Finding a gene for virulence in Trypanosoma brucei

Trypanosoma brucei is the protozoan parasite that causes sleeping sickness in humans and Nagana in cattle in sub-Saharan Africa. The genome of this diploid parasite has been sequenced and T. brucei has been shown to follow a Mendelian genetic system. This has made the use of classical genetic approaches to finding genes that confer phenotypic traits possible in T. brucei. A genetic map has already been assembled for the non-human infective sub-species of T. b. brucei and this has been used successfully for quantitative trait analysis to identify genetic loci that contribute to parasite specific traits. Variation in virulence between different species, sub-species and strains of trypanosomes is well reported in the literature but the genetic basis underlying these differences has not been extensively studied. However, it is clear that trypanosome virulence is influenced by both host and parasite genetic factors. One study examining the genetic basis of difference in virulence between T. brucei parasite strains was conducted by Morrison et al. (2009). This study found T. brucei genetic loci that were associated with different virulence phenotypes: anaemia, splenomegaly, hepatomegaly and reticulocytosis. The work detailed in this thesis is based on one of the Quantitative Trait Loci (QTLs) identified in the 2009 study, the QTL on chromosome 3 that is associated with enlargement of the host spleen (splenomegaly). This thesis describes the generation of new restriction fragment length polymorphism markers and adaptation of SNP sequencing for use in fine mapping the T. brucei virulence associated QTL, specifically the locus associated with splenomegaly (at that stage covering approximately 300 MB with 383 genes). These new markers were used in combination with existing markers to narrow down the splenomegaly QTL identified by Morrison et al. (2009) to a region 50 kb and 52 genes and from these identified a candidate gene. This thesis then goes on to describe the design and application of a novel allele replacement approach to studying the contribution of different alleles of the identified candidate to the splenomegaly phenotype. This approach has not been used before to examine T. brucei genes. Constructs were designed and produced to enable allele replacement of the candidate gene, and a genotyping validation pipeline established to enable evaluation of construct integration. Alleles of the candidate gene were replaced in two different T. brucei strains with a different allele of the same gene. Multiple clones of four different strains of allele replacement (AR) parasites were generated, two in two different progeny clone backgrounds (77 and 86). For each wild-type (WT) clone, a specific allele of the candidate gene was replaced with either a different allele of the same gene or, as a transfection control, with the same allele to rule out any contribution of the transfection process to the splenomegaly phenotype. This resulted in a panel of successfully transfected clones that are a platform for robustly testing the influence of the candidate gene alleles on the splenomegaly phenotype. These AR clones were then used in an in vivo experiment to assess any changes to the splenomegaly phenotype caused by the introduction of a different allele into the different genetic backgrounds. The resulting phenotypic measurements were unexpected and the implications of those results is discussed in the final discussion