Genome characterisation and mobility investigation in trypanosomes

Abstract: Trypanosomes are uni-flagellated protozoa responsible for some of the most debilitating human infections such as Chagas disease (Trypanosoma cruzi) and sleeping sickness (Trypanosoma brucei). Unravelling their genome organisation and their biology would improve our basic knowledge and could contribute to find new drug target specific to the species. Trypanosoma cruzi has previously been known to exhibit extensive chromosome size variation, sequence polymorphism and large clusters of tandemly repeated genes. These characteristics have made the elucidation of the genome structure a difficult task. As part of the Tritryp genome project, a large karyotyping study was performed on three strains of T. cruzi: the reference strain CL Brener (T. cruzi lle strain) and T cruzi I strains, Sylvio X10/7 and CAI/72. A total of 239 eDNA markers have been hybridised to PFGE separated chromosomes and their pattern of hybridisation revealed a highly complex genome organisation. Many homologous chromosomes show large size differences, up to 173% different in the case of CL Brener that may be explained by chromosome rearrangements. Because of a highly repetitive genome (over 50%), assembling the sequencing data is complicated. The draft of the T. cruzi genome could be improved by sequencing the parental strain of the hybrid CL Brener strain, Esmeraldo and by comparing the sequences with those obtained in the two other Tritryp genomes (T. brucei and L. major), which displayed a high degree of gene synteny despite the evolutionary distance. To finish the genome assembly, karyotyping data will be essential to anchor scaffolds to chromosomal bands as the number of chromosomes is still uncertain. Released trypanosomes sequencing data allowed scientists to explore the genome and perform large functional studies in trypanosomes. I focussed on the flagellum, an essential organelle for trypanosome survival. Flagellar genes were identified in the trypanosome date bases and functional studies were performed in T. brucei, for which potent reverse genetics are available in contrast to T.cruzi. Several inducible RNAi mutant cell fines were generated, resulting upon silencing in various affections of cell motility. Reducing or paralysing flagellum movement resulted in basal migration impairment, demonstrating for the first time that flagella can position their own basal body. As a result of basal body mis-positioning, cell cycle defect and morphogenesis default. Detailed analysis of the flagellum movement in wild type cells revealed that flagellum beating exhibits two waves, one propulsive, going from tip to base and one reverse that is used for changing directions. This striking behaviour is likely to be highly relevant in vivo during the course of a blood infection in T. brucei or for host cell invasion in T. cruzi. Silencing the TbDNAI1 gene (encoding the intermediate chain 1 of the outer dynein arm) resulted in absence of outer dynein arms. Remarkably, it is exactly the same phenotype as the one observed in human patients suffering of primary ciliary dyskinesia. This study reveals that studying the flagellum mobility in trypanosomes is important to improve our knowledge on the parasite biology but could also lead to the development of trypanosomes as a powerful model organism for functional analysis of genes implicated in human diseases.