Carbohydrate Metabolism in theToxoplasma gondiiApicoplast: Localization of Three Glycolytic Isoenzymes, the Single Pyruvate Dehydrogenase Complex, and a Plastid Phosphate Translocator

Abstract

Many apicomplexan parasites, such as Toxoplasma gondii and Plasmodium species, possess a nonphotosynthetic plastid, referred to as the apicoplast, which is essential for the parasites' viability and displays characteristics similar to those of nongreen plastids in plants. In this study, we localized several key enzymes of the carbohydrate metabolism of T. gondii to either the apicoplast or the cytosol by engineering parasites which express epitope-tagged fusion proteins. The cytosol contains a complete set of enzymes for glycolysis, which should enable the parasite to metabolize imported glucose into pyruvate. All the glycolytic enzymes, from phosphofructokinase up to pyruvate kinase, are present in the T. gondii genome, as duplicates and isoforms of triose phosphate isomerase, phosphoglycerate kinase, and pyruvate kinase were found to localize to the apicoplast. The mRNA expression levels of all genes with glycolytic products were compared between tachyzoites and bradyzoites; however, a strict bradyzoite-specific expression pattern was observed only for enolase I. The T. gondii genome encodes a single pyruvate dehydrogenase complex, which was located in the apicoplast and absent in the mitochondrion, as shown by targeting of epitope-tagged fusion proteins and by immunolocalization of the native pyruvate dehydrogenase complex. The exchange of metabolites between the cytosol and the apicoplast is likely to be mediated by a phosphate translocator which was localized to the apicoplast. Based on these localization studies, a model is proposed that explains the supply of the apicoplast with ATP and the reduction power, as well as the exchange of metabolites between the cytosol and the apicoplast.Up to 20 to 30% of the world population is estimated to be chronically infected with the apicomplexan parasite Toxoplasma gondii. The parasite differentiates within the human host between tachyzoites and bradyzoites, which display distinct physiological features. Mature bradyzoites are adapted for lifelong persistence in their hosts and display an extreme reduction in growth rate, up to a complete arrest of the cell cycle (7). In contrast, tachyzoites, which are present during the acute phase of infection, are characterized by a fast duplication time of 6 to 8 h, indicating that this stage possesses effective pathways for nutrient acquisition and energy metabolism.T. gondii possesses, like many other apicomplexan parasites, a nonphotosynthetic plastid, the so-called apicoplast. This organelle contains a 35-kb circular genome with similarities to plastid genomes from algae and is surrounded by four membranes. These features support the secondary endosymbiosis of a photosynthetic alga by the apicomplexan ancestor as the phylogenetic origin of the apicoplast (21,32,43,51). Numerous nucleus-encoded proteins are imported into the apicoplast, aided by a bipartite presequence which is composed of a signal peptide and an adjacent transit peptide (23,24,39,49).The apicoplast is the location of several anabolic pathways, such as type I...

Make It or Take It: Fatty Acid Metabolism of Apicomplexan Parasites

“…Importantly, this incorporation is sensitive to triclosan inhibition (63). The observed chain length is in agreement with the substrate preference of the PfFabB/F enzyme, which most effectively elongates C 6 to C 10 -ACP chains, with reduced activity for C 12 and C 14 chains and no activity for C 16 or longer chains (33). In T. gondii, acetate labeling equally resulted in robust incorporation of the label into fatty acids and a variety of lipids (5,37).…”

“…In most eukaryotes, PDH is an essential mitochondrial enzyme complex linking glycolysis to the tricarboxylic acid cycle. Apicomplexans appear to lack mitochondrial PDH (16,17), and one could speculate that metabolites might be exchanged between plastid and mitochondrion, which are closely apposed through much of the intracellular development of the parasites (69,70). However, the finding that an apicomplexan that lacks the FASII pathway, Theileria, equally lacks PDH could argue against this model and suggest that the critical role of PDH likely is to provide acetyl-CoA for fatty acid synthesis (19).…”

“…Although the ACP units failed to be activated to holo-ACP in this system (they are apparently not recognized by the E. coli phosphopantetheinyl transferase), the enzyme activities could be measured by using CoA as an alternative acceptor. Substrate inhibition studies of the recombinant loading unit indicated fatty acids with chain lengths of C 12 to C 24 as potential substrates, with a pronounced preference for C 16 . Further studies showed that all three elongation units are active.…”

Apicomplexan Energy Metabolism: Carbon Source Promiscuity and the Quiescence Hyperbole

“…Furthermore, the pyruvate dehydrogenase complex (PDH) responsible for conversion of pyruvate into acetyl-CoA -an obligate fuel for the TCA cycle -is only present in the apicoplast [10]. Consequently, there was no obvious entry point to allow catalysis of glycolytic pyruvate by the TCA cycle [10][11][12][13][14]. Despite this, there is overwhelming evidence that apicomplexans rely on a functional and canonical TCA cycle (as reviewed [4,5,15]), and apicomplexan genome annotations indicate the presence of all enzymes of the TCA cycle.…”

Type II NADH Dehydrogenase Inhibitor 1-Hydroxy-2-Dodecyl- 4(1H)Quinolone Leads to Collapse of Mitochondrial Inner- Membrane Potential and ATP Depletion inToxoplasma gondii

“…Although the T. gondii mitochondrion harbors the complete set of enzymes for the tricarboxylic acid cycle (15), it lacks the pyruvate dehydrogenase complex (7,14,18), which is typically a central enzyme in carbohydrate metabolism that catalyzes the decarboxylation from pyruvate to acetyl coenzyme A. Other mitochondrial pathways for mitochondrial acetyl coenzyme A generation, such as the 2-methylcitrate cycle, are currently under investigation (33).…”