TgKDAC4: A Unique Deacetylase of Toxoplasma's Apicoplast.

Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa and causes toxoplasmosis infections, a disease that affects a quarter of the world's population and has no effective cure. Epigenetic regulation is one of the mechanisms controlling gene expression and plays an essential role in all organisms. Lysine deacetylases (KDACs) act as epigenetic regulators affecting gene silencing in many eukaryotes. Here, we focus on TgKDAC4, an enzyme unique to apicomplexan parasites, and a class IV KDAC, the least-studied class of deacetylases so far. This enzyme shares only a portion of the specific KDAC domain with other organisms. Phylogenetic analysis from the TgKDAC4 domain shows a putative prokaryotic origin. Surprisingly, TgKDAC4 is located in the apicoplast, making it the only KDAC found in this organelle to date. Transmission electron microscopy assays confirmed the presence of TgKDAC4 in the periphery of the apicoplast. We identified possible targets or/and partners of TgKDAC4 by immunoprecipitation assays followed by mass spectrometry analysis, including TgCPN60 and TgGAPDH2, both located at the apicoplast and containing acetylation sites. Understanding how the protein works could provide new insights into the metabolism of the apicoplast, an essential organelle for parasite survival.

TgKDAC4: a unique deacetylase of toxoplasma's apicoplast

Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa and causes toxoplasmosis infections, a disease that affects a quarter of the world’s population and has no effective cure. Epigenetic regulation is one of the mechanisms controlling gene expression and plays an essential role in all organisms. Lysine deacetylases (KDACs) act as epigenetic regulators affecting gene silencing in many eukaryotes. Here, we focus on TgKDAC4, an enzyme unique to apicomplexan parasites, and a class IV KDAC, the least-studied class of deacetylases so far. This enzyme shares only a portion of the specific KDAC domain with other organisms. Phylogenetic analysis from the TgKDAC4 domain shows a putative prokaryotic origin. Surprisingly, TgKDAC4 is located in the apicoplast, making it the only KDAC found in this organelle to date. Transmission electron microscopy assays confirmed the presence of TgKDAC4 in the periphery of the apicoplast. We identified possible targets or/and partners of TgKDAC4 by immunoprecipitation assays followed by mass spectrometry analysis, including TgCPN60 and TgGAPDH2, both located at the apicoplast and containing acetylation sites. Understanding how the protein works could provide new insights into the metabolism of the apicoplast, an essential organelle for parasite survival.

Dichloroacetate and Pyruvate Metabolism: Pyruvate Dehydrogenase Kinases as Targets Worth Investigating for Effective Therapy of Toxoplasmosis.

Toxoplasmosis, a protozoan infection caused by Toxoplasma gondii, is estimated to affect around 2.5 billion people worldwide. Nevertheless, the side effects of drugs combined with the long period of therapy usually result in discontinuation of the treatment. New therapies should be developed by exploring peculiarities of the parasite's metabolic pathways, similarly to what has been well described in cancer cell metabolism. An example is the switch in the metabolism of cancer that blocks the conversion of pyruvate into acetyl coenzyme A in mitochondria. In this context, dichloroacetate (DCA) is an anticancer drug that reverts the tumor proliferation by inhibiting the enzymes responsible for this switch: the pyruvate dehydrogenase kinases (PDKs). DCA has also been used in the treatment of certain symptoms of malaria; however, there is no evidence of how this drug affects apicomplexan species. In this paper, we studied the metabolism of T. gondii and demonstrate that DCA also inhibits T. gondii's in vitro infection with no toxic effects on host cells. DCA caused an increase in the activity of pyruvate dehydrogenase followed by an unbalanced mitochondrial activity. We also observed morphological alterations frequently in mitochondria and in a few apicoplasts, essential organelles for parasite survival. To date, the kinases that potentially regulate the activity of pyruvate metabolism in both organelles have never been described. Here, we confirmed the presence in the genome of two putative kinases (T. gondii PDK [TgPDK] and T. gondii branched-chain α-keto acid dehydrogenase kinase [TgBCKDK]), verified their cellular localization in the mitochondrion, and provided in silico data suggesting that they are potential targets of DCA.IMPORTANCE Currently, the drugs used for toxoplasmosis have severe toxicity to human cells, and the treatment still lacks effective and safer alternatives. The search for novel drug targets is timely. We report here that the treatment of T. gondii with an anticancer drug, dichloroacetate (DCA), was effective in decreasing in vitro infection without toxicity to human cells. It is known that PDK is the main target of DCA in mammals, and this inactivation increases the conversion of pyruvate into acetyl coenzyme A and reverts the proliferation of tumor cells. Moreover, we verified the mitochondrial localization of two kinases that possibly regulate the activity of pyruvate metabolism in T. gondii, which has never been studied. DCA increased pyruvate dehydrogenase (PDH) activity in T. gondii, followed by an unbalanced mitochondrial activity, in a manner similar to what was previously observed in cancer cells. Thus, we propose the conserved kinases as potential regulators of pyruvate metabolism and interesting targets for new therapies.