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Characterizations of key phospholipid biosynthesis enzymes in Plasmodium falciparum - NU/LSHTM project

Supervisors

Nagasaki University

LSHTM 

Project

All malaria parasites, including P. falciparum, invade and replicate inside human erythrocytes. During this replication, the parasite requires large amounts of phospholipids to support the expansion of its cell membrane. The phospholipid content in infected erythrocytes increases sixfold compared to uninfected cells (1). While parasites absorb some lipids from the host cell and environment, they primarily rely on biosynthesis to meet their phospholipid requirements during rapid proliferation.

The major steps in lipid biosynthesis include fatty acid synthesis from glucose and the formation of phospholipids through the Kennedy pathway. Despite its significance, lipid metabolism in P. falciparum is poorly understood compared to mammalian systems (2). Only a few enzymes involved in P. falciparum phospholipid synthesis have been identified, leaving many of the parasite's lipid metabolic processes uncharacterized.

Our research aims to elucidate the biological functions of two critical enzymes in P. falciparum lipid metabolism: CDP-choline/ethanolamine phosphotransferase (PfCEPT) and phosphatidylserine synthase (PfPSS).  Both genes are considered to be essential in P. falciparum, and these enzymes are key players in the biosynthesis of essential phospholipids. Their unique characteristics in P. falciparum make them promising targets for novel antimalarial therapies. 

PfCEPT: This enzyme is involved in the synthesis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), both of which are essential components of the cell membrane. In P. falciparum, PfCEPT is the sole enzyme catalyzing the production of PC, which accounts for more than 40% of the parasite’s cell membrane (3). PC and PE are also crucial for protein trafficking and signal transduction, making PfCEPT a potential drug target. 

PfPSS: Unlike mammalian cells, which have two phosphatidylserine (PS) synthases, P. falciparum expresses only one prokaryote/yeast-type PSS. PS is a precursor to PE and plays a role in apoptosis and removal of apoptotic cells by phagocytes (1, 3). PS expression by parasites varies between developmental stages, suggesting the relative fractions of PS, PE, and PC are highly regulated (4). The study of PfPSS, particularly its localization in ER-mitochondria contact sites (5), could reveal novel insights into parasite survival and resistance mechanisms.

Our hypothesis is that PfCEPT and PfPSS play pivotal roles in maintaining P. falciparum membrane integrity, protein trafficking, and immune evasion. Targeting these enzymes could disrupt parasite growth and survival within the host environment, making them viable candidates for the development of new antimalarial therapies.

The specific objectives of our project are:

  • To characterize PfCEPT and PfPSS: Elucidate the biochemical pathways involving PfCEPT and PfPSS in P. falciparum lipid metabolism.
  • To investigate PfPSS and mitochondrial interactions: Explore the role of PfPSS in the transport of PS between the ER and mitochondria and its impact on parasite survival.
  • To evaluate potential drug targets: Assess the viability of PfCEPT and PfPSS as drug targets by analyzing the effects of enzyme inhibition on parasite growth and survival.
  • To investigate the roles of PfCEPT and PfPSS in immune response evasion: The mutant murine Plasmodium will be generated and the immune response of mice infected with these strains will be evaluated.  

Methodology 

  • Conditional gene knockout Studies: Use CRISPR-Cas9 to knockout the expression of PfCEPT and PfPSS and analyze the impact on phospholipid composition, membrane integrity, and parasite viability. Characterize impact of PfCEPT and PfPSS knockout on clear parasitemia and immune responses, including leukocyte composition and activation.  
  • Lipidomic Profiling: Conduct lipidomic analyses to compare the phospholipid composition of wild-type and enzyme-deficient parasites. 
  • Inhibitor Screening: Screen small molecule inhibitors of PfCEPT and PfPSS for their antimalarial activity in vitro. 

Expected Outcomes

  • Identification of PfCEPT and PfPSS as critical enzymes in P. falciparum phospholipid metabolism.
  • Demonstration of the role of PfPSS in mitochondrial interactions and immune evasion.
  • Development of PfCEPT and PfPSS inhibitors with potential as novel antimalarial therapies. 

Conclusion

This study aims to advance our understanding of P. falciparum lipid metabolism and identify new therapeutic targets in the fight against malaria. By focusing on the unique characteristics of PfCEPT and PfPSS, we hope to contribute to the development of more effective antimalarial treatments, addressing the growing threat of drug resistance, and the role of parasite lipid metabolism on host immune response. 

References

  1. Vial HJ, Ancelin ML. Malarial lipids. An overview. Subcell Biochem. 1992;18:259-306. Epub 1992/01/01. PubMed PMID: 1485354.
  2. Kennedy EP, Weiss SB. The function of cytidine coenzymes in the biosynthesis of phospholipides. J Biol Chem. 1956;222(1):193-214. Epub 1956/09/01. PubMed PMID: 13366993.
  3. Dechamps S, Shastri S, Wengelnik K, Vial HJ. Glycerophospholipid acquisition in Plasmodium - a puzzling assembly of biosynthetic pathways. Int J Parasitol. 2010;40(12):1347-65. Epub 2010/07/06. doi: 10.1016/j.ijpara.2010.05.008. PubMed PMID: 20600072.
  4. Sturm A, Amino R, van de Sand C, Regen T, Retzlaff S, Rennenberg A, et al. Manipulation of host hepatocytes by the malaria parasite for delivery into liver sinusoids. Science. 2006;313(5791):1287-90. Epub 20060803. doi: 10.1126/science.1129720. PubMed PMID: 16888102.
  5. Anwar MO, Islam MM, Thakur V, Kaur I, Mohmmed A. Defining ER-mitochondria contact dynamics in Plasmodium falciparum by targeting component of phospholipid synthesis pathway, phosphatidylserine synthase (PfPSS). Mitochondrion. 2022;65:124-38. Epub 20220524. doi: 10.1016/j.mito.2022.05.005. PubMed PMID: 35623558.

The role of LSHTM and NU in this collaborative project

This PhD project will be supervised by Dr. DKI at Nagasaki University (NU), a leader of a diverse research group investigating P. falciparum metabolism and drug development. The candidate will join a dynamic team with expertise in biochemistry and drug development, including high-throughput screening and the analysis of inhibitor-target interactions. Dr. Ken Daniel Inaoka will provide expert guidance in biochemical characterisation of PfCEPT and PfPSS, and if required, a high-throughput screening system will be developed to identify small molecules that inhibit their function.

Dr. Takaya Sakura, co-supervisor at NU, will contribute with expertise in molecular parasitology and omics-based techniques (genomics, transcriptomics, metabolomics). With extensive experience in using the CRISPR-Cas9 system and in the phenotypic characterisation of P. falciparum mutants, Takaya Sakura will support the functional investigation of PfCEPT and PfPSS in relation to parasite survival.

Dr. Julius Hafalla at LSHTM, will contribute with expertise in parasite biology and immunology-related studies. Julius Hafalla support work in the characterisation of mutant murine parasites, and the assessment of immune responses to these parasites. 

The candidate will also benefit from collaboration with Prof. Fuyuki Tokomasu at Gunma University, who specializes in advanced imaging systems and lipidomics. This collaboration will enrich the project by providing cutting-edge tools for the detailed study of lipid metabolism and cellular architecture of P. falciparum.

At NU, the doctoral student will have ample opportunities to develop essential molecular biology skills, including culturing P. falciparum, generating mutants, and performing various phenotypic assays. There will be flexibility for the student to integrate omics-based techniques, fostering the development of bioinformatics and statistical skills. 

Additionally, collaboration with the London School of Hygiene and Tropical Medicine (LSHTM) will allow the student to acquire core immunology skills, contributing to the investigation of PfCEPT and PfPSS in parasite immune evasion mechanisms.

Particular prior educational requirements for a student undertaking this project

The doctoral candidate should have completed an undergraduate and postgraduate degree related to infectious disease. Prior working experience in molecular biology and parasitology are recommended (but not mandatory). 

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

Molecular Biology – The doctoral student will have the opportunity to develop core molecular microbiology skills, learn how to grow Plasmodium falciparum, how to create mutants using Cas9 system, and how to perform several different phenotypic assays depending on the direction the doctoral student wishes to drive the project.

Bioinformatics – All aims can also utilise omics-based techniques (e.g., genomics, metabolomics, lipidomics) allowing the opportunity to run and develop bioinformatic pipelines and statistical analyses methods using R/Python(transcriptome) and LC-MS/MS(lipidomics).

As well as key scientific skills, development of core transferrable skills are core tenets of the NU and LSHTM doctoral programme. The doctoral student will join the Parasitology research group and be provided with world-class training that will lead to them becoming an independent scientist. They will have the option to attend MSc modules which may be relevant to their research project. In addition, the doctoral student will attend relevant courses/workshops to understand the value of knowledge transfer and the value of intellectual property (IP). Meetings and social events are organised at intervals throughout the year to encourage students to get to know each other and to develop a supportive environment.