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Understanding the Molecular Structures within Plasmodium Berghei

Introduction


Does the term RTS,S or Mosquirix ring a bell? This recombinant vaccine has recently been making rounds in news articles for the dreaded disease Malaria. As we all know, Malaria is a deadly infectious disease that takes hundreds of thousands of lives across the world every year. The 2018 WHO report shows that there was a whopping 219 million cases of Malaria and 435,000 related deaths in 2017 and most of these cases came from the African region. This is shocking, isn’t it? Although there has been a steady decline in the number of deaths through the years, the lack of efficient and affordable vaccines, as well as the ever-increasing resistance to existing drugs, begs for more research to understand the biology of the parasite Plasmodium. What makes the Plasmodium a complex parasite is that its life cycle uses two hosts- vertebrates (that’s us!) and mosquitoes. The figure below provides a quick glance at the Plasmodium life cycle.


Figure 1. Life cycle of Plasmodium. Learn more from : Enomoto, M., Kawazu, S., Kawai, S., Furuyama, W., Ikegami, T., Watanabe, J., and Mikoshiba, K. (2012) Blockage of spontaneous Ca2+ oscillation causes cell death in intraerythrocitic Plasmodium falciparum. PLoS One, 7 (7), e39499.

Nuclear Pore Complex


Our lab at Iowa State works with a model system in rodents with Plasmodium berghei. P. berghei is a relative of P. falciparum, which causes malaria in humans. We are focused on understanding the make-up of the nuclear pore complex (NPC). The NPC is the gatekeeper for nucleocytoplasmic transport in all eukaryotic cells and plays a major role in the regulation of gene expression. The NPC forms a basket-like structure that interacts with and controls the transport of molecules in and out of the nucleus.

Figure 2. Image of the nuclear pore. Original photo can be found at :https://www.the-scientist.com/infographics/infographic-the-nuclear-pore-complex-32456

P. berghei is a rodent malaria model whose nuclear pore complex components are poorly defined due to their evolutionary divergence. The parasite is part of the Alveolata, a clade distant from the Opisthokonta on the eukaryotic evolutionary tree, a group of organisms with flagellate cells. In Opisthokonts, for example yeast and humans, the nuclear pore complex (NPC) has been extensively studied defining its quaternary protein structure. However, Plasmodium parasites have little or no primary sequence conservation of these proteins which has prevented researchers from identifying these proteins by orthology. Recent research in this area, using genome scanning, has identified five phenylalanine-glycine nucleoporins (FG-NUPS) which were also shown by imaging to localize at the nuclear periphery in both sexual and asexual stages of the parasite life cycle. FG-NUPS are scaffolding proteins in the nuclear pore complex.


Methodologies for Investigating Protein Interactions


In order to identify the nucleoporins of the P.berghei NPC, our lab adopted the proximity labelling approach using BioID. This is a system developed by Kim et al that uses the bacterial enzyme biotin ligase. When this enzyme is fused to a bait protein, it biotinylates proximal proteins in the presence of biotin. We follow a simple workflow to identify novel nucleoporins in P.berghei. We begin with constructing a fusion protein containing plasmid with one of the FG-NUPS as a bait protein. Mutant parasites are then created which are grown in mice for about 7-10 days. The blood containing the parasites is collected and the proteins are isolated using Saponin lysis. This protein pool contains a mixture of biotinylated and non-biotinylated proteins from our blood sample. To purify the proteins of interest, we use magnetic streptavidin beads to isolate biotinylated proteins (proteins that were proximal to our bait FG-NUP in vivo). Mass spectrometry analysis of the biotinylated protein sample gives us more information about these proteins. Assessment of localization by immunofluorescence assays allows us to confirm that the newly identified proteins are localized to the nuclear periphery.


Figure 3. Workflow of protein constructs for the assessment of protein interactions.

I believe that this research will make an important impact in the field of malaria research, as it will provide us with potential vaccine targets for the parasite that are currently unknown. Identifying these nucleoporins will pave the path for understanding interactions of various nuclear components and molecules with these proteins through the life cycle of the parasite. I believe that only when we understand the disease completely, we can effectively work towards eradicating it.


Of course, check out these references:


1. Enomoto, M., Kawazu, S., Kawai, S., Furuyama, W., Ikegami, T., Watanabe, J., and Mikoshiba, K. (2012) Blockage of spontaneous Ca2+ oscillation causes cell death in intraerythrocitic Plasmodium falciparum. PLoS One, 7 (7), e39499.


2. Kim, D.I., Jensen, S.C., Noble, K.A., Kc, B., Roux, K.H., Motamedchaboki, K., and Roux, K.J. (2016) An improved smaller biotin ligase for BioID proximity labeling. Mol. Biol. Cell, 27 (8), 1188–1196.


3. Kehrer, J., Kuss, C., Andres-Pons, A., Reustle, A., Dahan, N., Devos, D., Kudryashev, M., Beck, M., Mair, G.R., and Frischknecht, F. (2018) Nuclear Pore Complex Components in the Malaria Parasite Plasmodium berghei. Sci. Rep., 8 (1), 11249.


For extra information, check out these websites:

https://www.mmv.org/malaria-medicines

https://www.the-scientist.com/infographics/infographic-the-nuclear-pore-complex-32456



Author Bio

Sushma Ambekar is a PhD Graduate student at Iowa State University in the Mair Laboratory. She can be found on Twitter (@sushma_ambekar) and on LinkedIn. Also check out her Blog at https://parasitelady.wordpress.com/ to learn more!


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