BBSome trafficking: investigating a novel pathway associated with virulence in Leishmania

The microscopic parasite Leishmania causes a tropical infectious disease called leishmaniasis, which is particularly found in regions of extreme poverty, war and population displacement. The disease affects millions of people worldwide and causes an estimated 50,000 deaths per year. The most severe form of leishmaniasis is known as kala-azar or black fever which develops when parasites invade internal organs including the liver and spleen, causing them to swell. Kala-azar is fatal unless treated but the handful of available drugs to treat this condition are highly toxic, extremely expensive or difficult to administer, and therefore inaccessible to many infected people. The disease is spread by the bite of a sand fly, which injects thousands of tiny parasites into the skin of a human or animal host. The parasites survive inside the hostile environment of the host by rapidly invading and hiding inside cells of the immune system called macrophages. These immune cells normally engulf and kill harmful microbes but Leishmania are able to block normal responses by the macrophages and instead are able to grow and multiply inside these cells. Understanding the survival strategies of the parasite would enable us to design ways of interfering with this process and may underpin new research into development of new drugs to treat the disease. My previous research has identified a group of proteins called the BBSome which are needed for the Leishmania parasite to survive inside the host. The same set of proteins are found in humans and are known to be important in structures called primary cilia. Cilia are long slender structures that protrude from some cells in the human body which act as 'antennae' for sensing the environment and communicating with other cells. Small changes in the BBSome proteins due to genetic mutations can have a big impact, preventing the primary cilia from transmitting signals. People with these mutations have a condition called Bardet-Biedl syndrome and develop blindness, kidney problems and obesity from an early age. My experimental results suggest that the parasite BBSome may be important in communicating important signals from the parasite in a similar way to the equivalent proteins in humans. Studying how these proteins work will give us specific insights into the biology of the parasite and may also provide valuable information that is generally applicable to primary cilia in mammals. I propose to study this group of proteins in detail using a range of cutting-edge technologies to find out which molecules they interact with in the cell. Genetic engineering will be used to make specific changes to, or completely remove, selected BBSome proteins in Leishmania. The resulting microbes will be studied to find out whether they have any obvious defects in cell shape and their ability to grow. Biochemical analysis will be done to give a detailed picture of where hundreds of different proteins, carbohydrates and lipids are found within these parasites. This information will help construct a model of how the BBSome proteins help to transport molecules (including those involved in communication) from where they are made to where they are needed. I will also develop new methods for studying these microbes in the laboratory, including a new way of testing if two proteins in the cell bind to each other. Findings from the proposed research will enable us to understand more about the mechanisms the Leishmania parasite uses in order to survive and may in the long term contribute to the development of new treatments for this deadly disease.

My previous research showed that Leishmania parasites lacking the orthologue of the human BBSome subunit BBS1 are unable to survive in a murine host. The proposed study is based on the hypothesis that the BBSome is involved in trafficking virulence factors in these parasites. The first objective is to identify changes in the surface molecules of amastigotes in which BBSome function has been disrupted by deletion of one subunit of the complex. Surface proteins will be isolated by biotinylation and streptavidin pulldown, followed by isobaric tag quantification. Surface carbohydrates and plasma membrane lipids will be identified by mass spectrometry-based techniques. The second objective will take a global approach to find differences in protein localization patterns in amastigotes of BBSome mutant lines. Localization of Organelle Proteins by Isotope Tagging (LOPIT) combines biochemical fractionation and shotgun proteomics with isobaric tag quantification and enables the localization of large numbers of proteins to be studied simultaneously. As this technique has not been used previously on Leishmania, initial studies will be performed to generate a LOPIT profile for wild-type L. mexicana amastigotes. Following extensive cell fractionation, Western blotting will be used to find fractions that represent peak organelle densities. Fractionations will be repeated for BBSome mutant lines and the composition of equivalent fractions from different lines will be compared using isobaric tag quantification. Identified differences will be validated by endogenous epitope tagging and immunofluorescence. The third objective is to identify interacting partners of the L. mexicana BBSome using proximity-dependent biotin identification (BIOID) and NanoLuc Binary Technology (NanoBiT) complementation assays. Findings from the proposed study will provide a clearer picture of how the Leishmania BBSome functions at the molecular level and the specific effects it exerts on the cell.

The proposed project has broad potential impact for biomedical research and in the long term may contribute to the development of new treatments for a neglected tropical disease. Beneficiaries are as follows:

1. Academic Community
The proposed project will generate new knowledge on a key protein complex linked to virulence in Leishmania. In addition, a considerable body of data will be produced on surface molecules and global protein distribution patterns in the infective amastigote stage of the parasite. Findings will have direct relevance to kinetoplastids and other ciliated eukaryotes. This work therefore has the potential for significant academic impact in multiple research areas related to infectious diseases, cell biology, primary cilia and genetic disorders. Novel methodologies will be developed to improve resources for comparative proteomics and protein-protein interaction studies in Leishmania. Rapid dissemination of datasets, protocols and reagents (e.g. plasmid vectors and cell lines) will ensure delivery of a positive impact across the kinetoplastid research community.

2. Staff and Students
As the lead applicant, I would greatly benefit from a New Investigator award as this would enable me to channel more time and resources into building my research team at a critical point in my career. The study will provide a training environment for the named PDRA and myself to develop a specialised combination of expertise in kinetoplastid biology, cell biology and proteomics. The co-investigator will also directly benefit through co-authorship on planned publications. The Price and Hart laboratories also provide training opportunities for undergraduate and postgraduate students and visiting researchers (e.g. Newton Fund travelling fellowship scheme, Summer studentships). The PDRA will be encouraged to undertake additional internal and externally provided training courses where appropriate, including the analysis of proteomic datasets, scientific writing and oral presentation skills.

3. Public Engagement
Research outcomes will be communicated to visiting schools and to the general public at events including the Keele Community Day and the Big Biology day at Staffordshire University. Events such as these enable the general public to improve their understanding of scientific issues and may improve widening participation and engagement with higher education.

4. Drug Discovery
The project aims to identify new molecular pathways which can be exploited for drug discovery. Translational exploitation of findings will require further studies, including validation of new targets, compound screening and testing in disease models. Collaborations will be sought during the course of the project in order to accelerate progress towards translational benefits, with applications for additional funding being submitted as appropriate.. The project outcomes could ultimately lead to new therapies for leishmaniasis. A new effective treatment for leishmaniasis would help to fulfill part of WHO's Sustainable Development Goal 3: to prevent epidemics of neglected tropical diseases. Long-term beneficiaries of an effective new treatment for leishmaniasis would therefore include patients, WHO and governmental health agencies in endemic areas, and industrial collaborators within the pharmaceutical industry. A significant long-term impact would be improved healthcare and better worldwide life quality, providing far-reaching benefits to society in endemic regions. Further, economic and societal impact will be generated in these geographical regions by enhancing quality of life and health, and by enhancing the effectiveness of public services (e.g. through the availability of new clinical treatments). New molecular information regarding the BBSome and its targets could also lead in the long term to novel treatments for diseases characterised by primary cilium dysfunction in humans.