Role of α-tubulin acetylation in the cell cycle of Trypanosoma cruzi

ALONSO, VICTORIA LUCIA; MARTINEZ PERALTA, GONZALO; MOTTA, MARIA CRISTINA M.; SERRA, ESTEBAN CARLOS

Caxambu

Congreso; XXXVIII Meeting of the Brazilian Society of Protozoology; 2023

Brazilian Society of Protozoology

The most abundant isoform in all microtubular structures of Trypanosomatids is acetylated α-tubulin. Acetylation on K40 of α-tubulin is conserved from lower eukaryotes to mammals and is associated with microtubule stability. It is also known that it occurs significantly on flagella, centrioles, cilia, basal body and the mitotic spindle. The primary acetyltransferase that delivers this modification was recently identified as Mec-17/ATAT, a Gcn5-related N-acetyltransferase. Despite evidence supporting a role for K40 acetylation in microtubule stability, its biological function in vivo is unclear. To study α-tubulin K40 acetylation we employed different genetic manipulation strategies in Trypanosoma cruzi. We analyzed the phenotypes of the resulting parasites using expansion, confocal and electron microscopy. Firstly we have expressed TcATAT in epimastigotes using the inducible vector pTcINDEX-GW. TcATAT is located in the cytoskeleton and flagella, colocalizes with acetylated α-tubulin in these structures and over-expression causes increased levels of the acetylated isoform and a halt in the cell cycle progression of epimastigotes. Also, these parasites become more resistant to microtubule tubulin depolymerizing drugs. Then we used the same system to over-express mutant versions of α-tubulin K40, which generates reduced levels of acetylated α-tubulin (measured by flow cytometry). The cell cycle progression is altered and an aberrant morphology is observed in these parasites. Finally we obtained TcATAT knock-out epimastigotes by CRISPR/Cas9, these parasite have undetectable levels of acetylated α-tubulin and severe defects in there replication rates, their motility is impaired and present a detached flagella. These evidence supports the idea that tubulin acetylation is crucial for T. cruzi replication and differentiation and that TcATAT is responsible for this posttranslational modification.