Functional analyses of sphingolipid biosynthesis in an apicomplexan parasite

Abstract

The phylum Apicomplexa includes many protozoan parasites that cause serious human and animal disease, for example Plasmodium, Eimeria and Toxoplasma. Treatments against these parasites are limited and novel solutions are urgently required. Recently, research has focused on parasite specific features of lipid biosynthesis as drug targets. In particular the biosynthesis of sphingolipids, which have essential roles in many processes, has been highlighted as a potential target.
Using the model apicomplexan Toxoplasma gondii we are studying the role of parasite and host sphingolipid biosynthesis in invasion and proliferation. Serine palmitoyltransferase (SPT) catalyzes the first step in sphingolipid biosynthesis, and our results demonstrated that the expression of host cell SPT is unaffected by Toxoplasma infection. In mammals the primary complex sphingolipid is sphingomyelin (SM), again our data demonstrated that the SM synthases (1 and 2) are not influenced by infection. Together these data indicated that parasite manipulation of host sphingolipid biosynthesis does not occur, supporting the hypothesis that Toxoplasma is dependant on de novo sphingolipid biosynthesis. To characterise this pathway, we showed that the Toxoplasma TgSPT1 and 2 are, like other eukaryotes, localised and active in the endoplasmic reticulum. However, uniquely, they have a prokaryotic origin. Metabolic labelling showed that several distinct complex sphingolipids are synthesized independently by the parasite. The fungal inositol phosphorylceramide (IPC) synthase inhibitor aureobasidin A (AbA) has been reported to target Toxoplasma IPC synthesis. However, our results demonstrated that whilst AbA, and an orthologue, are active against the parasite, their effect on Toxoplasma de novo sphingolipid biosynthesis is negligible. In addition, by using Leishmania major as a model we have analysed the global effect of compounds recognised as IPC synthase inhibitors in this kinetoplastid protozoan parasite. The results showed that ceramide levels increased in treated parasites, perhaps leading to parasite death via secondary signalling dysfunction. These data confirmed that the sphingolipid biosynthetic pathway is targeted by these anti-leishmanial compounds.
Finally, the anti-leishmanial drug miltefosine showed reduced activity against a transgenic strain of L. major lacking sphingolipid biosynthesis ΔLCB2 compared to wild type. This suggested the sphingolipid synthesis has a role in sensitivity to the drug, metabolomic analyses supported this.
Taken together, the present findings further characterised the T. gondii sphingolipid biosynthetic pathway and indicated the potential to target this in drug discovery efforts. In addition, metabolomic and lipidomic approaches confirmed that clemastine targets L. major IPCS.