Authors
S Teixeira2; V Grazilelle-Silva2; R Cardoso de Paiva2; M Santos-Cardoso2; R Mendonça-Neto2; C Junqueira1; G Burle-Caldas2; W daRocha3; R Gazzinelli1; 1 Centro de Pesuisas Rene Rachou, Brazil; 2 Universidade Federal de Minas Gerais, Brazil; 3 Universidade Federal do Paraná, BrazilDiscussion
Leishmania spp and Trypanosoma cruzi are intracellular protozoan parasites responsible for diseases that affect a large number of people in the tropical world. The membrane of both parasites contains glycoproteins named amastins that are highly abundant in the intracellular amastigote stage and are encoded by a multi gene family that has been expanded in the Leishmania genome. Infective, trypomastigote forms of T. cruzi also express a large family of polymorphic proteins that include a group of enzymes named trans-sialidades (TS). TS transfers sialic acid from host glycoconjugates to terminal β-galactopyranosyl residues of mucin-like molecules also present on the parasite's cell surface. By knocking down the expression of amastin genes in L. braziliensis, we showed evidences indicating that amastins are essential players in the tight interaction that occurs between the parasite surface and the parasitophorous vacuole membrane of the infected macrophage. Amastin knock down parasites showed impaired growth during in vitro infection of macrophages and completely failed to produce lesions when inoculated in BALB/c mice, an attenuated phenotype that was reverted by the re-expression of an RNAi-resistant amastin gene. By comparing the genomes of the avirulent CL-14 strain of T. cruzi with the virulent CL Brener strain, we also identified the C-terminal amino acid repeat, known as SAPA repeats, that is present in a group of TS genes, as T. cruzi virulence factor. TS-SAPA domain, absent in CL-14 parasites, consists of multiple overlapping B cell epitopes that may act as to control the host immune response. Expression in CL-14 of a TS containing a large SAPA domain partially reverts the CL-14 avirulent phenotype, indicating that the repeat domain of TS plays a major role related to the T. cruzi infection capacity. Finally, by demonstrating that we can use the CRISPR-Cas system to knock out multi gene families in the T. cruzi genome, we are now able to directly address the role of various parasite surface protein. 
