Abstract
The flagellar pocket (FP) of the pathogen Trypanosoma brucei is an important single copy structure that is formed by the invagination of the pellicular membrane. It is the unique site of endo- and exocytosis and is required for parasite pathogenicity. The FP consists of distinct structural sub-domains with the least explored being the flagellar pocket collar (FPC). TbBILBO1 is the first-described FPC protein of Trypanosoma brucei. It is essential for parasite survival, FP and FPC biogenesis. In this work, we characterize TbKINX1B, a novel TbBILBO1 partner. We demonstrate that TbKINX1B is located on the basal bodies, the microtubule quartet (a set of four microtubules) and the FPC in T. brucei. Down-regulation of TbKINX1B by RNA interference in bloodstream forms is lethal, inducing an overall disturbance in the endomembrane network. In procyclic forms, the RNAi knockdown of TbKINX1B leads to a minor phenotype with a small number of cells displaying epimastigote-like morphologies, with a misplaced kinetoplast. Our results characterize TbKINX1B as the first putative kinesin to be localized both at the basal bodies and the FPC with a potential role in transporting cargo along with the microtubule quartet.
Highlights
Trypanosoma brucei is a zoonotic pathogen and the etiological agent of sleeping sickness with 60 million people living in areas with a high risk of infection, even though fewer than 2000 cases are detected per year
The gene Tb927.7.3000 [6] was previously identified in a genomic yeast two-hybrid library screen with TbBILBO1 as bait (Hybrigenics) [21] and codes for a putative-kinesin that belongs to the paralog group, Kinesin-X1 of kinesins, as classified by Wickstead et al [45]
Using the TriTryDB BlastP tool, TbKINX1B orthologs were found in the parasites represented by Trypanosoma and Leishmania, and the neotropical porcupine parasite Porcisia hertigi (34% aa identity), Leptomonas seymouri (40% aa identity), the monoxenous parasites Crithidia fasciculata (41% aa identity), Blechomonas ayalai (44% aa identity), Paratrypanosoma confusum (38% aa identity), and in the free living kinetoplastid Bodo saltans (45% aa identity) [6]
Summary
Trypanosoma brucei is a zoonotic pathogen and the etiological agent of sleeping sickness with 60 million people living in areas with a high risk of infection, even though fewer than 2000 cases are detected per year. The procyclic form (PCF) and bloodstream form (BSF) T. brucei cell has a polarized organization and importantly, within its posterior end, it houses a unique structure called the flagellar pocket (FP) from which the flagellum emerges. This organelle-like structure is the exclusive site for endo- and exocytotic activity [32]. Transport of material often involves the activity of microtubule-dependent processes mediated by molecular motors such as kinesins (KIN) or dyneins. There are 17 kinesin families (kinesin-1 to -17), as well as 14 additional paralog groups that cannot be assigned to any of the kinesin families (kinesinX1-14). Functional characterization of kinesins in T. brucei has shown multiple, distinct and essential roles in flagellar construction, cell morphology, organelle segregation and mitosis [20, 27, 12, 13, 45]
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