Illuminating the life cycles of parasites
The new research, published in the September edition of the journal Molecular & Cellular Proteomics, describes how a team of researchers UK, managed to purify and analyse the parasite responsible for causing leishmaniasis, a disease that causes skin sores, fever, damage to the spleen and liver, as well as anaemia.
Many infectious pathogens, like those that cause malaria, toxoplasmosis or leishmaniasishave a complex life cycle that leads them to alternate between free-living promastigote creatures and cell-enclosed amastigote parasites.
While it is relatively simple to isolate and analyse the extracellular promastigotes it is notoriously difficult to separate the amastigote parasites from the host cells, making detailed proteomic analysis of the intracellular form of the parasites all but impossible.
However, using a combination of isopycnic density (equilibrium) centrifugation and florescent particle sorting, the researchers, led by Dr Toni Aebischer of the Institute of Immunology and Infection Research at the University of Edinburgh, managed to purify the amastigote form of Leishmania mexicana, one of the many leishmaniasis parasites.
The research team engineered transgenic parasites that express a fluorescent protein so they can be easily differentiated from host cell material.
After the transgenic parasites had been allowed to infect a number of host cells, they separated the cell components by weight using isopycnic density centrifugation before sorting the fluorescent parasites from the other cellular material using a fluorescence-activated cell sorting (FACS) techniques.
The approach gave the researchers access to the parasites at a purity of around 98 per cent.
Mass spectrometric (MS) proteomic analysis of the parasites enabled 509 different proteins to be identified, a number that corresponds to around 6 per cent of the gene products predicted by the reference genome of Leishmania major.
The proteome of the L. mexicana amastigotes was compared with that of the extracellular promastigotes and revealed that the intracellular amastigotes synthesised synthesised significantly more basic fatty-acid binding proteins and showed a greater abundance of enzymes of fatty acid catabolism.
Analysis of the subset of genes whose products were more abundant in amastigotes revealed characteristic sequence motifs in 3'-untranslated regions that have been linked to translational control elements, suggesting that proteome data sets may be used to identify regulatory elements in messenger RNA.
The identified proteins contained all vaccine antigens tested to date and should provide a valuable resource to aid the selection of future vaccine antigen candidates.
