Two apicoplast dwelling glycolytic enzymes provide key substrates for metabolic pathways in the apicoplast and are critical for Toxoplasma growth

Many apicomplexan parasites harbor a non-photosynthetic plastid called the apicoplast, which hosts important metabolic pathways like the methylerythritol 4-phosphate (MEP) pathway that synthesizes isoprenoid precursors. Yet many details in apicoplast metabolism are not well understood. In this study, we examined the physiological roles of four glycolytic enzymes in the apicoplast of Toxoplasma gondii. Many glycolytic enzymes in T. gondii have two or more isoforms. Endogenous tagging each of these enzymes found that four of them were localized to the apicoplast, including pyruvate kinase2 (PYK2), phosphoglycerate kinase 2 (PGK2), triosephosphate isomerase 2 (TPI2) and phosphoglyceraldehyde dehydrogenase 2 (GAPDH2). The ATP generating enzymes PYK2 and PGK2 were thought to be the main energy source of the apicoplast. Surprisingly, deleting PYK2 and PGK2 individually or simultaneously did not cause major defects on parasite growth or virulence. In contrast, TPI2 and GAPDH2 are critical for tachyzoite proliferation. Conditional depletion of TPI2 caused significant reduction in the levels of MEP pathway intermediates and led to parasite growth arrest. Reconstitution of another isoprenoid precursor synthesis pathway called the mevalonate pathway in the TPI2 depletion mutant partially rescued its growth defects. Similarly, knocking down the GAPDH2 enzyme that produces NADPH also reduced isoprenoid precursor synthesis through the MEP pathway and inhibited parasite proliferation. In addition, it reduced de novo fatty acid synthesis in the apicoplast. Together, these data suggest a model that the apicoplast dwelling TPI2 provides carbon source for the synthesis of isoprenoid precursor, whereas GAPDH2 supplies reducing power for pathways like MEP, fatty acid synthesis and ferredoxin redox system in T. gondii. As such, both enzymes are critical for parasite growth and serve as potential targets for anti-toxoplasmic intervention designs. On the other hand, the dispensability of PYK2 and PGK2 suggest additional sources for energy in the apicoplast, which deserves further investigation. Author summaryThe apicoplast is a unique organelle in most apicomplexan parasites, a large group of unicellular pathogens that infect humans and animals. It is essential for parasite growth as it hosts important metabolic pathways and provides critical metabolites. By endogenous gene tagging, here we found four glycolytic isoenzymes localized to the apicoplast of Toxoplasma gondii, including PYK2, PGK2, TPI2 and GAPDH2. Gene inactivation studies demonstrated that the two ATP generating enzymes PYK2 and PGK2 were dispensable for the growth of T. gondii, whereas TPI2 and GAPDH2 were essential. Combined genetic and metabolic analyses suggest that TPI2 generated glyceraldehyde-3-phosphate is critical for the MEP pathway to synthesize isoprenoid precursors, which are essential for parasites proliferation. On the other hand, GAPDH2 is probably a main source of reducing power for metabolic enzymes involved in isoprenoid precursor and fatty acid synthesis in the apicoplast. As such, both enzymes are required for optimal parasite growth.

Automated summary

This study investigates the physiological roles of glycolytic enzymes in the apicoplast of the parasite Toxoplasma gondii. It is found that TPI2 and GAPDH2 are critical for parasite growth, while PYK2 and PGK2 are dispensable. TPI2 provides carbon source for isoprenoid precursor synthesis, while GAPDH2 supplies reducing power for metabolic pathways in the parasite.