Abstract
The flagellum of Trypanosomatids is an organelle that contributes to multiple functions, including motility, cell division, and host–pathogen interaction. Trypanin was first described in Trypanosoma brucei and is part of the dynein regulatory complex. TbTrypanin knockdown parasites showed motility defects in procyclic forms; however, silencing in bloodstream forms was lethal. Since TbTrypanin mutants show drastic phenotypic changes in mammalian stages, we decided to evaluate if the Trypanosoma cruzi ortholog plays a similar role by using the CRISPR-Cas9 system to generate null mutants. A ribonucleoprotein complex of SaCas9 and sgRNA plus donor oligonucleotide were used to edit both alleles of TcTrypanin without any selectable marker. TcTrypanin −/− epimastigotes showed a lower growth rate, partially detached flagella, normal numbers of nuclei and kinetoplasts, and motility defects such as reduced displacement and speed and increased tumbling propensity. The epimastigote mutant also showed decreased efficiency of in-vitro metacyclogenesis. Mutant parasites were able to complete the entire life cycle in vitro; however, they showed a reduction in their infection capacity compared with WT and addback cultures. Our data show that T. cruzi life cycle stages have differing sensitivities to TcTrypanin deletion. In conclusion, additional work is needed to dissect the motility components of T. cruzi and to identify essential molecules for mammalian stages.
Highlights
Trypanosoma cruzi is a protozoan parasite of the Trypanosomatidae family, which bears a group of early diverging eukaryotes
We found that TcTrypanin −/− mutants have several phenotypic changes including reduced epimastigote growth and differentiation into metacyclic trypomastigotes, increased ratio of parasites with detached flagellum, motility defects, and reduced infection capacity
Phylogenetic analysis shows that the Trypanosoma rangeli SC58 strain (ID: TRSC58_03641) is the closet related Trypanin among trypanosomatids followed by the T. brucei sequence (ID: Tb927.10.6350), sharing 76.32% identity (Figure 1A)
Summary
Trypanosoma cruzi is a protozoan parasite of the Trypanosomatidae family, which bears a group of early diverging eukaryotes. This parasite is the etiological agent of Chagas disease, a potentially lifethreatening illness that affects about 6–8 million people around the world (Mills, 2020). Trypanosoma cruzi epimastigotes (a replicative form) differentiate into metacyclic trypomastigotes (MTs), the infective and non-replicative forms in triatomine insects. Once inside the host cells, they differentiate into amastigotes, a replicative form containing a short flagellum. After a period of multiplication intracellularly, the amastigote forms transform into bloodstream trypomastigotes; these cells egress the host cells, cross the extracellular matrix, and swim in a crowded and viscous environment (blood) to be able to reach different cell types of the mammalian organism (Ferri and Edreira, 2021). The contribution of parasite motility in this physiological process remains to be elucidated
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