Project Summary.

The protozoan parasite Leishmania is the causative agent of leishmaniosis which affects about 12 million people worldwide, the majority being located in underdeveloped countries. The disease is considered to be a problem of world-wide health by the World Health Organization. Biochemical and molecular biology studies can help in the understanding of the biology of the parasite and thereby contribute to strategies which combat the illness.

Our approach is conceptually quite simple; since all the surface proteins in the infectious form of Leishmania must be secreted by the parasite, then by analysing only these proteins we can gain a list of potential targets for drugs which might act to inhibit parasite invasion. In practice the specific separation of only the secreted proteins is complicated by the fact that each Leishmania cell contains thousands of other types of proteins. However,  protein secretion in Leishmania occurs by a transport system that uses a series of intracelular vesicles, so by isolating these vesicles, the secreted proteins can in principle be isolated. This approach reduces the number of proteins that we will need to analyze, and so simplifies our search. The initial stage of the study consisted of cell fractionation of the promastigote forms of Leishmania major in order to enrich the samples with the vesicular sub-cellular fraction. 

Even using this stategy, dozens of vesicle proteins have to analyze , and the quickest way to do this is by using proteomic techniques, which combine 2D-electrophoresis with mass-spectrometry. After rupture of the vesicles, the soluble proteins derived from the vesicle contents were separated by 1D and 2D gels, and the protein spots were identified by mass spectrometry. ESI-MS/MS and MALDI-TOF/MS were used to identify the proteins by peptide mass fingerprinting. Using this technique for the analysis of 74 spots resulted in the identification of 28 proteins. Initial results showed that the cell coat proteins gp45 and gp63 could be identified in our vesicle extracts, demonstrating that this strategy is viable for the identification of secreted proteins.

The final objective is to identify, clone and express proteins of the vesicular fraction of the promastigote forms of L. major. After identification of proteins in the soluble extract of vesicles, we have selected three proteins as tagets for further studies in the biology of L. major. The selected proteins were: the nucleoside diphosphate kinase b (NDKb), calpain-like protease (CLP) and thermostable carboxypeptidase (TCP). The genomic DNA sequences encoding theses proteins have been amplified from total L. major genomic DNA by PCR, and cloned into the vectors pT7T3, pET28a. The pET28a constructs have been used for expression of the heterologous protein in E. coli BL21(DE3)pLysS.

The strategy applied in this study is based on the characteriztion of a subcellular proteome, and suggests that molecular biology and biochemistry can contribute to the understanding of the processes involved in the mechanism of host invasion by parasite and, thus contribute to the development of drugs or strategies to fight leishmaniasis.