Mechanisms and significance of inter-organelle pyruvate transport in Toxoplasma gondii

• Applicant: Dr. Nishith Gupta
• Subject Area: Parasitology and Biology of Tropical Infectious Disease Pathogens ; Metabolism, Biochemistry and Genetics of Microorganisms
• Term: from 2020 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 432249451

Toxoplasma gondii is a ubiquitous intracellular pathogen infecting a variety of animals and humans, which imposes significant socioeconomic burden globally. Our previous work has shown that T. gondii displays exceptional metabolic flexibility, being able to utilize glucose, glutamine, lactate and amino acids to fuel energy and macromolecule synthesis. We also showed that metabolism of these nutrients converges at pyruvate, which is primarily derived from glycolysis in the cytosol, but is used in at least three compartments, mitochondrion, apicoplast (a chloroplast relict) and cytoplasm. In mitochondrion, pyruvate is converted to acetyl-coA to drive TCA cycle, whereas in the apicoplast it serves as a substrate for de novo synthesis of fatty acids and isoprenoid precursors. Our work suggests that both organelles harbor specialized pyruvate carriers to import this key metabolite from the cytosol, but the molecular identities of these transporters are not known. Using bioinformatic and localization analyses, we found 4 genes, which very likely encode for the mitochondrial pyruvate carrier (MPC1/2) and apicoplast pyruvate carriers (APC1/2). Going forward, our collaborative Sino-German proposal aims to examine the pyruvate transport activity and physiological relevance of these proteins by biochemical and genetic engineering approaches. In particular, we will assess the membrane topology and complex formation by genomic tagging of MPC and APC proteins, as well as evaluate the phenotypic impact of their single or combinatorial deletions in the parasite. Selected mutants will be subject to metabolic assays, and biosensor-based detection of subcellular pools of pyruvate. Specificity of the phenotypes will be further endorsed by complementing the chosen mutants with corresponding MPC/APC, and well-characterized homologs from yeast and mammals. The parasite work will be consolidated by functional complementation and transport assays using yeast and bacterial mutants lacking the pyruvate transport. Not least, we will assay recombinant yeast or bacterial strains to determine the selectivity, specificity and ion/pH-dependence of the transport by MPC/APC proteins. Successful completion of this work shall reveal the mechanism and importance of cross-compartment pyruvate transport for the carbohydrate metabolism of T. gondii. It should also aid the identification and validation of novel anti-parasitic targets.

• DFG Programme: Research Grants
• International Connection: China
• Partner Organisation: National Natural Science Foundation of China
• Cooperation Partner: Professor Bang Shen, Ph.D.