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Characterisation of the Plasmodium PHIST protein family

Periodic Reporting for period 1 - PfPHIST (Characterisation of the Plasmodium PHIST protein family)

Reporting period: 2018-05-01 to 2020-04-30

Malaria is one of the most devastating human diseases, with almost a third of the world’s population at risk of infection. The most severe form of the disease is caused by Plasmodium falciparum and while there have been considerable efforts to control this parasite, there is still relatively little known about exactly how the parasite causes disease. What is known is that parasite is the ultimate home renovator, and once it has infected human red blood cells it can drastically alter their structure and function, making them rigid and sticky, and in turn cause the more severe malaria symptoms. While the association between these modifications and disease is well described, it has only recently come to light that the parasites capacity to renovate its home may also be important for allowing the parasite to transmit to mosquitoes. The possibility that this ability is vital for both the severity and spread of the disease is incredibly exciting and this is what this project set out to uncover. To begin this task the focus was on one unique family of parasite proteins that we believe could be the Rosetta Stone that finally decodes the mechanisms underlying the parasites ability to renovate the human red blood cells that leads to both severe disease and transmission, with the hope that this information could be used in future vaccine or drug development. The overall conclusion from this project was that the results point to the fact that parasites ability to renovate its home is not only important for disease but also for it spread in the mosquito vector, and as such represent good targets for malaria intervention strategies.
The research objective of this project was to characterise proteins important to the malaria parasite to renovate its home. This was done by first developing new tools and techniques to obtain and analyse mutant parasites that help to answer the question of how the parasite is able to alter the host cell. The work performed with this objective in mind and the results that were obtain are outlined below.

Genetic manipulation of the selected malaria genes.
We set out to first develop new tools to better and more quickly obtain critical mutant parasites that can be used to help analyse the function of unique and unknown parasite proteins. In order to do this while not altering the drug sensitivity of the parasite, we selected fluorescent proteins as a marker of new parasite mutants. This gave us the ability to not only visually confirm the presence of the new mutant parasite lines but also to speed up the selection process. The overall result of this new method was allowing us to obtain critical mutant parasite lines in 3 weeks as opposed to the normal 3 months. This in turn allowed us to delete, mutate, and tag several of our selected parasite proteins that then underwent further functional analysis.

Develop new rheological analyses based on microfluidic devices.
We also set out to develop an entirely new way to analyse the properties of the human red blood cell with and without a parasite infection. Normal human red blood cells are extremely deformable, something that is required to get through the very small capillaries in the human body. This is significantly altered by the parasite and being able to measure these changes in minute detail is critical to understand how the parasite is able to achieve this change. Throughout the course of the project we were able to design useful devices and begin to record normal and malaria infected red blood cells travelling through microscopic channels. Due to the high level of data this high throughput new method generated we also began to develop new software that was able to identify red blood cells in all frames of the recorded videos. The overall result of this was the first ever high speed video of a malaria parasite infected red blood cell travelling through microchannels which gives a high level of data to help analyse the parasite ability to alter the red blood cell.

Functional analysis of mutant parasite lines.
We analysed a number of mutant parasites with one in particular providing the most interesting outcome. While it had originally been thought to be important for altering red blood cells we were able to see the protein in the parasite in an infected mosquito and also in the liver stage of infection. The overall result from this alone revealed that the proteins that are important for the parasite to modify the human red blood cell could also be important for the parasite to modify liver cells or even mosquito cells and in this particular case it is more transmission than disease progression that appears to be the key. It is projects like this that underscore the importance of looking at all parasite stages and is one of just a handful to link the parasites ability to modify human red blood cells to the spread of disease and to suggest that the parasites ability to renovate its home maybe more diverse than previously thought.

The overall outcome from the results obtained in this two year project showed that this family of parasite proteins not only helps to uncover critical aspects of the parasites renovation capacity but also that this is important for the spread of malaria. This information will has been disseminated at a malaria scientific conference and will be further compiled into a scientific publication. Furthermore these data will be exploited through applying for larger project grants and the further attempts at developing a service platform for scientific research using our new techniques.
The finding of this project will have relevance to the wider malaria community as the vast amount of research surround the parasites ability to alter its host cell is conducted solely on the blood stage of the parasite and it is becoming increasingly clear that the other stages play just as an important role and could also be targeted as a control measure. On a more technical side we were able to develop a new system to create parasite mutant lines in a relatively short timeframe which will have significant impact on projects aimed at understanding this parasite. These results were presented with some interest at the international Molecular Approaches to Malaria conference in Lorne, Australia in 2020. Furthermore our other technique developments has encouraged us to look further into modeling disease with the future aim of providing a platform that would be available to the wider research field and has been the subject of an Radboudumc internal grant application. Ultimately this project helped to highlight the role of parasite ‘renovator’ proteins and their importance not just in the human red blood cell but to all life-cycle stages on the malaria parasite. As this was fundamental research there is limited capacity for wider socio-economic impact, and while there is not yet a direct societal impact of this project there is a scientific impact that helps to inform on the future development of anti-malaria strategies which in turn has a significant impact on the wider society through the control of a deadly pathogen.
Fluorescent image of a malaria parasite infecting a human liver cell