Structure - Function Relationships in Protozoan Parasites Utilising High Resolution 3D Bioimaging

The family Trypanosomatidae contains a number of important human-infective protozoan parasites. These include Trypanosoma cruzi which causes Chagas Disease in Central and South America and there are reported to be ~18 million people infected in some of the poorest communities with no long term effective drug treatment, particularly for chronically infected patients. Trypanosoma brucei is a closely related parasite that causes African sleeping sickness and kills ~50,000 people annually in Sub-Saharan Africa. The cattle species of Trypanosoma brucei has a devastating economic impact, with large areas of countryside that cannot be used due to the prevalence of the Tsetse fly, which spreads the disease. Both parasites are on the World Health Organisation list of 17 neglected Tropical Diseases and more efforts are required to understand the biology of these parasites to help find effective treatments.

There are two aim objectives with 4 main research areas that will be developed within a context of modern molecular and cell biology of parasites and the use of novel three dimensional electron microscopy.These parasites have an indentation in the cell surface called the flagellar pocket, which is the major site of entry and exit of molecules, nutrients and entry of drugs and is a critical factor in the pathogenicity of these parasites. It has a complex three dimensional structure and we aim to use a range of three dimensional electron microscopy techniques to really understand how the flagellar pocket and associated structures divides and functions. These parasites have completed genome projects and a range of genetic tools to allow in-depth analysis of individual genes. The partners in both the UK and Brazil are experts in understanding the flagellar pocket structure of the two parasites. However, important differences are emerging through concerted efforts by both partners. These differences and similarities are important to understand, particularly as the flagellar pocket is a major site for entry of drugs and nutrients to these cells. Our objective is to run a fully collaborative project to understand these differences in further detail and the design of the visits between staff and students over the 3 years is aimed at maximising contact and discussion between the partners in the context of 4 experimental research areas. We will also organise a mini-symposium during year 2 to ensure that the outcomes of the experiments and conclusions can be disseminated to a wide academic audience, including those working with drug discovery, endocytosis (nutrient uptake in cells) and immunology.

In order to really understand how living cells divide and interact with their surroundings we need to understand a detailed view of their three dimensional spatial organisation. This has been difficult to achieve using existing electron microscopes due to their inherent limitations. Recently, there have been significant developments in three dimensional electron microscopy to image the detailed structure of cells and tissues of organisms in an automated manner and the partners on this proposal are leading centres of excellence for these 3D microscopy techniques. The number of trained scientists is low both nationally and internationally and during the 3 year grant our aim is to train as many staff and students as possible as we believe these techniques will revolutionise general cell biology in the next 10 years.

There are 4 main research areas covered by this partnership. The partners will collaborate to produce an in-depth analysis of the flagellar pocket of T. cruzi and T. brucei. We think this is important as it is becoming clear from work that has been carried out by the UK and Brazilian partners that there are important differences, such as the presence of the contractile vacuole in T. cruzi, but also to share our knowledge on potential similarities such as the discovery of microtubule connections with the kinetoplast and this will be important to understand as the kinetoplast has been a focus for potential drug therapy.

We will use a range of 3D microscopy techniques that are available at both partners including cellular electron tomography to produce a high resolution map of flagellar pocket structure and biogenesis. Serial block face scanning electron microscopy (SBF-SEM) and FIB-SEM will also be used to produce whole individual cell reconstructions, which is a very recent application development by the UK-PI, so that we can map division into the context of the whole cell (SBF-SEM) and obtain larger areas of the flagellar pocket at higher resolution (FIB-SEM). The UK-PI discovered 3 sets of microtubule connections to the kinetoplast and this will be investigated in both parasites to analyse if this is a common mechanism for the maintenance of the kinetoplast in these parasites. The UK partners will also develop new molecular tools for use in T. cruzi that has already been developed in T. brucei for high through-put endogenous tagging of cells and a novel genetic tag (APEX) for use in both parasites. In T. cruzi the contractile vacuole is associated with the flagellar pocket and undergoes dynamic changees during osmotic stress and is assoociated to acidocalcisomes. These 3D microscopy techniques also allow a dissection of the spatial organisation and quantification of acidocalcisomes following osmotic stress.

Partners on this proposal have identified a number of ways in which this work will have impact. The first is in the potential for improving health and well-being as these protozoan parasites are highly complex and safe treatments are severely lacking. This proposal is aimed at gaining a better understanding of a key organelle of these parasites, which are involved in interacting and obtaining nutrients from the environment. This study is important and it allows researchers to gain a better understanding of how this organelle functions and can inform those working to discover drug to combat these diseases. This proposal also has the potential for general economic impact in the partner countries in terms of developing applications for three dimensional microscopy and ensuring that both economies remain at the cutting edge of these techniques, which have the potential for wealth creation.