Authors
S L Berry2; S Hart1; H Price2; 1 Institute for Science and Technology in Medicine, Keele University, UK; 2 School of Life Sciences, Keele University, UK Discussion
The neglected tropical disease leishmaniasis is caused by infection with the protozoan parasite Leishmania spp, with an estimated 1 million new cases per year. The Leishmania parasite cycles between a procyclic promastigote stage in the sandfly vector; a host-infective metacyclic promastigote stage which is transferred from vector to mammalian host during a bloodmeal; and an intracellular amastigote stage which resides inside host macrophages.
The BBSome is a protein complex which is associated with molecular trafficking to/from primary cilia and flagella in other eukaryotes. Previous work (Price et al 2013) showed that deletion of one of the subunits, BBS1, from L. major severely reduces parasite virulence in mice. We hypothesise that the Leishmania BBSome is involved in the transportation of macromolecules to the parasite cell surface. We are testing this hypothesis by creating transgenic parasite cell lines with disrupted BBSome function. We will analyse the effect these changes have on the distribution of macromolecules, including proteins, on the cell surface. This work will initially involve biotinylation and streptavidin pull down of cell surface proteins, which will be analysed by mass spectrometry for differences in protein expression. This will lead on to the analysis of global protein distribution within the cell using LOPIT (Localisation of Organelle Proteins by Isotope Tagging) – a mass spectrometry-based technique that allows the subcellular location of large numbers of different proteins to be mapped. 
