Molecular analysis of insect stage Trypanosoma brucei

Abstract

This project used both a genomic and proteomic approach to investigate the molecular and biochemical changes that occur in the insect stage of Trypanosoma brucei. The project had three main aims, the first of which was the application of an already established approach RADES -PCR to investigatc genes expressed during the maturation of established mid -gut infections. The method consisted of two steps, 1) reverse transcription of first strand DNA using oligo (dT) strech and 2) enrichment of parasite material over host material using the splice leader (SL) the miniexon. It was found that the high prevalence of artefacts and the presence of bacterial material from symbionts in the tsetse midgut hampered this investigation. The application of a low and high stringency approach using random anchored primers did not improve the situation. This feature of T brucei maturation could therefore not be investigated further by this approach.Our second aim was to investigate the effect that 8 Br -cGMP has on T brucei procyclic forms. It had been observed in our laboratory that the addition of this chemical to an infected bloodmeal increased the level of T brucei establishment within the fly population to 100 %. An enriched subtractive cDNA library was made to study the effect that this compound has on T brucei, this library provided a full set of the differentially expressed genes resulting from this 8 Br -cGMP treatment. Analysis of over 1000 recombinant clones highlighted the involvement of the polyamine and trypanothione synthesis pathway, in particular the potential role of the antioxidant response.Our third aim was to investigate the protein profile of T brucei in response to this treatment. Protein profiles were characterised using surface enhanced laser desorption/ionization time of flight mass spectrophotometry (SELDI- TOF -MS) where proteins are separated according to their biochemical properties. This approach revealed a number of potential differences that resulted in response to the treatment. Therefore there is a response at the proteomic level that may reflect those genes identified by the subtractive library.