Thiamine pyrophosphate-dependent enzymes in Toxoplasma gondii and functional significance of an apicoplast-localized pyruvate dehydrogenase.

Thiamine pyrophosphate (TPP)-dependent enzymes are central to carbon metabolism and metabolic plasticity in intracellular protozoan parasites. Toxoplasma gondii harbors at least five TPP-dependent proteins, namely pyruvate dehydrogenase (PDH) and 1-deoxy-D-xylulose-5-phosphate synthase (DXPS) in the apicoplast (a plastid relict), branched-chain α-ketoacid dehydrogenase (BCKDH) and α-ketoglutarate dehydrogenase (α-KGDH) in the mitochondrion, and transketolase (TKT) in the cytosol. These enzymes mutually regulate metabolic flux via glycolysis, tricarboxylic acid (TCA) cycle, pentose phosphate pathway (PPP) for nucleotide biogenesis, methylerythritol 4-phosphate pathway (MEP) for isoprenoid synthesis and fatty acid synthase 2 (FAS2) pathway to support lipid biosynthesis. Among these, the PDH complex represents a unique evolutionary adaptation that generates acetyl-CoA from glycolysis-derived pyruvate for the FAS2 pathway in the apicoplast. This chapter provides a methodological framework for functional characterization of TPP-dependent enzymes, exemplified by the PDH complex in T. gondii. We describe protocols for protein expression, antibody preparation, 3'-genomic tagging, subcellular localization, CRISPR/Cas9-mediated knockout, parasite phenotyping, metabolic profiling and animal virulence assays. These approaches reveal that PDH facilitates myristate and palmitate synthesis in the apicoplast. Its genetic disruption impairs parasite development and membrane biogenesis due to a reduction in glucose-derived fatty acid synthesis. Notably, PDH-deficient parasites are virulent in mice, and exogenous fatty acids can restore the mutant's growth, underscoring the salvage of host fatty acids and remarkable metabolic plasticity of T. gondii. The experimental strategies described herein are applicable to investigating other metabolic enzymes and molecular determinants of intracellular parasitism in T. gondii as a model pathogenic protist.