Reproduce, divide and invade: microtubule cytoskeletons in Plasmodium parasites

Malaria caused by Plasmodium parasites represents a major public health problem. In order to develop new strategies to overcome infections, it is necessary to understand fundamental processes relevant for parasite proliferation and dissemination. The parasite cytoskeletons and associated structures are essential at multiple developmental stages that are critical in the pathogenesis and propagation of Plasmodium. This includes structures conserved across eukaryotes such as the mitotic spindle, the spindle pole and the kinetochore for cell division or the basal body nucleating axonemes for sexual reproduction. Due to evolutionarily divergence of Plasmodium parasites from well-studied organisms, the molecular organisation and the regulation of these structures remain elusive. The parasitic life style is also associated with the emergence of the apical complex, an organelle based on a specific cytoskeleton required for motility and host cell invasion that is only found in apicomplexan parasites. The apical complex has mainly been studied in another related parasite, Toxoplasma gondii, but little is known about this structure in Plasmodium. Ultrastructure expansion microscopy (U-ExM) has recently been implemented to study the nanoscale organisation of the Plasmodium cytoskeletons. Now this powerful approach will be combined with deep molecular phenotyping and cutting-edge reverse genetics to tackle the molecular organisation and the regulation of these structures. The intention is also to use the resulting knowledge to test whether they can be targeted by small molecules to block multiple stages of the parasite development.

Using a limited number of conserved proteins, the study will first determine the protein repertoire of this MTOC by immuno-precipitation followed by semi-quantitative mass spectrometry (MS). The localisation and function of these proteins will then be determined by combining reverse-genetic approaches with U-ExM. Finally, previous results indicate that four kinases, and three ubiquitin ligases are important to regulate the homeostasis of this MTOC. Combining quantitative phosphoproteomics and ubiquitinomics with functional analyses, how phosphorylation, ubiquitination and their interplay, regulate replication and segregation of this MTOC to sustain mitotic division and axoneme nucleation will be defined.  A study recently identified and characterised the molecular organisation of the Plasmodium kinetochore to an unprecedented level using U-ExM. This allowed the identification that entry into anaphase is likely controlled by a putative spindle assembly checkpoint (SAC). Such checkpoint was thought to be lost in Plasmodium in the absence of adequate markers and of most conserved regulators involved in the eukaryotic SAC. The unpublished results indicate that two divergent aurora kinases (ARK), a phosphatase and a ubiquitin ligase may be involved the regulation of chromosome segregation. Combining interactomic analyses, conditional protein depletion, phosphoproteomics, ubiquitinomics and U-ExM, the study will identify and characterise SAC proteins and other signalling pathways regulating chromosome segregation along the mitotic spindle. As the two identified ARKs are essential at multiple proliferative stages during the Plasmodium lifecycle, recombinant proteins will be produced to screen small molecules and identify inhibitors. For the most promising leads, their mechanism of action and potential as multistage anti-Plasmodium will be assessed. It will then define the role of selected proteins in the formation, function and regulation of the apical complex to sustain intercellular dissemination of Plasmodium in the blood or its transmission to the mosquito. Altogether, this project will reveal the molecular organisation and regulation of cytoskeletal structures that have been neglected despite their essentiality for the pathogenesis and dissemination of parasites causing malaria. It will also bring new insights into the diversity of cytoskeletal structures in eukaryotes and how they evolved in response to a parasitic life style.

Project details

Basic Science

Apr 2022 — Mar 2026

$977,025

Switzerland