Generation of inositol polyphosphates through a phospholipase C-independent pathway involving carbohydrate and sphingolipid metabolism in .

Unlabelled: Inositol phosphates are involved in a myriad of biological roles and activities such as Ca signaling, phosphate homeostasis, energy metabolism, and disease pathogenicity. In synthesis of inositol phosphates occurs through the phosphoinositide phospholipase C (PLC)-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP) into inositol 1,4,5-trisphosphate (IP) and diacylglycerol and further IP phosphorylation by additional kinases that leads to the formation of highly phosphorylated inositol derivatives, known as inositol pyrophosphates. Inositol-tetrakisphosphate 1-kinase (ITPK1) is an enzyme that mediates a PLC-independent inositol polyphosphate synthesis through phosphorylation of inositol monophosphates and other intermediates in the cytosol. In this work, we identified and characterized a ITPK1 (TcITPK1) homolog. The ability of TcITPK1 to act as the mediator for this alternative pathway was established through Δ and Δ Δ yeast complementation assays and SAX-HPLC analyses of radioactively labeled inositol. TcITPK1 localizes to the cytosol, and knockout attempts of revealed that only one allele was replaced by the DNA donor cassette at the specific locus, suggesting that alleles may have lethal effects in epimastigotes. Ablation of phosphoinositide phospholipase C 1 () affected the synthesis of IP from glucose 6-phosphate but did not affect the synthesis of inositol polyphosphates, while ablation of inositol phosphosphingolipid phospholipase () affected the synthesis of inositol polyphosphates, thus revealing that the PLC-independent pathway using either glucose 6-phosphate or inositol phosphoceramide is involved in the synthesis of inositol polyphosphates, while the PLC-dependent pathway is involved in IP formation needed for Ca signaling.

Importance: Millions of people are infected with and the current treatment is not satisfactory. Inositol pyrophosphates have been established as important signaling molecules. Our work demonstrates the presence of a phospholipase C-independent pathway for the synthesis of inositol pyrophosphates in . Furthermore, we demonstrate that this pathway starts with the synthesis of inositol monophosphates from glucose 6-phosphate or from inositol phosphoceramide, linking it to carbohydrate and sphingolipid metabolism. The essentiality of the pathway for the survival of infective stages makes it an ideal drug target for treating American trypanosomiasis.