rypanin Disruption Affects the Motility and Infectivity of the Protozoan Trypanosoma cruz

Author(s)

Saenz-Garcia, Jose L. ^[1] ; Borges, Beatriz S. ^[2] ; Souza-Melo, Normanda ^{[3, 4]} ; Machado, Luiz V. ^[1] ; Miranda, Juliana S. ^[1] ; Pacheco-Lugo, Lisandro Alfonso ^[5] ; Moretti, Nilmar S. ^[3] ; Wheleer, Richard ^[6] ; Soares Medeiros, Lia C. ^[2] ; DaRocha, Wanderson D. ^[1]

Affiliation

^[1] Univ Fed Parana, Lab Gen Func Parasitos GFP, Curitiba - Brazil

^[2] Fundacao Oswaldo Cruz Fiocruz, Lab Biol Celular, Inst Carlos Chagas, Curitiba - Brazil

^[3] Univ Fed Sao Paulo, Dept Microbiol Imunol & Parasitol, Lab Biol Mol Patogenos LBMP, Escola Paulista Med, Sao Paulo - Brazil

^[4] Univ Fed Rio De Janeiro UFRJ, Lab Ultraestrutura Hertha Mayer, Rio De Janeiro - Brazil

^[5] Univ Simon Bolivar, Fac Ciencias Bas & Biomed, Barranquilla - Colombia

^[6] Univ Oxford, Nuffield Dept Med, Oxford - England

• Document type: Journal article
• Source: FRONTIERS IN CELLULAR AND INFECTION MICROBIOLOGY; v. 11, JAN 7 2022.
• Web of Science Citations: 0

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. (AU)

• FAPESP's process: 18/09948-0 - Study of protein acetylation in Leishmania
• Grantee: Nilmar Silvio Moretti
• Support Opportunities: Regular Research Grants
• FAPESP's process: 20/07870-4 - Mechanisms of trypanosomatid adaptation to hosts through the control of transcription, protein synthesis and secretion of extracellular vesicles
• Grantee: Ana Claudia Trocoli Torrecilhas
• Support Opportunities: Research Projects - Thematic Grants