Deciphering the molecular mechanisms of PI3P-mediated vesicular transport to the apicoplast in apicomplexan parasites – APICO-PIP

Transgenic parasites are generated to study candidate proteins potentially involved in PI3P-dependent functions. The localization and the functional link of those proteins with the apicoplast are analyzed by microscopy. The complete Plasmodium life cycle is followed in red blood cells, mosquito stages and liver cells.

PI3P lipid is present on small vesicles. Genetic and pharmacological tools interfering with PI3P production or availability lead to the accumulation of vesicles and disrupt apicoplast biogenesis. Effectors proteins possess specific PI3P-binding domains. Transgenic parasites targeting these proteins are produced. The analysis of their localization and function is in progress.

Identifying and characterizing new proteins targeted to the apicoplast through PI3P-mediated vesicle transport. Our project will allow increasing knowledge on the composition of this particular organelle. Collected data should lead to apicoplast-specific drug design to block

First results were presented at a poster session of a malaria conference held in Australia (Molecular Approaches to Malaria, Lorne, Australia, February 2012).

The phylum Apicomplexa encompasses many protozoan parasites that cause devastating diseases of humans and farm animals, including Plasmodium spp causing malaria and Toxoplasma gondii causing toxoplasmosis.
A particular feature of most apicomplexan parasites is the presence of a plastid-like, non-photosynthetic organelle of algal origin, the apicoplast that has become a promising target for the development of new anti-apicomplexan drugs because of its original and essential metabolic pathways. The apicoplast has been acquired by secondary endosymbiosis and in consequence is bounded by four membranes. The apicoplast proteome is almost entirely encoded by the nucleus and must thus be post-translationally imported. While information on apicoplast targeting sequences and some molecular mechanisms of how proteins cross the three inner membranes have been obtained, how proteins accomplish the first step, i. e. trafficking from the endoplasmic reticulum to the apicoplast outermost membrane remains enigmatic.
There is some evidence that proteins destined to the apicoplast surface might travel via a vesicular route and two recent studies by Partner 1 conducted in parallel in Plasmodium and T. gondii discovered intriguing evidence for the involvement of the lipid phosphatidyinositol-3 monophosphate (PI3P) in this traffic. In eukaryotic cells in general PI3P exerts its function in vesicle trafficking and endocytic fusion events through the binding of effector proteins that contain specific PI3P-binding domains (FYVE- and PX-domains). In P. falciparum and T. gondii PI3P is present at the apicoplast and on small vesicles, which in T. gondii additionally carry proteins destined for the apicoplast surface. Interference with PI3P-function in T. gondii by drug treatment or PI3P-sequestration led to vesicle accumulation and to severe plastid biogenesis defects. These results revealed a function of PI3P in a cellular process in apicomplexan parasites that is different from classical endosomal traffic observed in other organisms including the human host. Interestingly, conventional endocytic traffic has not been found in Apicomplexa. We postulate that apicomplexan parasites have reshaped the classical PI3P-dependent endocytic machinery found in other eukaryotic organisms to target proteins (and possibly also lipids) to the apicoplast.
The objective of this proposal is to dissect the molecular mechanisms of protein traffic to the apicoplast in Plasmodium and Toxoplasma. One original aspect of this proposal lies in our ability to study this important and most likely essential cell biological process in both parasites. Rapid transposition of results from one system to the other allows advancing rapidly with a maximum output on the subject for two important human pathogens.
The first aim is to establish the functional link between PI3P and apicoplast biogenesis in Plasmodium mainly by analysing PI3P-dependent functions in stages without a food vacuole where PI3P might additionally play a role. These experiments will be performed with the rodent malaria parasite P. berghei and Partner 2 has been selected for their expertise in the analysis of in vitro liver stages and their interest in organelle biogenesis during these stages. The second aim is to identify players of the vesicular traffic to the outermost membrane of the apicoplast in both parasites. Finally, the third aim is to identify and characterize new apicoplast proteins through the purification of vesicles destined to fuse with the organelle.
Our long term objective is to interfere with the biogenesis of this unique organelle, which is essential for intracellular parasite survival and in this way open new ways in the fight against apicomplexan parasites and their major impediment to public health and economy.