O papel da histona desacetilase 4 (TgHDAC4) na regulação da Expressão Gênica de Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite member of the phylum Apicomplexa and is responsible for toxoplasmosis, a disease that affects a quarter of the world’s population and which has no effective cure. In order to pass immune barriers, T. gondii differentiates between different stages, which is tightly regulated by controlling gene expression. Epigenetic regulation is one of the mechanisms controlling gene expression that seems to play an important role in T. gondii. One category of enzymes that act as epigenetic regulators are histone deacetylases (HDACs). T. gondii has seven histone deacetylases (HDACs and Sir2), which are classified into four classes according to their domains, cofactors and sequence. The aim of the present dissertation was to characterize TgHDAC4, a histone deacetylase of class IV, a class of which the only well characterized member is HDAC11, found only in metazoans. TgHDAC4 has two predicted signals: a possible nuclear export signal and a cleavage, whose functions have yet to be confirmed. Searches for similar sequences showed that TgHADC4 is conserved in some members of the phylum Apicomplexa, and the only region common to other species is the typical HDAC domain. In order to characterize TgHDAC4, we focused on construction of epitope tagged lines and knockouts. The construction of these tools was delayed due to misannotation of the C-terminal portion of tghdac4 gene present on ToxoDB (the T. gondii database). According to ToxoDB, TgHDAC4 is a large protein with an incomplete HDAC domain at the C-terminal domain, resulting in a predicted molecular mass of 98 kDa. After resequencing the final portion of tghdac4, we identified the correct stop codon, which resulted in two more exons than predicted, and an approximate protein size of 124 kDa. With the correct sequence, we constructed endogenous tagged lines, adding a HA tag to the C-terminal portion of TgHADC-4. By indirect immunofluorescence and co-localization assays, we verified that TgHDAC4 is located in the apicoplast, also co-localizing with FDR protein (Ferredoxin-NADP+ reductase). Western blot assays identified a protein smaller than expected, of about 100 kDa. Several attempts to construct knockout lines failed, despite successful drug selection. This is likely due to a misannotation problem. As an alternative, we used the CRISPR/Cas9 system (constructs provided by Dr. Hakimi-University of Grenoble Alpes, France). Plasmids were transfected and used for immunofluorescence assays. Parasites that stained positively for GFP showed atypical morphology, especially in nuclei. On the other hand, so far it was not possible to identify parasite clones with specific mutations on tghdac4. Our data indicate for the first time the presence of a histone deacetylase in the T. gondii apicoplast, which seems to be unique to Apicomplexa parasites. The apicoplast is an organelle acquired from endosymbiotic origin, considered the “Achilles’ Heel" of the parasite, due to its importance for parasite survival. Understanding how this protein works could provide new insights into the metabolism of the apicoplast and in addition reveal a potential drug target for the effective treatment of toxoplasmosis.