA. Most interestingly, at least 2 independent TSSAII-KO clones were significantly less infective in non-macrophagic cell monolayers than control cell lines in in vitro infection assays (p ˂ 0,05, one-way ANOVA, Bonferroni post-test). In vivo infection assays using immunocompetent and immunodeficient mice are underway. Altogether, our results shed new light on the role of TSSAII on the interaction between T. cruzi and the host cell. Elucidation of this interaction and the molecules and signals involved is essential for the discovery of new drugable targets and development of new tools to fight against Chagas Disease.