The role of de novo myo-inositol synthesis and matabolism in Leishmania parasites

© 2018 Dr Tim Chung-Ting LiuLeishmania are protozoan parasites responsible for a spectrum of sandfly-borne diseases known as the leishmaniases that cause substantial morbidity and mortality in some of the most impoverished countries in the world. Conventional anti leishmanials are limited by toxicity, high cost, and increasing resistance, highlighting an urgent need to identify novel drug targets. Leishmania parasites require the nutrient myo-inositol for a multitude of crucial cellular processes that relate to its role as an integral building block for inositol lipids, including phosphatidylinositol (PI), inositol phosphorylceramide (IPC), free and anchoring glycosylphosphatidylinositol (GPI), and organelle-specific phosphoinositides. This work investigates the extent to which Leishmania parasites are dependent on de novo myo-inositol synthesis versus salvage from the extracellular milieu both in vitro and in vivo for growth and pathogenesis. By generating defined genetic deletion mutants in Leishmania mexicana lacking two key putative enzymes in this pathway, namely myo inositol 3-phosphate synthase (INO1) and myo-inositol 3 phosphate monophosphatase 1 (IMP1), the importance of de novo myo-inositol synthesis was directly addressed. As expected, the L. mexicana ∆ino1 mutant lacked the capacity for de novo myo inositol synthesis and was an inositol auxotroph. Strikingly, this mutant could only grow in ex vivo infected macrophages which had been supplied with an exogenous sources of myo-inositol and was avirulent in the susceptible BALB/c mouse model, suggesting that myo-inositol levels in Leishmania-induced granulomas are low. Analysis of the steady state polar metabolite profile of ∆ino1 mutant and 13C glucose labelling experiments failed to reveal key differences compared to wild type, demonstrating that L. mexicana does not enter self preserving quiescence in response to myo-inositol starvation. This, combined with the profound changes in lipid composition revealed by lipidomic analysis, may underlie the rapid death phenotype induced by myo inositol starvation. A second L. mexicana mutant lacking IMP1 was also generated. Unexpectedly, deletion of imp1 did not lead to disruption of de novo myo-inositol synthesis, but resulted in a mutant that displayed a modest growth defect in vitro that was unrelated to exogenous myo inositol levels. Despite the presence of de novo synthesis, the Δimp1 mutant was unable to proliferate in ex vivo macrophages or cause disease in mice. Global metabolomic and lipidomic analysis of this mutant revealed subtle changes in central carbon metabolism and lipid profile. Further studies showed that the loss of virulence of this mutant was linked to increased sensitivity to elevated temperatures encountered in the mammalian host, suggesting an unexpected role for IMP1 in the development of thermotolerance and differentiation to the amastigote stage. Genomic analysis also revealed a second putative IMPase isoform, IMP2, which may explain why loss of IMP1 does not lead to pertrubation of de novo myo-inositol synthesis in the Δimp1 mutant. In summary, this work highlights the importance of de novo myo-inositol synthesis for Leishmania growth and survival, providing a better understanding of a vital pathway. This improved understanding of the metabolic mechanisms essential for Leishmania provides further insights into their pathogenesis and validates enzymes in this pathway as new targets for the development of new strategies to combat the rising drug resistance