Parasite Genetics and Adaption - Project B2

Characterising Lipid droplets (LD) in various life cycle stages of Plasmodium sp.

Host lab: Melanie Rug (ANU)

HUB partner: Andreas Hermann, Martin Blume, Kai Matuschewski 

ANU partner: Alexander Maier and Lara Malin

State of the art and previous own work:

Many stages across the Plasmodium cycle have changing demands in storage and mobilisation of lipids. We have identified that the profile of lipid groups, including neutral lipids, changes significantly between blood stage Plasmodium falciparum parasites (1). 

Furthermore, the ATP-binding cassette transporter gABCG2, which has been suggested to transport neutral lipids into a lipid storage compartment (2), is primarily transcribed in sexual stages and accumulates in a single lipid-rich dot in female gametocytes, reminiscent of a large lipid droplet (LD), organelles known to accumulate neutral lipids. Our gABCG2 ko cell line produces more gametocytes of both sexes than wild type parasites and neutral lipids are significantly reduced in gABCG2-knockout gametocytes indicating that the transporter plays a crucial role in regulation of gametocyte numbers and the accumulation of neutral lipids in gametocytes, which are likely important for parasite development of insect stages (2). Here, we set out to study the biogenesis, morphology and composition of Plasmodium LDs, which have not been explored in detail. Identifying proteins involved in lipid metabolism, stress responses and cell signalling and functional studies of these regulatory molecules might reveal new drug targets to combat malaria.

Hypothesis and work plan:

We hypothesise that LD numbers, size and distribution and their composition of neutral lipid classes and surface proteins are different in various stages of the Plasmodium life cycle. Furthermore, we propose that gABCG2 is instrumental in LD formation and impacts LD lipid composition. To address these aspects, we will apply click chemistry to label various lipid species used as building blocks or ‘cargo’ for LDs with biologically inert tags that will allow us to study the morphology, biogenesis and distribution of LDs in the asexual and sexual life cycle stages via superresolution microscopy in wild type and gABCG2 knock-out cell lines. We will also generate a cell line that co-expresses an APEX (soybean ascorbate peroxidase)-GFP-binding protein (GBP) protein together with the gABCG2-GFP. The GBP indicates the presence of gABCG2 within the cell, with the APEX part forming an electron dense precipitate under certain conditions visible in electron microscopy (3). This precipitate allows to apply volume imaging and segmentation to gain insight into the 3D ultrastructural details of the dynamics of LDs in the Plasmodium life cycle. 

Structural analysis in situ via cryo EM might give further insights into the molecular association of gABCG2 with LDs. These studies on LD biogenesis in the presence and absence of gABCG2 are anticipated to reveal the function of gABCG2 in the process of lipid accumulation or mobilisation in these storage organelles. 

Interlinkages: The above project will be performed in close collaboration with the Maier lab, who have laid the groundwork to this study. A bioinformatics approach combined with purification and omics analysis of LDs from asexual stages performed in Alex Maier’s (ANU) and Martin Blume’s (HUB) lab in this cohort will provide further protein candidates associated with LDs which might play vital roles in Plasmodium lipid metabolism and these projects are complementing each other. We will tag these proteins with click chemistry (Lara Malin’s group, ANU) and generate deletion mutants in order to study the functions of LD proteins as described above. With Andreas Herrmann’s group (HUB), we will generate proteoliposomes with LD marker proteins (including gABCG2) to study their function in lipid uptake and mobilisation experiments. With Kai Matuschewski’s (HUB) and Elena Levashina’s (MPI) groups we will perform functional characterisation studies of LDs in Plasmodium berghei and morphological characterisation in insect stages of Plasmodium falciparum, respectively.