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Investigation of dense granule proteins in the human malaria parasites Plasmodium falciparum and Plasmodium knowlesi

Title of PhD project

Investigation of dense granule proteins in the human malaria parasites Plasmodium falciparum and Plasmodium knowlesi

Supervisory team

LSHTM  

Lead: Dr Robert Moon (Rob.Moon@lshtm.ac.uk, Faculty of Infectious and Tropical Diseases) 

Dr Christiaan van Ooij (Christiaan.vanOoij@lshtm.ac.uk)  

Nagasaki University 

Prof. Kiyoshi Kita (kitak@nagasaki-u.ac.jp) 

Prof. Osamu Kaneko (okaneko@nagasaki-u.ac.jp)                                                                 

Brief description of project

The symptoms of malaria are the result of invasion of malaria parasites into the host’s red blood cells (RBCs). To enter the host RBC, the parasite relies on three organelles, together referred to as the apical organelles. Two of these organelles, the micronemes and rhoptries, have been studied in detail and their functions have become clear. In contrast, the function of the third organelle, the dense granule (DG), has received very little attention in malaria parasites even though its function is essential to growth of the parasite. It has been investigated in detail in the related parasite Toxoplasma gondii, but differences between the intracellular lifestyles of the parasites do not allow the knowledge of the T. gondii DG to be translated to the malaria parasite. In the human malaria parasite Plasmodium falciparum, the DG are produced late in the 48-hour intraerythrocytic cycle; preliminary evidence from the van Ooij lab has indicated that the organelles are produced only around 30 minutes before the end of the cycle. This would be consistent with the proposed role for DG: delivery of proteins that modify the host RBC after the parasite has invaded a new RBC. However, the protein content, the mechanisms underlying the formation and release of the organelle, the signals required for targeting proteins to this organelle and the function of its cargo remain unclear.  

The aim of this studentship is to gain insight into the role of the DG in malaria parasites, with a focus on the role of DG proteins in the growth and survival of the parasites in the host RBC. A deeper knowledge of this will greatly increase our understanding of the ways in which this important pathogen interacts with and survives within its host RBC. The supervisor, Dr Moon, studies a less characterized human malaria parasite, Plasmodium knowlesi. The side-by-side investigation of the two species will allow the DG to be studied two malaria parasites, providing information about the species-specific and genus-specific roles of the DG, deepening our understanding of the role of the DG.  

The van Ooij lab has recently undertaken the first effort to identify the protein content of the DG in P. falciparum through bioinformatics and proximity-labeling approaches using the APEX technology [1]. Although these studies are still in progress, we have been able to identify several new DG proteins. However, there is surprisingly little conservation of exported proteins (proteins that modify the host RBC and that are expected to be the bulk of the cargo of DG) between species of malaria parasites. Therefore, the student will first identify the DG content in a different Plasmodium species, P. knowlesi, using the same proximity-labeling technique that have successfully been applied in P. falciparum. P. knowlesi is also a human pathogen (although it most commonly infects old world monkeys) and can be cultured and genetically manipulated in vitro [2, 3]. A comparison of the DG content between these two malaria parasites will allow identification of DG cargo that is conserved in all Plasmodium species, and hence may perform essential basic functions, and species-specific cargo that mediates modification of the host for each species’s specific growth requirements. The student will confirm that the proteins identified in the proximity-labeling experiments are indeed transported to the DG and subsequently identify the localization of the proteins after the DG contents have been released, thereby aiming to discover the function of the proteins. This will be done by fusing the putative DG protein to a fluorescent reporter protein and expressing these in a parasite strain in which the DG are marked with a different fluorescent protein, allowing colocalization of the markers to be determined in live parasites. Protein function will be determined by producing parasite strains in which the genes encoding DG proteins can be removed through the addition of a chemical, a well-established technique in Plasmodium parasites [3, 4]. In this part, the student will focus primarily on the proteins shared between the malaria parasites to uncover basic principles of the pathogen-host interaction that are applicable to all malaria parasites. However, 2-4 of the most abundant species-specific proteins will also be investigated.  

In summary, the aims of the studentship are: 

  1. Produce a genetically modified P. knowlesi parasite line to allow APEX-based proximity labeling, perform proximity labeling and identify putative DG proteins in P. knowlesi. 

  1. Confirm prediction of DG proteins in P. knowlesi (data from Aim 1) and P. falciparum (data already generated) using fusions of putative DG proteins to a fluorescent protein. 

  1. Produce P. knowlesi and P. falciparum parasite lines in which conserved DG proteins can be inducibly removed to determine the phenotype of parasite lacking the protein. 

We are confident that as the APEX-based proximity-labeling technique has been clearly established in Plasmodium parasites [5] and has been used successfully in Dr Robert Moon’s lab in P. knowlesi and in the van Ooij lab in P. falciparum, the student will be able to complete Aim 1 and use the findings in Aims 2 and 3. However, in the unlikely case that Aim 1 cannot be completed successfully, the data from proximity-labeling experiments in P. falciparum will provide the starting point for the confirmation and analysis of DG proteins in Aims 2 and 3. Bioinformatic analysis can be used to identify DG proteins shared by P. falciparum and P. knowlesi and the localization and function of the P. knowlesi proteins can be determined using the methods described. 

Together, these experiments will provide fundamental insight into the interaction of malaria parasites with their host RBCs. Identification of the shared and essential DG proteins may subsequently open avenues of drug development targeting DG proteins.  

The selected student will perform most of the experiments at LSHTM, under supervision by Dr Robert Moon. The student will visit Nagasaki occasionally to conduct experiments under supervision by Prof. Osamu Kaneko.  

References 

  1. Martell JD, Deerinck TJ, Sancak Y, Poulos TL, Mootha VK, Sosinsky GE, Ellisman MH, Ting AY. Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy. Nat Biotechnol. 2012;30:1143-8. doi: 10.1038/nbt.2375. 

  1. Moon RW, Hall J, Rangkuti F, Ho YS, Almond N, Mitchell GH, Pain A, Holder AA, Blackman MJ. Adaptation of the genetically tractable malaria pathogen Plasmodium knowlesi to continuous culture in human erythrocytes. Proc Natl Acad Sci U S A. 2013;110(2):531-6. doi: 10.1073/pnas.1216457110.  

  1. Mohring F, Hart MN, Rawlinson TA, Henrici R, Charleston JA, Diez Benavente E, Patel A, Hall J, Almond N, Campino S, Clark TG, Sutherland CJ, Baker DA, Draper SJ, Moon RW. Rapid and iterative genome editing in the malaria parasite Plasmodium knowlesi provides new tools for P. vivax research. Elife. 2019;8:e45829. doi: 10.7554/eLife.45829. 

  1. Collins CR, Das S, Wong EH, Andenmatten N, Stallmach R, Hackett F, Herman JP, Müller S, Meissner M, Blackman MJ. Robust inducible Cre recombinase activity in the human malaria parasite Plasmodium falciparum enables efficient gene deletion within a single asexual erythrocytic growth cycle. Mol Microbiol. 2013;88(4):687-701. doi: 10.1111/mmi.12206. 

  1. Kehrer J, Ricken D, Strauss L, Pietsch E, Heinze HM, Frischknecht F. APEX-based proximity labeling in Plasmodium identifies a membrane protein with dual functions during mosquito infection. bioRxiv. doi: 10.1101/2020.09.29.318857. 

The role of LSHTM and NU in this collaborative project

The selected student will conduct most of the experiments in London. However, owing to the difficulty of using monkey RBCs in London, the human RBC-adapted P. knowlesi A1/H1 strain is used with human RBCs in London. P. knowlesi grows better in monkey RBCs than human RBCs, thus we expect the phenotypes of P. knowlesi inducible knockout lines will be more clearly characterized with monkey RBCs (Aim 3). In the Kaneko lab in Japan, P. knowlesi is maintained with both monkey and human RBCs, which provides opportunities to examine phenotypes using monkey RBCs. In addition, comparison of the behaviours of released DG proteins in human and monkey RBCs may provide insights why P. knowlesi A1-H1 line grows slower with human RBCs. Furthermore, the electron microscopy lab in Nagasaki University has extensive expertise in electron microscopy of malaria parasites, which will be an invaluable technique for the analysis of the mutant parasites.  

Particular prior educational requirements for a student undertaking this project

The student is expected to have a basic molecular biology education with a degree in a relevant subject and experience working in a laboratory at a Masters' student level.  

Skills we expect a student to develop/acquire whilst pursuing this project

We expect the student to learn the following skills:

  • basic molecular biology skills (including PCR, plasmid construction, genomic DNA isolation and immunoblotting)
  • fluorescent microscopy techniques (including live imaging of parasites and immunofluorescence assays)
  • bioinformatics (genome-level comparisons of the data sets for the two malaria parasites investigated)
  • malaria parasite culture techniques
  • analysis of mass spectrometry data