Analysing the roles of peptidases in Leishmania infectivity and pathogenicity

Leishmaniasis is a severe disease of humans and animals in the tropics and sub-tropics (including Southern Europe) and 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, on the changes in cell shape mediated by 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 now aim to elucidate the key roles and molecular mechanism of autophagy in Leishmania under different stimuli, such as starvation and stress. We will determine the importance of autophagy for the multiplication of amastigotes of Leishmania (the parasite form that lives in humans) and so whether it can be effectively targeted by drug intervention. We will also investigate whether Leishmania modulates autophagy in its host cell as a survival strategy. Secondly, on the crucial role of the parasite?s internal membranous structures for interactions with the mammalian host, and how factors that make the parasite virulent are released from the cell. Thirdly, we will 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. One outcome from this study will be 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 to discover inhibitors of peptidases, which should be leads in the development of new drugs.

We have shown that peptidases play central roles in a variety of biological processes of the parasitic protozoon Leishmania, are necessary for them, and that they thus 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, on the cellular remodelling processes, such as autophagy, which lead to the development of infectious parasites and transformation within the mammalian host. We now aim to elucidate the key roles and molecular mechanism of autophagy in L. major, in particular the role of multiple ATG4 and ATG8 proteins that are activated under different stimuli, such as starvation and stress, and the involvement of other peptidases in the formation and maturation of autophagosomes. We will determine the importance of autophagy and lysosomal components for the multiplication of amastigotes of Leishmania and so whether they are targets for drug intervention. We will investigate whether Leishmania modulates autophagy in its host cell as a survival strategy. Secondly, on the crucial role of the parasite?s endosomal and lysosomal pathways in interactions with the mammalian host, and the route of intracellular trafficking of key virulence factors such as cysteine peptidases. A key approach will be to investigate the outcome of modulating lysosomal function through genetic manipulation of key genes, such as LAMP and AP-3, in L. major. Thirdly, on the function of the metacaspase cysteine peptidase ? its potential role in cell cycle-dependent organelle duplication and possible involvement in some form of programmed cell death. We will investigate its function by expression of recombinant enzyme and analysis of its activity and specificity, determination of its localisation during the cell cycle, identification of its in vivo substrates and interacting partners. One outcome from this study will be 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 outcome of this study will be knowledge on which peptidases are valid drug targets and this information will be exploited by collaborating to discover potent inhibitors of peptidases, which should be leads in the development of novel antileishmanial drugs. As a third outcome of these studies we shall develop, and make available for the Leishmania research community, new tools for studying Leishmania, such as methods for genetic manipulation and in vivo imaging of amastigotes in macrophages.