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.

Toxoplasma gondii is a widespread parasitic pathogen that infect humans and all warm-blooded animals, causing abortion and stillbirth in pregnant women and animals, as well as life threatening toxoplasmosis in immune compromised individuals. Felines are the only definitive hosts of Toxoplasma and oocysts shed by infected felines are the major source of infection for humans and other animals. Given the critical role of felines for T. gondii transmission, control of feline toxoplasmosis has significant impacts on reducing the overall prevalence of animal and human toxoplasmosis. However, reliable diagnosis of feline toxoplasmosis is still challenging. In this study, we found that the putative micronemal protein 17A (MIC17A) that was abundantly expressed in Toxoplasma merozoites is a good diagnostic marker for serological diagnosis of Toxoplasma infection in felines. T. gondii encodes four paralogs of MIC17A in total and the expression of three of them is drastically upregulated in merozoites than in tachyzoites. In contrast, when proteins like GRA1 and MIC3 that are more abundantly expressed in tachyzoites than in merozoites were used as diagnostic antigens to test feline toxoplasmosis, they reacted with Toxoplasma specific IgG antibodies poorly. Taken together, these results suggest that merozoite antigens are better suited for the diagnosis of feline toxoplasmosis than antigens that are highly expressed at tachyzoite or bradyzoite stages.

Apicomplexan parasites are a group of intracellular pathogens of great medical and veterinary importance, including Toxoplasma gondii and Plasmodium, which cause toxoplasmosis and malaria, respectively. Efficient and accurate manipulation of their genomes is essential to dissect their complex biology and to design new interventions. Over the past several decades, scientists have continually optimized the methods for genetic engineering in these organisms, and tremendous progress has been made. Here, we review the genetic manipulation tools currently used in several apicomplexan parasites, and discuss their advantages and limitations. The widely used CRISPR/Cas9 genome editing technique has been adapted in several apicomplexans and shown promising efficiency. In contrast, conditional gene regulation is available in only a limited number of organisms, mainly Plasmodium and Toxoplasma, thus posing a research bottleneck for other parasites. Conditional gene regulation can be achieved with tools that regulate gene expression at the DNA, RNA or protein level. However, a universal tool to address all needs of conditional gene manipulation remains lacking. Understanding the scope of application is key to selecting the proper method for gene manipulation.