Proteome analysis of Trypanosoma brucei with emphasis of prostaglandin metabolism

Abstract

African trypanosomiasis is caused by extra cellular parasitic protozoa that can be transmitted by the bite of a tsetse fly. The disease is caused by sub-species of Trypanosoma brucei, an extra-cellular eukaryotic flagellated parasite. There is no prophylactic chemotherapy or prospect of a vaccine and current treatment is inadequate. Drugs for late-stage disease are highly toxic. Livestock trypanosomiasis is caused by closely related Trypanosoma species. It has the greatest impact in sub-Saharan Africa, where the tsetse fly vector is common (WHO, 2002).

The complication of genomics results and analysis caused a big shift of interest from genomics to proteomics. The main analytical tool used for protein separation in proteomics is two-dimensional gel electrophoresis (2DGE) (O'Farrell, 1975 and Fey and Larsen, 2001). This technique separates proteins according to their masses and charges. These independent attributes enables separation of thousands of proteins in a single run. Since numerous proteins of a whole cell (its 'proteome') connect the genotype with the phenotype, we set out to study the proteome of the bloodstream form in T brucei which are infectious and known to cause diseases in human and animals. Deviating protein patterns between the different stages could direct the attention to disease-specific proteins and genes, which might be involved in the expression of infection and cause of disease. After separation, most proteins can be identified by mass spectroscopy. In studies performed to identify specific proteins related to a given metabolic process or disease it is, however, much more efficient to detect and identify only differentiated protein groups of samples. In order to understand the molecular basis of the parasites differentiation, we were interested in characterization of specific proteins expressed in bloodstream forms. However, the vast evolutionary distance between trypanosomes and the higher eukaryotes presents significant problems with functional assignment based on sequence similarities, and frequently homologues cannot be identified with sufficient confidence to be informative. Direct identification of proteins in isolated organelles has the potential of providing robust functional insight and is a powerful approach for initial assignment. The cytoplasm protein fraction and membrane protein fraction of T. brucei were used in this work to analyse the protozoan proteomics.

Proteomics is a rapidly developing technique, which allows the efficient isolation of multiple protein families and it is a valuable tool for global patterns of gene expression. It allows the studies of membrane as well as cytosolic proteins. The rapidly growing development of bioinformatics, for example the use of software's like The JVirGel will also transform the handling of the multitude of data accumulating in proteomic experiments. The JVirGel software creates and visualizes virtual two-dimensional (2D) protein gels based on the migration behaviour of proteins in dependence of their theoretical molecular weights in combination with their calculated isoelectric points. Although, 9068 protein-coding genes and many pseudo genes have been identified, (science 2005) a number of potential new proteins are yet to be characterized. Analysis of T. brucei proteins using two-dimension gel electrophoresis, may offer prognostic analysis and information on disease mechanism.

These results suggest the first step towards the generation of proteome profiles for use in future studies on protein expression, especially those accompanying the differentiation of the parasite. Comparison of the different stage-specific protein profiles will allow us to identify candidates as targets for drug action. Protein data using JVirGel software together with the provision of the complete genome sequence for T. brucei, the application of the genomic and post-genomic technologies should provide advances in the understanding of the biology of this parasite and the identification of key factors for virulence, drug resistance and infectivity. In this study the slender bloodstream form of T. brucei 221 were used because they are easy to grow in rats. These parasites can equally be cultivated at 37°C to grow to a cell density of about 1x106/ml. This approach allows the cultivation of high cell numbers without excessive expenditure of work and cost.

I first had to establish a protocol for separating trypanosomal proteins by iso-electric focusing to produce a reference map for the first time. In this project, it was possible to identify many cytoplasmic and membrane proteins on the proteome-map of T. bruce. In proteomics the combination of the high-resolution two-dimensional electrophoresis and matrix assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOFMS) is currently the method of choice for protein identification. More than 300 protein spots were detected on a silver-stained two-dimensional gel. Analysis of 50 spots among them those highly expressed when T. brucei when grown in the presence of arachidonic acid was carried out. The protein spots from gel were digested with trypsin in-gel digestion followed by subsequent MALDI-TOF mass spectrometry in a process termed 'peptide mass fingerprinting'. Following a database search, 27 protein spots were identifield (belonging to different functional groups of proteins). 20 of the identified proteins are components of the main biological and cell regulation pathways located in T. brucei With this study, I established a partial data base and reference proteome map of the bloodstream form T. bruce. These data will facilitate further addition of information to T. brucei proteome, which may aid drug design in the treatment of patients with and control of trypanosomiasis and the control of infection. Future research based on this dissertation should mainly focus in the identification of different proteins between the bloodstream forms, the procyclic forms and the short stumpy forms of T. brucei.

Trypanosoma brucei ist die Ursache für die afrikanische Schlafkrankheit (Trypanosomiasis), welche hauptsächlich die ärmsten Teile der Bevölkerung in den Entwicklungsländern in Zentralafrika und der Subsahara-Region bedroht. Trypanosoma brucei hat einen Mechanismus entwickelt, um der Immunabwehr des Wirts zu entkommen, der auf der Antigenvariation der über Glycosylphosphatidylinositol-Anker verbundenen Oberflächenproteine beruht. Die Genexpression ist hauptsächlich auf post-transkriptioneller Ebene reguliert.

Die vollständige Aufklärung dieses Organismus wurde immer wieder erschwert durch Herausforderungen technischer Art. Dennoch ermöglicht der Fortschritt verschiedener Disziplinen wie Genomik, Proteomik und der Erstellung und Analyse von 2D-Gelen verbunden mit Methoden wie der matrix-assisted laser desorption/ionisation-time-of-flight (MALDI-TOF) mass spectrometry (MS) einen neuen Ansatz um schnell funktionelle Daten über Proteine zu bekommen.

Im Gegensatz zu konventionellen biochemischen Ansätzen, die auf die Beobachtung einiger weniger Proteine abzielen, lässt sich durch proteomische Methoden die globale Proteinexpression im großen Maßstab untersuchen. Aus der direkten Messung der verschiedenen Proteinexpressionslevel lassen sich Rückschlüsse auf die Aktivität relevanter Proteine unter verschiedenen Bedingungen ziehen. Die Methoden der Proteomik bieten ein wichtiges Werkzeug um differentielle Expression von Proteinen während der verschiedenen Lebenscyclus-Phasen und verschiedenen Kulturbedingungen des Parasiten zu beobachten.