TbKINX1B: a novel BILBO1 partner and an essential protein in bloodstream form Trypanosoma brucei.

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.

Title: TbKINX1B, un nouveau partenaire de BILBO1, et une protéine essentielle dans la forme sanguine de Trypanosoma brucei. Abstract: La poche flagellaire (PF) de l'agent pathogène Trypanosoma brucei est une structure importante à copie unique formée par l'invagination de la membrane pelliculaire. Elle est le site unique de l'endo- et de l'exocytose et est nécessaire à la pathogénicité du parasite. La PF est constituée de sous-domaines structurels distincts, le moins exploré étant le collier de poche flagellaire (CPF). TbBILBO1 est la première protéine du CPF décrite. Elle est essentielle pour la survie du parasite et la biogenèse de la PF et du CPF. Dans ce travail, nous caractérisons TbKINX1B, un nouveau partenaire de TbBILBO1. Nous démontrons que TbKINX1B est localisée au niveau des corps basaux, du quartet de microtubules (un ensemble de quatre microtubules) et du CPF chez T. brucei. La diminution de l'expression de TbKINX1B par ARN interférence dans les formes sanguines est létale, induisant une perturbation globale du réseau endomembranaire. Dans les formes procycliques, l'ARN interférence conduit à un phénotype mineur avec un petit nombre de cellules présentant des morphologies de type épimastigote, avec un kinétoplaste mal placé. Nos résultats caractérisent TbKINX1B comme la première kinésine putative à être localisée à la fois au niveau des corps basaux et du CPF avec un rôle potentiel dans le transport de cargaison le long du quartet de microtubules.

Microtubule polyglutamylation is important for regulating cytoskeletal architecture and motility in Trypanosoma brucei.

The shape of kinetoplastids, such as Trypanosoma brucei, is precisely defined during the stages of the life cycle and governed by a stable subpellicular microtubule cytoskeleton. During the cell cycle and transitions between life cycle stages, this stability has to transiently give way to a dynamic behaviour to enable cell division and morphological rearrangements. How these opposing requirements of the cytoskeleton are regulated is poorly understood. Two possible levels of regulation are activities of cytoskeleton-associated proteins and microtubule post-translational modifications (PTMs). Here, we investigate the functions of two putative tubulin polyglutamylases in T. brucei, TTLL6A and TTLL12B. Depletion of both proteins leads to a reduction in tubulin polyglutamylation in situ and is associated with disintegration of the posterior cell pole, loss of the microtubule plus-end-binding protein EB1 and alterations of microtubule dynamics. We also observe a reduced polyglutamylation of the flagellar axoneme. Quantitative motility analysis reveals that the PTM imbalance correlates with a transition from directional to diffusive cell movement. These data show that microtubule polyglutamylation has an important role in regulating cytoskeletal architecture and motility in the parasite T. bruceiThis article has an associated First Person interview with the first author of the paper.

Anti-trypanosomal activity of cationic N-heterocyclic carbene gold(I) complexes.

Two gold(I) N-heterocyclic carbene complexes 1a and 1b were tested for their anti-trypanosomal activity against Trypanosoma brucei parasites. Both gold compounds exhibited excellent anti-trypanosomal activity (IC50=0.9-3.0nM). The effects of the gold complexes 1a and 1b on the T. b. brucei cytoskeleton were evaluated. Rapid detachment of the flagellum from the cell body occurred after treatment with the gold complexes. In addition, a quick and complete degeneration of the parasitic cytoskeleton was induced by the gold complexes, only the microtubules of the detached flagellum remained intact. Both gold compounds 1a and 1b feature selective anti-trypanosomal agents and were distinctly more active against T. b. brucei cells than against human HeLa cells. Thus, the gold complexes 1a and 1b feature promising drug candidates for the treatment of trypanosome infections such as sleeping sickness (human African Trypanosomiasis caused by Trypanosoma brucei parasites).