Formation and function of the parasitophorous vacuole membrane in Plasmodium falciparum-infected erythrocytes

Malaria parasites cause disease by infecting red blood cells. During the process of invading these cells, the parasites become surrounded by a membrane, referred to as the parasitophorous vacuole membrane (PVM). Throughout the entire replication cycle inside the red blood cell, this membrane separates the parasite from the cytosol of the red blood cell, which consists primarily of haemoglobin. However, the membrane also makes the uptake of nutrients and the transport of proteins from the parasite to the red blood cell much more difficult. It is not understood why the parasite would maintain such a barrier, whereas related organisms that can also replicate in red blood cells can remove it and live free in the cytosol of the red blood cell. It is likely that the PVM performs an essential function, or multiple functions, for the parasites, but that function is entirely unclear. Also, it is not known how the PVM is made, maintained and expanded as the parasite grows inside the red blood cell. The aim of this research programme is to determine how the PVM is made and determine the role of the proteins that normally reside in the PVM in its development and maintenance, and ultimately answer the question how this membrane helps the parasite survive inside the red blood cell.

Previous research has identified a protein, named PFA0210c, which may be involved in the formation of the PVM soon after the parasite has entered the red blood cell and again at later stages of the growth inside red blood cell. PFA0210c is a phospholipid transfer protein that can transfer phospholipids, the building blocks of cellular membranes, from one membrane to another. The PVM of parasites that do not produce this protein are much smaller than normal, indicating that potentially the role of PFA0210c is to transport phospholipids from the parasite to the PVM. Part of this research is aimed at discovering exactly how this protein functions and what its role in the formation of the PVM is.

Another aspect of this study is the investigation of proteins that are known to reside in the PVM. Only about fifteen of such proteins are known, and twelve of them will be investigated: eleven members of a family of proteins called ETRAMPS and a protein called EXP1. By investigating these proteins, initially through the creation of parasites in which the genes that encode these proteins can be rapidly deleted through the addition of a small molecule, it may be possible to determine the function of these proteins. This will provide insight into the function of the PVM and hence the ability of the parasite to survive inside the red blood cell.

Lastly, part of the research is aimed at the PVM itself. Parasites will be produced in which the synthesis of proteins that can break down phospholipids or produce large holes in membranes can be turned on. If this results in the loss of the PVM, then the role of the PVM can be observed directly by determining what happens to the parasite when it no longer has the protective coat that the PVM offers.

Ultimately the study of these proteins and the PVM could be used to search for more anti-malarial compounds. PFA0210c has been shown to be essential for the survival of the parasite and several PVM proteins, including EXP1, could be essential proteins and do not resemble proteins in the human host - important characteristics for proteins that are the targets of anti-malaria drugs. Hence, not only could this research lead to much deeper insight into the interaction of the parasite with its host, it could stimulate the search for anti-malaria drugs that target these proteins.

The proposed research aims to understand the formation of of the membrane of the parasitophorous vacuole (PVM) and the essential functions of the proteins present in this membrane during the growth of the malaria parasite Plasmodium falciparum in erythrocytes. During entry into the erythrocyte, the parasite becomes surrounded by a membrane that forms the PV, the PV membrane (PVM), that envelops the parasite until the very last stages of the erythrocytic cycle. As the parasite grows, the PVM expands to accommodate the growing parasite. The PVM separates the parasite from the cytosol of the erythrocyte, but also forms a significant barrier to the export of parasite proteins to the host cell that are required for the survival of the parasite and the uptake of haemoglobin and nutrients from the erythrocyte. The proposed research addresses the following questions: what is the origin of the phospholipids that make up the PVM when it is made and when it expands and how does the parasite phospholipid transfer protein PFA0210c affect this process; what are the functions of the proteins in the PVM; and how does the PVM allow survival of the parasite inside the erythrocyte.
These questions will be addressed using a combination of genetic techniques newly adapted for use in Plasmodium parasites (Cas9-mediated gene modification and inducible gene removal using the rapamycin-inducible diCre system), biochemical approaches, and automated EM microscopy that allows 3D models of parasites to be produced.
Together, the proposed experiments will provide insight into the function of the PVM and will reveal the essential functions of PVM proteins. Furthermore, this research could reveal entirely new targets for use in anti-malarial drug development.

This research in this project focuses on the interaction of the malaria parasite Plasmodium falciparum with erythrocytes. It is this stage of the Plasmodium lifecycle that causes the symptoms of malaria. In particular, the aims of the proposed research are to investigate the formation and expansion of the parasitophorous membrane, the membrane that separates the parasite from the cytosol of the erythrocyte, the role of the parasite protein PFA0210c in PVM formation, the function of PVM proteins and the effect of artificially removing the PVM. This research will provide insight into the interaction of the pathogen with the host cell. As PFA0210c has already been shown to be essential and other protein that will be investigated are also very likely to be essential, this will identify new drug targets.
These results will directly benefit other members of the Plasmodium research community. The P. falciparum strains, plasmids and genetic strategies that are developed as a result of the work in this proposal are likely to be beneficial for other researchers. Furthermore, this work could lead to the discovery of novel anti-malarials. This would be done in collaboration with Pharma or a biotech company. Development of new anti-malarials would have obvious benefits, as the malaria still claims over 400,000 lives annually and resistance to the front-line anti-malarial is spreading.