Function and therapeutic potential of transketolase enzymes in parasitic protists

Transketolase (TKT), the rate-limiting enzyme in the non-oxidative branch of the pentose phosphate pathway (PPP), catalyzes the reversible transfer of glycolaldehyde moieties between sugar phosphates. Here, we present wide-ranging methods to study TKT function in a clinically-relevant and globally-prevalent model intracellular pathogen, Toxoplasma gondii. The experimental framework utilizes heterologous expression of recombinant TgTKT proteins in a bacterial system, anti-TgTKT antibody production, CRISPR/Cas9-assisted genome editing to generate TgTKT-null mutants, and in-depth phenotyping, including ¹³C-based metabolomic analysis to determine the carbon flux in the acutely-infectious tachyzoite stage of T. gondii. The phenotypic studies comprise the parasite proliferation in vitro and virulence assessment in a murine model of acute toxoplasmosis. The data suggest TgTKT as a metabolic rheostat regulating nucleotide biosynthesis and enabling adaptation of this highly promiscuous parasite in different nutritional environments. Last but not least, the work presents the therapeutic potential of the TgTKT-null mutant as a metabolically-attenuated vaccine, highlights the evolutionary divergence of TgTKT proteins, and provides a robust foundation to investigate TgTKT orthologs in other pathogenic protozoan organisms.