Alpha tubulin acetylation is mediated by ATAT in Trypanosoma cruzi

ALONSO VL; PEZZA A; SERRA EC; CARLONI ME; CHESTA ME; MOTTA CM; MARTINEZ PERALTA G

Congreso; XXXI Molecular Parasitology Meeting; 2020

The cytoskeleton of trypanosomatids has a simpler arrange than most eukaryotic cells. However, it is precisely organized and constituted by stable microtubules. Such microtubules compose the mitotic spindle, the basal body, the flagellar axoneme and the subpellicular microtubules, which are connected to each other and also to the plasma membrane, thus forming a helical arrangement along the central axis of the parasite cell body. Subpellicular, mitotic and axonemal microtubules are extensively acetylated in Trypanosoma cruzi.Acetylation on K40 of α-tubulin is conserved from lower eukaryotes to mammals and is associated with microtubule stability. It is also known that K40 acetylation occurs significantly on flagella, centrioles, cilia, basal body and the mitotic spindle. Several tubulin PTMs, including acetylation of K40, have been catalogued in Trypanosomatids, but the functional importance of these modifications for microtubule dynamics and parasite biology remains largely undefined. The primary tubulin acetyltransferase that delivers this modification was recently identified in several eukaryotes 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. We have expressed T. cruzi ATAT with an HA tag in epimastigotes using the inducible vector pTcINDEX-GW. Over-expressing parasites present a growth defect and also, we observed a diminished infectivity and an alteration in the differentiation from amastigotes to trypomastigotes. TcATAT is located in the cytoskeleton and flagella of T. cruzi as determined by western blot and confocal microscopy. Moreover, TcATAT colocalizes with acetylated alpha-tubulin in these structures and over-expression causes increased levels of the acetylated isoform and several morphological defects (cells with an abnormal DNA content and a mitochondrial morphology). Also, over-expression causes a halt in the cell cycle progression of epimastigotes determined by flow cytometry and SEM. We observed that TcATAT acetylates a-tubulin in vitro and in vivo and is able to auto-acetylate. Finally, when ATAT is over-expressed we observed that parasites become more resistant to microtubule depolymerizing drugs. These evidence supports the idea that tubulin acetylation is crucial for T. cruzi replication and differentiation and that TcATAT is responsible for this postranslational modification.