Project description:Trypanosoma brucei Lister 427 bloodstream forms were cultured in HMI-11 medium. Total RNA was prepared using Qiagen RNAeasy kits for single sample RNAseq to estimate VSG mRNA abundance (and not to reconstruct the transcriptome). The cDNA libraries were prepared and sequenced at the Beijing Genomics Institute (Shenzhen, China). Polyadenylated RNA was purified from total RNA, converted to cDNA using random hexamer primers sheared and size selected for fragments ~200 bp in length using the Illumina TruSeq RNA Sample Preparation Kit v2. RNAseq of the resulting libraries was used for the determination of transcript abundances. Sequencing was performed on an Illumina Hiseq 2000 (Illumina, CA) platform and 90 base paired end reads obtained. Four samples were analysed: 1. Trypanosoma brucei Lister 427 expressing VSG2 2. Trypanosoma brucei Lister 427 expressing VSG6 3. Trypanosoma brucei Lister 427 expressing VSG6 and a VSG2 transgender located in the active bloodstream expression site 28 days after electroporation 4. Trypanosoma brucei Lister 427 expressing VSG6 and a VSG2 transgender located in the active bloodstream expression site 44 days after electroporation.
Project description:Trypanosoma brucei gambiense is the causative agent of the fatal human disease African sleeping sickness. Using Digital Gene Expression we have compared the transcriptome of two T.b.brucei (STIB 247)xT.b.gambiense (STIB386) hybrids.
Project description:Trypanosoma brucei gambiense is the causative agent of the fatal human disease African sleeping sickness. Using Digital Gene Expression we have compared the transcriptome of a group 1 T.b.gambiense (Eliane) and a T.b.brucei (STIB 247).
Project description:The host range of African trypanosomes is influenced by innate protective molecules in the blood of primates. A subfraction of human high-density lipoprotein (HDL) containing apolipoprotein A-I, apolipoprotein L-I, and haptoglobin-related protein is toxic to Trypanosoma brucei brucei but not the human sleeping sickness parasite Trypanosoma brucei rhodesiense. It is thought that T. b. rhodesiense evolved from a T. b. brucei-like ancestor and expresses a defense protein that ablates the antitrypanosomal activity of human HDL. To directly investigate this possibility, we developed an in vitro selection to generate human HDL-resistant T. b. brucei. Here we show that conversion of T. b. brucei from human HDL sensitive to resistant correlates with changes in the expression of the variant surface glycoprotein (VSG) and abolished uptake of the cytotoxic human HDLs. Complete transcriptome analysis of the HDL-susceptible and -resistant trypanosomes confirmed that VSG switching had occurred but failed to reveal the expression of other genes specifically associated with human HDL resistance, including the serum resistance-associated gene (SRA) of T. b. rhodesiense. In addition, we found that while the original active expression site was still utilized, expression of three expression site-associated genes (ESAG) was altered in the HDL-resistant trypanosomes. These findings demonstrate that resistance to human HDLs can be acquired by T. b. brucei. Keywords: Trypanosoma, VSG, antigenic switching, HDL-resistance
Project description:Trypanosoma brucei gambiense is the causative agent of the fatal human disease African sleeping sickness. Using Digital Gene Expression we have compared the transcriptome of two isogenic T.b.gambiense lines that are either sensitive or resistant to human serum.
Project description:Eukaryotes have an array of diverse mechanisms for organising and using their genomes, but the histones that make up chromatin are highly conserved. Unusually, histones from Kinetoplastids are highly divergent. The structural and functional consequences of this variation are unknown. Here, we have biochemically characterised nucleosome core particles (NCPs) from the Kinetoplastid parasite Trypanosoma brucei. A structure of the T. brucei NCP reveals that global histone architecture is conserved, but specific sequence alterations lead to distinct DNA and protein interaction interfaces. The T. brucei NCP is unstable and has weakened DNA binding overall. However, dramatic changes at the H2A-H2B interface introduce local reinforcement of DNA contacts. The T. brucei acidic patch has altered topology and is refractory to known binders, indicating that the nature of chromatin interactions in T. brucei may be unique. Overall, our results provide a detailed molecular basis for understanding evolutionary divergence in chromatin structure.
Project description:We examine the function of the TbRAP1 DB domain in gene expression regulation in Trypanosoma brucei that causes human African trypanosomiasis. TbRAP1 is required for normal VSG monoallelic expression, a key aspect of antigenic variation that is used by T. brucei to evade the host immune response.
