Structural and functional characterization of Plasmodium falciparum rhoptries and its proteins

  • Anton, Leonie L. (PI)
  • University, Department Of Microbiology And Immunology Hhsc Columbia (CoI)

Project: Research project

Project Details

Description

Malaria is caused by protozoan parasites of the Plasmodium species, which during their lifecycle infect human erythrocytes. Like all apicomplexan parasites, P. falciparum has specialized organelles that are essential to infectivity. One of these organelles are the rhoptries, which eject proteins, lipids and membranes into the erythrocyte upon invasion that aid in establishing an exomembrane system and building a new trafficking network to acquire the nutrients necessary for intracellular replication of the parasite. The molecular processes mediating rhoptry content expulsion and nutrient acquisition remain poorly understood. The first aim of this proposal is to leverage the latest developments in cryo electron tomography and cryo focused ion beam milling to achieve sub nanometer resolution of rhoptry ultrastructures during different stages of invasion. Investigation of the molecular mechanisms underlying rhoptry function and the accompanying changes in ultrastructure have been restricted to sub-micrometer visualization by electron microscopy due to challenges in sample preparation, especially control of sample thickness, which limit achievable resolution. To overcome these challenges, I will use cryo focused ion beam milling, a cutting edge technique for creating sections of vitrified cells thin enough to achieve sub-nanometer resolution 3D reconstructions of malaria parasite rhoptries. In the second aim, I propose to determine the near-atomic structure of the high-molecular-weight rhoptry complex known as the RhopH complex, which inserts into the erythrocyte membrane and is essential for nutrient uptake. The RhopH complex was one of the first described protein complexes contained in the rhoptries. This complex has been linked to the invasion of erythrocytes, establishment of the parasitophorous vacuole and nutrient acquisition by the parasite during intracellular replication. However, there is no known structure of the soluble or membrane bound form of any of the contained proteins. The lack of genetic tools for manipulation of parasites and the difficulty in recombinant expression of malaria proteins have made structure determination by conventional methods challenging. I will use the CRISPR-Cas gene-editing to add an epitope tag to the endogenous locus of RhopH complex proteins, which will enable me to purify the native membrane bound form directly from P. falciparum malaria parasites. By using cryo electron microscopy and single particle analysis, I can circumvent the need for large amounts of pure protein and achieve a near-atomic resolution 3D reconstruction of the native RhopH complex. I also aim to characterize additional novel rhoptry proteins by using the new cryoID method, which enables the identification of proteins from complex samples by their structure. The molecular characterization of rhoptry ultrastructure pre-, mid- and post-infection, combined with near-atomic structures of essential rhoptry proteins will deepen our understanding of how the parasite invades and manipulates the host-cell to establish intracellular infection. Elucidating the essential mechanisms of invasion and nutrient acquisition in malaria parasites will pave the way for developing drug mediated interventions to block parasite survival. Additionally, the novel approaches developed in the pursuit of the proposed research will provide much-needed tools for overcoming longstanding barriers to high resolution structural studies across the field of parasitology.

StatusFinished
Effective start/end date1/1/216/30/22

Funding

  • Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung

ASJC Scopus Subject Areas

  • Parasitology
  • Molecular Biology
  • Microbiology
  • Biochemistry

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