CREditing: a tool for gene tuning in Trypanosoma cruzi

The genetic manipulation of Trypanosoma cruzi continues to be a challenge, mainly due to the lack of available and efficient molecular tools. The CRE-lox recombination system is a site-specific recombinase technology, widely used method of achieving conditional targeted deletions, inversions, insertions, gene activation, translocation, and other modifications in chromosomal or episomal DNA. In the present study, the CRE-lox system was adapted to expand the current genetic toolbox for this hard-to-manipulate parasite. For this, evaluations of whether direct protein delivery of CRE recombinase through electroporation could improve CRE-mediated recombination in T. cruzi were performed. CRE recombinase was fused to the C-terminus of T. cruzi histone H2B, which carries the nuclear localization signal and is expressed in the prokaryotic system. The fusion protein was affinity purified and directly introduced into epimastigotes and tissue culture-derived trypomastigotes. This enabled the control of gene expression as demonstrated by turning on a tdTomato (tandem dimer fluorescent protein) reporter gene that had been previously transfected into parasites, achieving CRE-mediated recombination in up to 85% of parasites. This system was further tested for its ability to turn off gene expression, remove selectable markers integrated into the genome, and conditionally knock down the nitroreductase gene, which is involved in drug resistance. Additionally, CREditing also enabled the control of gene expression in tissue culture trypomastigotes, which are more difficult to transfect than epimastigotes. The considerable advances in genomic manipulation of T. cruzi shown in this study can be used by others to aid in the greater understanding of this parasite through gain- or loss-of function approaches

Engineering a single-chain antibody against Trypanosoma cruzi metacyclic trypomastigotes to block cell invasion.

Trypanosoma cruzi is a flagellate protozoan pathogen that causes Chagas disease. Currently there is no preventive treatment and the efficiency of the two drugs available is limited to the acute phase. Therefore, there is an unmet need for innovative tools to block transmission in endemic areas. In this study, we engineered a novel recombinant molecule able to adhere to the T. cruzi surface, termed scFv-10D8, that consists of a single-chain variable fragment (scFv) derived from mAb-10D8 that targets gp35/50. The synthetic gene encoding scFv-10D8 was cloned and fused to a 6×His tag and expressed in a prokaryotic expression system. Total periplasmic or 6xHis tag affinity-purified fractions of scFv-10D8 retained the capacity to bind to gp35/50, as shown by Western blot analyses. Pre-incubation of metacyclic trypomastigotes with scFv-10D8 showed a remarkable reduction in cell invasion capacity. Our results suggest that scFv-10D8 can be used in a paratransgenic approach to target parasites in insect vectors, avoiding dissemination of infective forms. Such advances in the development of this functional molecule will surely prompt the improvement of alternative strategies to control Chagas disease by targeting mammalian host stages

Engineering a single-chain antibody against Trypanosoma cruzi metacyclic trypomastigotes to block cell invasion

Trypanosoma cruzi is a flagellate protozoan pathogen that causes Chagas disease. Currently there is no preventive treatment and the efficiency of the two drugs available is limited to the acute phase. Therefore, there is an unmet need for innovative tools to block transmission in endemic areas. In this study, we engineered a novel recombinant molecule able to adhere to the T. cruzi surface, termed scFv-10D8, that consists of a single-chain variable fragment (scFv) derived from mAb-10D8 that targets gp35/50. The synthetic gene encoding scFv-10D8 was cloned and fused to a 6×His tag and expressed in a prokaryotic expression system. Total periplasmic or 6xHis tag affinity-purified fractions of scFv-10D8 retained the capacity to bind to gp35/50, as shown by Western blot analyses. Pre-incubation of metacyclic trypomastigotes with scFv-10D8 showed a remarkable reduction in cell invasion capacity. Our results suggest that scFv-10D8 can be used in a paratransgenic approach to target parasites in insect vectors, avoiding dissemination of infective forms. Such advances in the development of this functional molecule will surely prompt the improvement of alternative strategies to control Chagas disease by targeting mammalian host stages