A2 Sphingolipid synthesis in the obligate intracellular parasite Toxoplasma gondii (Gupta)
|Research Group:||Metabolism and interactions between parasites and hosts|
|Address:||Humboldt-Universität zu Berlin, Department of Biology, Molecular Parasitology, Philippstr. 13, House 14, 10115 Berlin|
|Supervisor:||PD Dr. Nishith Gupta|
|Doctoral Researcher:||Dimitrios Alexandros Katelas|
State of the art:
Lipids are generally regarded as building blocks of biological membranes. They have evolved
for multifaceted roles, such as membrane integrity, protein trafficking, enzymatic regulation,
ion homeostasis and cell signaling. The main lipid classes in biological membranes include
phospholipid, glycolipid and sterol. The diversity and functional repurposing of lipids is a
fascinating yet sparsely studied area of research in the protozoan parasites. Toxoplasma
gondii is a parasite, which inflicts opportunistic infections in diverse warm-blood organisms
including humans. The acute disease, i.e. tissue necrosis, occurs by rapid intracellular
proliferation of T. gondii in host cells, which demands extensive membrane biogenesis within
the parasite, as well as scavenging of host-derived lipids.
Previous own work:
Our earlier work has shown that T. gondii and the related parasite Eimeria falciformis contain
a repertoire of exclusive phospholipids and sphingolipids. Notably, T. gondii contains a
high content of ceramide phosphoethanolamine (CPE), which differs from Eimeria enriched in
inositol-phosphorylceramide (IPC), and from mammalian cells harboring ceramide
phosphocholine (alias sphingomyelin, SM). CPE contains an ethanolamine moiety donated by
either phosphatidylethanolamine (PtdEtn) or by CDP-ethanolamine, in contrast to
phosphatidylinositol-derived inositol in IPC, or phosphatidylcholine (PtdCho)-derived choline
in SM. We have also characterized pathways producing these three donor phospholipids in T.
gondii. Given the lack of CPE in mammalian cells, the parasite is unlikely to import it.
Accordingly, we have identified two genes potentially involved in autonomous synthesis of
CPE, one of which encodes a CPE synthase (CPES; TGGT1_246490), and the other one for
a CDP-ethanolamine ceramide phosphotransferase (CECPT; TGGT1_261760).
Hypothesis and work plan:
CPE accounts for a major fraction (≥5%) of membrane lipids in T. gondii; however genetic
basis of its synthesis, physiological importance and function in the parasite remain unknown.
In the proposed work, (1) parasites will be subjected to metabolic labeling and lipidomic
analyses to establish de novo synthesis of CPE in T. gondii. We will test whether the parasite
generates CPE from PtdEtn or CDP-ethanolamine. We will establish the gene function by
expressing CPES and CECPT in HEK and CHO cells. (2) CPES and CECPT will be epitopetagged
by 3’-genomic tagging to monitor their natural expression during the lytic cycle. The
eventual transgenic strains will also allow gene deletion by CRISPR-Cas9 strategy. In case of
a lethal phenotype, we will undertake auxin-regulated conditional knockdown in T. gondii.
Metabolic labeling, lipidomics and enzyme assays will be performed to evaluate the impairment
of CPE in mutants. They will be phenotyped to discern the effect of CPE depletion on the
replication, egress, invasion and motility. Finally, we will test the ‘vaccination/immunological’
potential of growth-attenuated strains in a mouse model. (3) We will assess the impact of
sphingolipid synthesis inhibitors (e.g. aureobasidin) on the asexual reproduction of the parental
and mutant parasites. A genetic and/or pharmacological inhibition of the parasite virulence will
ascertain CPE synthesis as a validated therapeutic target. In an opposing scenario, the
nonessential and redundant roles of CPES and CECPT proteins shall uncover the metabolic
plasticity in T. gondii addressed by generating a double mutant at a later stage.