AUTHOR=Saenz-Garcia Jose L. , Borges Beatriz S. , Souza-Melo Normanda , Machado Luiz V. , Miranda Juliana S. , Pacheco-Lugo Lisandro Alfonso , Moretti Nilmar S. , Wheleer Richard , Soares Medeiros Lia C. , DaRocha Wanderson D. 

TITLE=Trypanin Disruption Affects the Motility and Infectivity of the Protozoan Trypanosoma cruzi

JOURNAL=Frontiers in Cellular and Infection Microbiology

VOLUME=Volume 11 - 2021

YEAR=2022

URL=https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2021.807236

DOI=10.3389/fcimb.2021.807236

ISSN=2235-2988

ABSTRACT=The flagellum of Trypanosomatids is an organelle that performs multiple functions, including motility, cell division, and host-pathogen interaction. Trypanin was first described in Trypanosoma brucei as part of a dynein regulatory complex. TbTrypanin knockdown parasites showed motility defects in procyclic forms, however, silencing in bloodstream forms is 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 total displacement, speed, and tumbling propensity. The epimastigote mutant also showed decreased efficiency on 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 to WT and addback cultures. Our data show that, differently from T. brucei, TcTrypanin plays a contrasting function in replicative and non-replicative forms of mammalian stages. In conclusion, additional work is needed to dissect the motility components of T. cruzi, and to identify essential molecules for mammalian stages.