Leishmania metabolism and virulence

© 2013 Dr. Wai Hung William NgProtozoan parasites belonging to the genus Leishmania are the cause of human leishmaniasis, which comprises a spectrum of diseases. Leishmaniasis affects 12 million people worldwide, resulting in more than 50,000 deaths annually. Leishmaniasis is a major public health problem in many developing countries in the tropics and subtropics with limited healthcare. There is no safe and effective vaccine for the disease, and the conventional therapeutics for leishmaniasis are hampered by side effects, high cost and resistance. There is therefore an urgent need to develop new therapies against the disease, based on improved understanding of the molecular basis of parasite pathogenesis. Leishmania spp. have a complex digenetic life cycle that involves two major developmental stages: the free living promastigote stages that develop in the digestive tract of the sandfly vector and the non-motile amastigote stage, that proliferates within the phagolysosome compartment of macrophages and other phagocytic cells in the mammalian host. An improved understanding of the metabolism of these stages, particularly of the amastigotes stages, is crucial to the development of new therapies. While many aspects of promastigote metabolism have been studied in detail, much less is known about the metabolism of amastigotes. The major aim of this thesis is to identify key pathways in Leishmania amastigote carbon metabolism that are crucial for survival in macrophage host cells. This was achieved using gas chromatography-mass spectrometry based 13C-stable isotope resolved metabolomics to trace pathways of carbon utilization in both promastigote and amastigote stages and to investigate the functional importance of specific pathways using chemical inhibitors of enzymes identified to be active in these stages. In Chapter 3, 13C-glucose labeling experiments were used to confirm previous studies showing that Leishmania promastigotes are highly dependent on the aerobic fermentation of glucose for energy. While a major fraction of catabolized glucose is secreted as fermentation end-products (succinate, acetate, alanine), glycolytic end-products such as phosphoenolpyruvate and pyruvate are also catabolized in a canonical oxidative TCA cycle. Under glucose-replete conditions, the main function of the TCA cycle is the provision of α-ketoglutarate (αKG) for glutamate synthesis, while the role in energy generation is minor. Sodium fluoroacetate (NaFAc), an aconitase inhibitor, disrupts the continuity of the TCA cycle and results in growth arrest which can be rescued by glutamate supplementation in culture medium. In chapter 4, the potential role of mitochondrial metabolism in in vitro cultivated and intracellular amastigotes was explored. Metabolic labeling studies showed that amastigotes exhibit a complete TCA cycle in which carbon derived from glucose is converted to α-ketoglutarate and glutamate (Saunders EC, unpublished data). Both axenic and intracellular amastigotes were found to exhibit greater sensitivity to NaFAc inhibition than promastigotes, indicating that the TCA cycle may be used primarily to generate precursors for glutamate synthesis. Inhibitors of glutamine synthesis (MSO, PPI) were also found to prevent amastigote growth in macrophages, and enhanced clearance. Together, these findings suggest that Leishmania amastigote are highly dependent on de novo synthesis of glutamate and glutamine from carbon backbones derived from glucose metabolism, accounting for the essentiality of glucose uptake/metabolism for intracellular amastigote growth. As glutamine synthetase inhibitors are of low toxicity to human, the inhibitors used in this study can serve as a starting point for designing anti-leishmaniasis therapeutics. Our group has previously shown that Leishmania parasites are unable to scavenge all of their hexose requirements from the mammalian host, as they are dependent on key gluconeogenesis enzymes for infectivity. In Chapter 5, the role of different amino acids in gluconeogenesis was investigated. Glutamate was found to be essential for promastigote growth in glucose-free medium. Metabolite profiling and labeling studies showed that glutamate provides a source of αKG needed for the assimilation of other non-essential amino acids, such as alanine and aspartate in gluconeogenesis. Under glucose starvation, TCA cycle serves to assimilate the carbon skeleton of non-essential amino acids into gluconeogenesis. Overall, these studies highlight the importance of the TCA cycle and mitochondrial metabolism in parasite growth under both glucose replete and starvation conditions, consistent with increasing evidence that mitochondrial function is the target of existing anti-Leishmania drugs and a promising target for new therapeutics