Genome analysis and DNA marker-based characterisation of pathogenic trypanosomes.

The advances in genomics technologies and genome analysis methods that offer new leads for accelerating discovery of putative targets for developing overall control tools are reviewed in Chapter 1. In Chapter 2, a PCR typing method based on restriction fragment length polymorphism analysis of the internal transcribed sequence (ITS) rDNA region was used to reveal distinct fingerprinting patterns that characterise human- and animal-derived Trypanosoma brucei gambiense and T. b. brucei isolates. Although these results also highlighted doubts about the uniformity of T. brucei subspecies, the limitation of such a typing technique that is based on a single genetic locus is obvious. As a result, the studies were extended to include all T. brucei subspecies in a more global amplified fragment length polymorphism (AFLP) genotyping (Chapter 3). This approach permitted an unbiased estimate of the total genome variance and revealed closer genetic relatedness between, and higher variability within, T. b. brucei and T. b. rhodesiense subspecies, compared to T. b. gambiense strains. However, it was clear from these studies that a finer-scale genotyping tool with enhanced resolution power was required. Chapter 4 describes such an advanced tool, a multiplex-endonuclease genotyping analysis (MEGA) approach that simultaneously accesses multiple independent restriction enzyme-based polymorphisms within the genome. It offered a robust and detailed genotyping tool and was, therefore, used to study the population genetic structure of T. brucei isolates, for epidemiological and cladistic analysis (Chapter 5). The MEGA approach envisages the application of genotyping to identify genetic profiles that are associated with specific (parasite) traits. Therefore, in Chapter 6, genotypes were constructed and correlated with human serum response traits of T. brucei rhodesiense clones and strains, to further provide a general method for measuring differential phenotypes and an objective assessment of such differences. Also, such fine-scale approach can be used to rapidly enrich for identifiable polymorphisms in a set of known DNA sequences known to be associated with a phenotypic trait of interest. Using sets of four endonucleases selected on the basis of the concept defined by the MEGA approach, the role of differential DNA methylation patterns in the human serum response properties of trypanosomes was evaluated (Chapter 6), which proved to be insignificant. Furthermore, we clarified the genetic relationships between T. equiperdum and other Trypanozoon species (Chapter 7). In Chapter 8, a general discussion of the data is presented. In summary, three main applications of molecular marker systems in trypanosomes were described in this thesis. These involve genomic studies for (1) generating sensitive tools for molecular typing of strains, (2) elucidating taxonomy of Trypanozoon, and (3) the analysis of the relationships between genetic variations and their consequent functional effects that may enhance our understanding of important traits. These applications have permitted fine-scale genotypic characterisation of the parasites, and offered a template for phenotypic correlations of the genotype data.