CRISPR/Cas9: an approach to its use in a trypomastigote-specific multicopy gene

BALOUZ VIRGINIA

Mar del Plata, Buenos Aires

Congreso; Reunión Anual de SOCIEDADES DE BIOCIENCIA 2019; 2019

Trypanosoma cruzi is the etiological agent of Chagas disease. TSSA (Trypomastigote Small Surface Antigen) is encoded by a multicopy mucin-like gene showing 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 TSSAvariant 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 TSSA displayed improved adhesion and infectivity towards non-macrophagic cell lines in vitro. To get further insights into the functional significance of TSSA, we applied CRISPR/Cas9 technique in the RA strain (TcVI)to obtain TSSA-KO parasites. After antibiotic selection, epimastigotes were cloned and genotypified by PCR and PacBIO WGS. Clones of interest, i.e. those bearing partial or complete tssa genetandem ablation, were cycled in vitro and the expression of TSSA protein in trypomastigotes was assessed by Western blot, IFA and flow cytometry. TSSA-KO clones were significantly less infective in non-macrophagic cell monolayers and in a three-dimensional cell array as compared to control cell lines (wild-type and/or TSSA-partial KO) in in vitro infection assays. Interestingly, TSSA-KOparasites showed an attenuated phenotype in vivo evidenced by a decreased parasitemia and virulence in BALB/c mice as compared to control cell lines. Altogether, our results shed new light on the interaction between T. cruzi and the mammalian host. Elucidation of this interphase and the molecules and signals involved is essential for the discovery of novel targets of intervention inChagas disease.