Discussion

Trypanosoma brucei is a protist parasite. It belongs to a genus of pathogens that cause life-threatening and economically important diseases of human and animals in Sub-Saharan Africa. Exo-/endocytotic trafficking in these cells occurs via a single copy organelle called the flagellar pocket (FP). The FP is maintained and enclosed around the flagellum by the flagellar pocket collar (FPC). To date, the most important cytoskeletal component of the FPC is an essential, calcium-binding, polymer-forming protein called TbBILBO1. In searching for novel immune-tools to study this protein, we raised nanobodies (Nb), against TbBILBO1. Nanobodies were selected according to their binding properties to TbBILBO1, and were tested as immunofluorescence tools and expressed as intrabodies (INb). Nb48, proved to be the most robust nanobody and intrabody. We further demonstrate that inducible, cytoplasmic expression of INb48 was lethal to these parasites, producing abnormal phenotypes resembling those of TbBILBO1 RNAi knockdown.  Our results validate the feasibility of generating functional single-domain antibody derived intrabodies to target trypanosome cytoskeleton proteins and potentially other non-trypanosome proteins.