Global Parasitology Seminars
Title: “DRiF-Seq: Quantitative cartography of gene silencing across trypanosome genomes in culture and in the host"
Speaker: Catarina Gadelha, PhD
Lecturer in Molecular Cell Biology
School of Life Sciences
Date: Monday, May 24, 2021
Time: 12-1 pm EST
Abstract: High-throughput methods for gene function analysis such as BarSeq and RIT-seq are powerful modern tools to understand parasite biology. In African trypanosomes, genome-scale screening has to date only been possible in a culture-adapted monomorphic strain, and the system employed works well only when strong positive selection can be applied, meaning that significant aspects of parasite biology and disease cannot be directly addressed.
To understand the biology of the parasite both in culture and in contact with the host, we developed Direct RNAi-Fragment Sequencing (DRiF-Seq), a novel genome-scale approach to create highly parallel libraries of inducible RNAi mutants in monomorphic and also pleomorphic bloodstream-form EATRO1125 T. brucei (“AnTat”). The method can be used to quantitatively follow 100,000s of barcoded mutants as individual clones over >20 generations, and robustly estimate clone and gene specific competitive growth rates both in vitro and in vivo. In pleomorphic cells, ~50% of the non-VSG genes are associated with a detectable loss-of-fitness – similar to that seen by knock-outs in Plasmodium. Further genes are necessary for survival in vivo and ~25% of mutants in non-VSG surface genes differ significantly in their behaviour in libraries in culture versus mouse models of chronic infection.
Parallel phenotyping over multiple timepoints reveals the characteristic timings and effect sizes associated with specific cell processes and allows the functional dissection of essentiality within cellular structures/complexes. Moreover, the method can be quickly modified for application to trypanosome species causing animal disease, and intra- and inter-species DRiF-Seq comparisons reveal unexpected differences in specific biology between strains of T. brucei, and also substantial differences between T. brucei and the major agent of animal African trypanosomiasis, T. congolense. These data exemplify the power of robust quantitation of mutant fitness at genome-scale in vitro and in the host, and point to aspects of parasite biology that are not well represented by common culture-adapted lines. They also point to a much more effective means to study gene function in both acute and chronic models of trypanosomiasis that would remove many confounding factors from analysis and massively reduces animal usage.
Please contact Sue Waddington-Pilder (email@example.com) to arrange to speak with Dr. Le Roch.