Combining structural biology and genetics to understand the function of a multi-gene family expanded in neglected human malaria parasites
Lead Research Organisation:
University of Cambridge
Department Name: Cambridge Institute for Medical Research
Abstract
More than a third of the world's population is at risk of contracting malaria and there are more than 200 million cases each year, leading to nearly half a million deaths. Malaria is caused by multiple species of Plasmodium parasite, which are spread from person to person by Anopheles mosquitoes. Most malaria research has focussed on Plasmodium falciparum, the dominant species in Africa and the one that causes the majority of malaria deaths. However, P. falciparum is only relatively distantly related to the species that cause most malaria cases outside Africa. These fall into a different evolutionary group which includes both Plasmodium vivax, the second most significant cause of malaria globally, and Plasmodium knowlesi, a parasite that predominantly infects monkeys in Southeast Asia but can also be transmitted to humans where it can cause severe disease and death. The biology of this group of parasites, collectively referred to as the Plasmodium subgenus, is significantly underexplored, in part because only P. knowlesi can be routinely grown and experimentally manipulated in a lab setting.
In this project we will focus on a group of proteins, Tryptophan-rich antigens (TRAgs) that are found in all Plasmodium parasites but are present in significantly higher numbers in species within the Plasmodium subgenus. The P. vivax genome contains 38 genes that encode TRAgs, while P. knowlesi contains 29 TRAgs - by contrast the major African malaria parasite P. falciparum contains only 3. The function of this protein family is unknown, but the fact that they are expanded in the Plasmodium subgenus suggests that they are particularly important in these neglected human malaria parasite species.
We have recently solved the three-dimensional protein structure of one Plasmodium vivax TRAG, which revealed similarities between it and proteins in other organisms that bind to the lipids that make up the membranes of all cells, including the red blood cells that Plasmodium parasites grow inside. We subsequently confirmed that both P. vivax and P. knowlesi TRAgs can bind directly to lipids, and in the case of one P. knowlesi TRAg, that this interaction is involved in the process by which these parasites recognise and invade human red blood cells. This for the first time raises a clear hypothesis for TRAg function - that they bind to lipids and are involved in manipulating human red blood cell membranes, either during or after the process of invasion.
We will explore this hypothesis by carrying out the first systematic study of a large number of TRAgs in a Plasmodium subgenus parasite species. We will focus on P. knowlesi, which can be grown in the lab and genetically manipulated, which makes it possible to probe the location and function of individual TRAgs. We will extend our structural studies, exploring whether different TRAgs have specificity for different lipids, and solving the structure of TRAg proteins in complex with lipids found in the membrane of human red blood cells. We will identify which TRAg genes are expressed in the lab strain of P. knowlesi and establish the specific localisation of the most abundant TRAg proteins using advanced microscopy. We will also leverage recent technical advances to create P. knowlesi parasite lines in which these highly expressed TRAg genes have been deleted using CRISPR-Cas9 genome editing and monitor the effect on parasite growth and invasion.
Overall, this research will provide critical information about the function of an understudied protein family in a neglected malaria parasite species.
In this project we will focus on a group of proteins, Tryptophan-rich antigens (TRAgs) that are found in all Plasmodium parasites but are present in significantly higher numbers in species within the Plasmodium subgenus. The P. vivax genome contains 38 genes that encode TRAgs, while P. knowlesi contains 29 TRAgs - by contrast the major African malaria parasite P. falciparum contains only 3. The function of this protein family is unknown, but the fact that they are expanded in the Plasmodium subgenus suggests that they are particularly important in these neglected human malaria parasite species.
We have recently solved the three-dimensional protein structure of one Plasmodium vivax TRAG, which revealed similarities between it and proteins in other organisms that bind to the lipids that make up the membranes of all cells, including the red blood cells that Plasmodium parasites grow inside. We subsequently confirmed that both P. vivax and P. knowlesi TRAgs can bind directly to lipids, and in the case of one P. knowlesi TRAg, that this interaction is involved in the process by which these parasites recognise and invade human red blood cells. This for the first time raises a clear hypothesis for TRAg function - that they bind to lipids and are involved in manipulating human red blood cell membranes, either during or after the process of invasion.
We will explore this hypothesis by carrying out the first systematic study of a large number of TRAgs in a Plasmodium subgenus parasite species. We will focus on P. knowlesi, which can be grown in the lab and genetically manipulated, which makes it possible to probe the location and function of individual TRAgs. We will extend our structural studies, exploring whether different TRAgs have specificity for different lipids, and solving the structure of TRAg proteins in complex with lipids found in the membrane of human red blood cells. We will identify which TRAg genes are expressed in the lab strain of P. knowlesi and establish the specific localisation of the most abundant TRAg proteins using advanced microscopy. We will also leverage recent technical advances to create P. knowlesi parasite lines in which these highly expressed TRAg genes have been deleted using CRISPR-Cas9 genome editing and monitor the effect on parasite growth and invasion.
Overall, this research will provide critical information about the function of an understudied protein family in a neglected malaria parasite species.
Technical Summary
The Tryptophan Rich Antigens (TRAgs) are found in all Plasmodium species, but are significantly expanded in the Plasmodium subgenus, implying that they have a critical function in these parasite species, including both P. vivax, the cause of most malaria in Latin America, and P. knowlesi, a zoonotic parasite that causes thousands of cases of malaria each year in Southeast Asia. The function of TRAgs has never been explored in detail. We have recently experimentally solved the structure of the conserved trypotphan/threonine rich C-terminal domain which defines the TRAg family. This revealed homology to the lipid-binding BAR domain, and we have confirmed that multiple P. vivax and P. knowlesi TRAg domains are able to directly interact with lipids. This presents for the first time a unifying hypothesis for TRAg domain function, that they interact with lipids to modulate cell membranes, and may play roles in host-parasite interactions both during and after the invasion of human erythrocytes.
In this interdisciplinary project we will combine genetic, biochemical and structural approaches to systematically explore the function of TRAgs in P. knowlesi, the only member of the Plasmodium subgenus that can be grown in vitro and experimentally manipulated. We will use biochemical and structural biology tools to explore the extent of and structural basis for TRAg-lipid specificity. We will combine antibody generation and epitope tagging to establish the location of multiple TRAgs using advanced microscopy. Finally, we will use CRISPR/Cas9 editing to carry out standard, conditional and combinatorial knockout approaches to delete individual/combinations of TRAgs and explore the impact on growth and invasion. This will be the first systematic study of TRAgs in the Plasmodium subgenus and will provide critical information about the blood-stage biology of these neglected human malaria parasites.
In this interdisciplinary project we will combine genetic, biochemical and structural approaches to systematically explore the function of TRAgs in P. knowlesi, the only member of the Plasmodium subgenus that can be grown in vitro and experimentally manipulated. We will use biochemical and structural biology tools to explore the extent of and structural basis for TRAg-lipid specificity. We will combine antibody generation and epitope tagging to establish the location of multiple TRAgs using advanced microscopy. Finally, we will use CRISPR/Cas9 editing to carry out standard, conditional and combinatorial knockout approaches to delete individual/combinations of TRAgs and explore the impact on growth and invasion. This will be the first systematic study of TRAgs in the Plasmodium subgenus and will provide critical information about the blood-stage biology of these neglected human malaria parasites.
