Proteomic analysis of Leishmania mexicana by permeabilization of different cell membranes with digitonin

Abstract

The leishmaniasis are a group of diseases caused by the genus Leishmania. These affect 350 million people in the world in its two main forms, the cutaneous leishmaniasis and visceral leishmaniasis. After the complete sequencing of the genome of Leishmania major and the breakthrough in sequencing other species (Leishmania braziliensis, L. mexicana and L. infantum), much has been advanced regarding the study of proteomic platform companies of its parasites forms (amastigote and promastigotas) in order to know better interaction with host, bringing important information on the basic biology of these organisms, with potential application in the improvement or development of diagnostic and therapeutic techniques, avoiding thus cross reactions or treatment failures due to intricacies resistance mechanisms, respectively. In this way, the aim of this project is the training of subcellular fractionation technique for cell permeabilization by digitonin, in which it is possible, by using immunoblotting techniques, characterize proteins under study as to their locations in different subcellular compartments, constituting important information about their function in the cell. Such methodological approach will be applied in undergraduate research project (process FAPESP no 2011/06995-9) or in a future Master Degree project, for functional characterization of genes under study, as well as direct application of the same in different laboratory research projects related to the understanding of the mechanism of action of new compounds with leishmanicidal activity. The overall objective is to introduce the biochemical and cell biological approaches of (i) cell fractionation by digitonin extraction and (ii) fluorescence microscopy of epitope-tagged proteins to study protein localisation (and thus function) in trypanosomatid parasites, studying adenylate kinase function, as well as cytoskeletal dynamics in Leishmania. The adenylate kinases provide an unusually large gene family in trypanosomatid parasites with gene products localised to a variety of sub-cellular compartments, mitochondrion, cytosol, glycosomes, nucleus, and flagellum (where different isoforms are found selectively in either the axoneme or paraflagellar rod). Within these compartments the nucleotide homeostasis function provided by adenylate kinases serves a number of roles in energy management and cell motility (J. Biol. Chem 280: 11781-9; work in prep). The objective is to use the digitonin fractionation to determine the sub-organellar localization of an essential mitochondrial adenylate kinase and use fluorescence microscopy to study life-cycle expression in Leishmania of an adenylate. This isoform is selectively up-regulated in specific life-cycle stages and the single cell analysis of parasites expressing green fluorescent protein (GFP)-tagged adenylate kinase from an endogenous locus provides opportunity to not only test this hypothesis but to also teach the application of live cell imaging, through the use of fluorescence recovery after photobleaching (FRAP) to study protein turnover and the mobility of protein complexes within trypanosomatid cells. (AU)