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

• Published in: Frontiers in cellular and infection microbiology Vol. 11; p. 807236
• Main Authors: 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
• Format: Journal Article
• Language: English
• Published: Switzerland Frontiers Media S.A 07-01-2022
• Subjects: Animals; Cellular and Infection Microbiology; Chagas Disease; CRISPR-Cas9; detached flagellum; Flagella - genetics; metacyclogenesis; motility; Protozoan Proteins - genetics; trypanin; Trypanosoma brucei brucei; Trypanosoma cruzi (T. cruzi); Trypanosoma cruzi - genetics; metacyclogenesis; Trypanosoma cruzi (T. cruzi); CRISPR-Cas9; trypanin; detached flagellum; motility; SaCas9
• ISSN: 2235-2988; 2235-2988
• DOI: 10.3389/fcimb.2021.807236

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
and is part of the dynein regulatory complex.
Trypanin knockdown parasites showed motility defects in procyclic forms; however, silencing in bloodstream forms was lethal. Since
Trypanin mutants show drastic phenotypic changes in mammalian stages, we decided to evaluate if the
zi 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
Trypanin without any selectable marker.
Trypanin -/- 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
metacyclogenesis. Mutant parasites were able to complete the entire life cycle
; however, they showed a reduction in their infection capacity compared with WT and addback cultures. Our data show that
life cycle stages have differing sensitivities to
Trypanin deletion. In conclusion, additional work is needed to dissect the motility components of
and to identify essential molecules for mammalian stages.