Ultrastructural characterization of hyperacetylated epimastigotes of Trypanosoma cruzi

GONÇALVES CS; ALONSO VL; SERRA, EC; MOTTA, MARIA CRISTINA M.

Woods Hole

Congreso; XXXII Molecular Parasitology Meeting; 2021

Trypanosomatids have a cytoskeleton that is precisely organized and constituted by stable microtubules. Such microtubules compose the mitotic spindle during mitosis, the basal body, the flagellar axoneme and the subpellicular microtubules. These structures are extensively acetylated in Trypanosoma cruzi. The functional importance of K40 acetylation for microtubule dynamics and parasite biology remains largely undefined although it was described more that 20 years ago. We have over-expressed TcATAT, the primary tubulin acetyltransferase, with an HA tag using the inducible vector pTcINDEX-GW. We have studied the defects caused by hyperacetylation in epimatigotes of T. cruzi using confocal microscopy and transmission electron microscopy (TEM). Hyperacetylation induces the formation of a round refringent structure in epimastigotes that grew with induction time. This round structure is electrondense, not delimited by membrane, and sometimes seen in continuity with the endoplasmic reticulum, resembling an inclusion body. These results correlate with the accumulation of TcATAT-HA observed in association to isolated cytoskeletons, where this structure is seen connected to the flagellum. Hyperacetylation causes mitochondrial cristaes welling and sometimes cells presented a kinetoplast containing multiple networks that are very condensed indicating kinetoplast division impairment. Also, when the kDNA duplicates but the kinetoplast does not divide, the network became curved, folded over itself, acquiring a round shape with an atypical condensation. Such kDNA alterations were also observed by confocal microscopy in parasites stained with Mitotracker and DAPI. These results support the idea that -tubulin acetylation levels need to be finely regulated for the normal progression of T. cruzi cell cycle.