Characterization of the apicoplast-localized enzyme TgUroD in Toxoplasma gondii reveals a key role of the apicoplast in heme biosynthesis

Abstract

Apicomplexan parasites such as <i>Toxoplasma gondii</i> possess an unusual heme biosynthesis pathway whose enzymes localize to the mitochondrion, cytosol, or apicoplast, a nonphotosynthetic plastid present in most apicomplexans. To characterize the involvement of the apicoplast in the <i>T. gondii</i> heme biosynthesis pathway, we investigated the role of the apicoplast-localized enzyme uroporphyrinogen III decarboxylase (<i>Tg</i>UroD). We found that <i>Tg</i>UroD knockdown impaired parasite proliferation, decreased free heme levels in the parasite, and decreased the abundance of heme-containing <i>c</i>-type cytochrome proteins in the parasite mitochondrion. We validated the effects of heme loss on mitochondrial cytochromes by knocking down cytochrome <i>c</i>/<i>c</i>₁ heme lyase 1 (<i>Tg</i>CCHL1), a mitochondrial enzyme that catalyzes the covalent attachment of heme to <i>c</i>-type cytochromes. <i>Tg</i>CCHL1 depletion reduced parasite proliferation and decreased the abundance of <i>c</i>-type cytochromes. We further sought to characterize the overall importance of <i>Tg</i>UroD and <i>Tg</i>CCHL1 for both mitochondrial and general parasite metabolism. <i>Tg</i>UroD depletion decreased cellular ATP levels, mitochondrial oxygen consumption, and extracellular acidification rates. By contrast, depletion of <i>Tg</i>CCHL1 neither diminished ATP levels in the parasite nor impaired extracellular acidification rate, but resulted in specific defects in mitochondrial oxygen consumption. Together, our results indicate that the apicoplast has a key role in heme biology in <i>T. gondii</i> and is important for both mitochondrial and general parasite metabolism. Our study highlights the importance of heme and its synthesis in these parasites.