Proteolysis and life cycle progression in Leishmania

Leishmaniasis is a severe disease of humans and one of the world's most neglected diseases, primarily affecting the poor in developing countries. 350 million people are at risk of contracting the disease and it has severe costs in both health and economic terms and drains resources that could be used to promote growth of developing nations. There is no effective vaccine against the disease and chemotherapy is the prime means for reducing the leishmaniasis burden. Unfortunately the drugs available have many limitations and new drugs are desperately needed. Our aim in this programme of work is to characterise key biological processes of Leishmania, the parasite that causes leishmaniasis, and so identify and validate potential drug targets. We have shown that peptidases, enzymes that digest proteins, play central roles in the development of Leishmania and are important in the infectivity and pathogenicity of the parasite. Our work will concentrate on 3 key areas of the biology of the parasite. Firstly, we will identify peptidases that are important for the pathogenesis of the disease and investigate key pathways involved in peptidase function. In particular we shall investigate a process called autophagy (self-cannibalism), which leads to the development of infectious parasites and allows the parasite to live within a mammalian host. We will also investigate the function of a parasite-specific peptidase, metacaspase, which is essential for growth of the parasite but the function of which is unclear. A key approach will be to genetically manipulate important Leishmania genes to find out what role the encoded proteins play in the parasite's infectivity and virulence and to determine whether they might be exploited as drug targets. To achieve this we will develop new methods and research tools for the genetic manipulation of Leishmania. Secondly, we shall investigate the genome variation that occurs in different strains and species of Leishmania. We will sequence the genomes of Leishmania parasites isolated from patients in the Sudan and identify genes that influence the outcome of disease. Finally, we shall use a closely related parasite, the African trypanosome, as a model for investigating the regulation of peptidase function. The African trypanosome is suitable for large scale studies, so we can utilise information obtained in trypanosomes to study Leishmania. Overall, we expect one outcome from this study to be a greatly improved understanding on the roles of these biological processes in Leishmania, and the molecular mechanisms of the processes themselves - which will be relevant to many areas of biology. Another expected outcome will be knowledge on which peptidases are valid drug targets and this information will be exploited by collaborating with chemists to make inhibitors of peptidases, which might be used in the development of new drugs.

This multidisciplinary research programme will exploit state-of-the-art techniques in parasite genetic manipulation, genomics, quantitative proteomics and molecular parasitology and will lead to the elucidation of key mechanisms that enable Leishmania to undergo the cellular remodelling required for successful life cycle transition.
We will
1. use standard molecular genetics and biochemistry approaches to the study of a few selected Leishmania peptidases.

2. develop a new approach for genetic manipulation of Leishmania using a modified cre-lox system - this will allow the investigation of essential peptidases in both promastigotes and amastigotes.

3. apply quantitative proteomics (such as SILAC and TAILS) to identify secreted peptidases (potential virulence factors) and peptidase substrates in Leishmania.

4. Use high content cell imaging to carry out phenotype-directed, peptidase-wide or kinome-wide RNAi screens using specific reporter lines. This will identify protein kinases involved in regulating key cellular processes such as autophagy, endocytosis and differentiation.

5. Use high content cell imaging to carry out novel, target-based and phenotype-directed screens with kinase focussed or peptidase-focussed chemical libraries. This will identify hits suitable for further drug discovery efforts, as well as chemical tools for investigating protein kinase or peptidase function.

6. Sequence 150-250 clinical isolates of Leishmania from the Sudan and then use an in house bioinformatics pipeline to identify structural variation that might contribute to disease tropism.

The work we propose to undertake will contribute to delivery of Aim 3 of the MRC Strategic Plan 2009-14 by accelerating progress in international health research. The research falls within the remit of the MRC's recent Neglected Tropical Diseases highlight.

Academic impact
1. The attainment of new knowledge and scientific advancement on the molecular genetics and biochemistry of parasitic protozoa that cause neglected infectious disease.
2. A deeper understanding of the interaction of trypanosomatids parasites and their hosts, which will help to provide solutions to the control of neglected diseases endemic in developing countries.
3. The provision of new tools, innovative methods for genetic manipulation of Leishmania and reagents to academic partners for the study of the molecular genetics, biochemistry and cell biology of trypanosomes and Leishmania.
4. Delivery and training of highly skilled researchers in parasitology, molecular genetics, genomics, proteomics and cell biology.

Economic and societal impact
1. Enhancing the quality of life in countries endemic for Human African Trypanosomiasis and leishmaniasis
2. Potential to change the effectiveness of clinical practice in endemic areas (through the development of predictive diagnostics for PKDL)
3. To enhance the capacity of the Institute of Endemic Diseases in Khartoum, Sudan to carry out basic and applied research into leishmaniasis.