Microtubule polyglutamylation is an essential regulator of cytoskeletal integrity in Trypanosoma brucei.

Tubulin polyglutamylation, catalysed by members of the tubulin tyrosine ligase-like (TTLL) protein family, is an evolutionarily highly conserved mechanism involved in the regulation of microtubule dynamics and function in eukaryotes. In the protozoan parasite Trypanosoma brucei, the microtubule cytoskeleton is essential for cell motility and maintaining cell shape. In a previous study, we showed that T. brucei TTLL6A and TTLL12B are required to regulate microtubule dynamics at the posterior cell pole. Here, using gene deletion, we show that the polyglutamylase TTLL1 is essential for the integrity of the highly organised microtubule structure at the cell pole, with a phenotype distinct from that observed in TTLL6A- and TTLL12B-depleted cells. Reduced polyglutamylation in TTLL1-deficient cells also leads to increased levels in tubulin tyrosination, providing new evidence for an interplay between the tubulin tyrosination and detyrosination cycle and polyglutamylation. We also show that TTLL1 acts differentially on specific microtubule doublets of the flagellar axoneme, although the absence of TTLL1 appears to have no measurable effect on cell motility.

Evaluation of the Antiparasitic and Antifungal Activities of Synthetic Piperlongumine-Type Cinnamide Derivatives: Booster Effect by Halogen Substituents.

A series of synthetic N-acylpyrrolidone and -piperidone derivatives of the natural alkaloid piperlongumine were prepared and tested for their activities against Leishmania major and Toxoplasma gondii parasites. Replacement of one of the aryl meta-methoxy groups by halogens such as chlorine, bromine and iodine led to distinctly increased antiparasitic activities. For instance, the new bromo- and iodo-substituted compounds 3 b/c and 4 b/c showed strong activity against L. major promastigotes (IC50 =4.5-5.8 µM). Their activities against L. major amastigotes were moderate. In addition, the new compounds 3 b, 3 c, and 4 a-c exhibited high activity against T. gondii parasites (IC50 =2.0-3.5 µM) with considerable selectivities when taking their effects on non-malignant Vero cells into account. Notable antitrypanosomal activity against Trypanosoma brucei was also found for 4 b. Antifungal activity against Madurella mycetomatis was observed for compound 4 c at higher doses. Quantitative structure-activity relationship (QSAR) studies were carried out, and docking calculations of test compounds bound to tubulin revealed binding differences between the 2-pyrrolidone and 2-piperidone derivatives. Microtubules-destabilizing effects were observed for 4 b in T. b. brucei cells.

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.

Novel Cytoskeleton-Associated Proteins in Trypanosoma brucei Are Essential for Cell Morphogenesis and Cytokinesis.

Trypanosome brucei, the causative agent of African sleeping sickness, harbours a highly ordered, subpellicular microtubule cytoskeleton that defines many aspects of morphology, motility and virulence. This array of microtubules is associated with a large number of proteins involved in its regulation. Employing proximity-dependent biotinylation assay (BioID) using the well characterised cytoskeleton-associated protein CAP5.5 as a probe, we identified CAP50 (Tb927.11.2610). This protein colocalises with the subpellicular cytoskeleton microtubules but not with the flagellum. Depletion by RNAi results in defects in cytokinesis, morphology and partial disorganisation of microtubule arrays. Published proteomics data indicate a possible association of CAP50 with two other, yet uncharacterised, cytoskeletal proteins, CAP52 (Tb927.6.5070) and CAP42 (Tb927.4.1300), which were therefore included in our analysis. We show that their depletion causes phenotypes similar to those described for CAP50 and that they are essential for cellular integrity.

New Pyrano-4H-benzo[g]chromene-5,10-diones with Antiparasitic and Antioxidant Activities.

New pyranonaphthoquinone derivatives were synthesized and investigated for their activity against Trypanosoma brucei, Leishmania major, and Toxoplasma gondii parasites. The pentafluorophenyl derivative was efficacious against T. brucei with single digit micromolar EC50 values and against T. gondii with even sub-micromolar values. The 3-chloro-4,5-dimethoxyphenyl derivative showed an activity against amastigotes of Leishmania major parasites comparable to that of amphotericin B. In addition, antioxidant activities were observed for the bromophenyl derivatives, and their redox behavior was studied by cyclovoltammetry. Anti-parasitic and antioxidative activities of the new naphthoquinone derivatives appear uncorrelated.

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.

Evaluation of the antiparasitic activities of imidazol-2-ylidene-gold(I) complexes.

A series of cationic gold(I)-carbene complexes with various 4,5-diarylimidazolylidene ligands were either newly prepared or repurposed for testing against protozoal Leishmania major, Toxoplasma gondii, and Trypanosoma brucei parasites. The syntheses of the new complexes 1b and 1c were described. Ferrocene compound 1a showed the highest activities against L. major amastigotes and T. gondii and distinct selectivity for T. gondii cells when compared with the activity against nonmalignant Vero cells. The ferrocene derivatives 1a-c are generally more active against the L. major amastigotes and the T. gondii tachyzoites than the other tested anisyl gold complexes and the approved drugs atovaquone and amphotericin B. Compounds 1a and 1e showed the highest selectivities for L. major amastigotes. Compounds 1d and 1f showed the highest selectivities for L. major promastigotes; 1f was the most active compound against L. major promastigotes of this series of compounds. The 3,4,5-trimethoxyphenyl analog 1b also exhibited a much greater selectivity for T. b. brucei cells when compared with its activity against human HeLa cells.

New Antiparasitic Bis-Naphthoquinone Derivatives.

A series of bis-naphthoquinone derivatives prepared by condensation of aryl aldehydes with lawsone were tested for antiparasitic activities against Toxoplasma gondii and Trypanosoma brucei parasites. Monofluorophenyl derivative 1a, 3,4-difluorophenyl analog 1c and furyl compound 1l exhibited significant activity against T. gondii cells and appear to be new promising drug candidates against this parasite. The 3,4,5-trifluorophenyl derivative 1g and the isovanillyl derivative 1j displayed selective activity against Leishmania major amastigotes.

Antiparasitic activities of new lawsone Mannich bases.

A series of new lawsone Mannich bases derived from salicylaldehydes or nitrofurfural were prepared and tested for their activities against Leishmania major, Toxoplasma gondii, and Trypanosoma brucei brucei parasites. The hydrochloride salts 5a and 6a of the Mannich bases 2a and 3a, derived from unsubstituted salicylaldehyde and long-chained alkyl amines, were selectively and strongly active against T. gondii cells and appear to be new promising drug candidates against this parasite. Compound 6a showed an even higher activity against T. gondii than the known lawsone Mannich base 1b. Compound 4a, derived from salicylaldehyde and 2-methylaminopyridine, was also distinctly active against T. gondii cells. The derivatives 3a (salicyl derivative), 3b (3,5-dichloro-2-hydroxyphenyl derivative), and 3d (5-nitrofuranyl derivative) as well as the hydrochlorides 6a and 6b were also efficacious against T. b. brucei cells with compounds 3a and 3b being more selective for T. b. brucei over Vero cells when compared with the known control compound 1b. The derivatives 5a, 5c, 6a, and 6c proved to be up to five times more active than 1b against L. major promastigotes and up to four times more efficacious against L. major amastigotes.

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).

Improved anticancer and antiparasitic activity of new lawsone Mannich bases.

Substituted lawsone Mannich bases 2a-e, 3a-e and 4a-e were prepared and tested for their biological activities. The new fatty alkyl substituted compounds 2a-c exhibited strong and selective growth inhibitory activities in the low one-digit micromolar and sub-micromolar range against a panel of human cancer cell lines associated with ROS formation. In addition, compounds 2a-c revealed sub-micromolar anti-trypanosomal activities against parasitic Trypanosoma brucei brucei cells via deformation of the microtubule cytoskeleton. The N-hexadecyl compound 2c was also highly active against locally isolated Entamoeba histolytica parasite samples exceeding the activity of metronidazole.

Nuclear architecture, genome and chromatin organisation in Trypanosoma brucei.

The nucleus of the human pathogen Trypanosoma brucei not only has unusual chromosomal composition, characterised by the presence of megabase, intermediate and minichromosomes, but also chromosome and gene organisation that is unique amongst eukaryotes. Here I provide an overview of current knowledge of nuclear structure, chromatin organisation and chromosome dynamics during interphase and mitosis. New technologies such as chromatin immunoprecipitation, in combination with new generation sequencing and proteomic analysis of subnuclear fractions, have led to novel insights into the organisation of the nucleus and chromatin. In particular, we are beginning to understand how universal mechanisms of chromatin modifications and nuclear position effects are deployed for parasite-specific functions and are centrally involved in genomic organisation and transcriptional regulation. These advances also have a major impact on progress in understanding the molecular basis of antigenic variation.

NLP is a novel transcription regulator involved in VSG expression site control in Trypanosoma brucei.

Trypanosoma brucei mono-allelically expresses one of approximately 1500 variant surface glycoprotein (VSG) genes while multiplying in the mammalian bloodstream. The active VSG is transcribed by RNA polymerase I in one of approximately 15 telomeric VSG expression sites (ESs). T. brucei is unusual in controlling gene expression predominantly post-transcriptionally, and how ESs are mono-allelically controlled remains a mystery. Here we identify a novel transcription regulator, which resembles a nucleoplasmin-like protein (NLP) with an AT-hook motif. NLP is key for ES control in bloodstream form T. brucei, as NLP knockdown results in 45- to 65-fold derepression of the silent VSG221 ES. NLP is also involved in repression of transcription in the inactive VSG Basic Copy arrays, minichromosomes and procyclin loci. NLP is shown to be enriched on the 177- and 50-bp simple sequence repeats, the non-transcribed regions around rDNA and procyclin, and both active and silent ESs. Blocking NLP synthesis leads to downregulation of the active ES, indicating that NLP plays a role in regulating appropriate levels of transcription of ESs in both their active and silent state. Discovery of the unusual transcription regulator NLP provides new insight into the factors that are critical for ES control.

The FACT subunit TbSpt16 is involved in cell cycle specific control of VSG expression sites in Trypanosoma brucei.

The African trypanosome Trypanosoma brucei monoallelically expresses one of more than 1000 Variant Surface Glycoprotein (VSG) genes. The active VSG is transcribed from one of about 15 telomeric VSG expression sites (ESs). It is unclear how monoallelic expression of VSG is controlled, and how inactive VSG ESs are silenced. Here, we show that blocking synthesis of the T. brucei FACT subunit TbSpt16 triggers a G2/early M phase cell cycle arrest in both bloodstream and insect form T. brucei. Segregation of T. brucei minichromosomes in these stalled cells is impaired, implicating FACT in maintenance of centromeres. Strikingly, knock-down of TbSpt16 results in 20- to 23-fold derepression of silent VSG ES promoters in bloodstream form T. brucei, with derepression specific to the G2/M cell cycle stage. In insect form T. brucei TbSpt16 knock-down results in 16- to 25-fold VSG ES derepression. Using chromatin immunoprecipitation (ChIP), TbSpt16 was found to be particularly enriched at the promoter region of silent but not active VSG ESs in bloodstream form T. brucei. The chromatin remodeler FACT is therefore implicated in maintenance of repressed chromatin present at silent VSG ES promoters, but is also essential for chromosome segregation presumably through maintenance of functional centromeres.

Functional characterisation and drug target validation of a mitotic kinesin-13 in Trypanosoma brucei.

Mitotic kinesins are essential for faithful chromosome segregation and cell proliferation. Therefore, in humans, kinesin motor proteins have been identified as anti-cancer drug targets and small molecule inhibitors are now tested in clinical studies. Phylogenetic analyses have assigned five of the approximately fifty kinesin motor proteins coded by Trypanosoma brucei genome to the Kinesin-13 family. Kinesins of this family have unusual biochemical properties because they do not transport cargo along microtubules but are able to depolymerise microtubules at their ends, therefore contributing to the regulation of microtubule length. In other eukaryotic genomes sequenced to date, only between one and three Kinesin-13s are present. We have used immunolocalisation, RNAi-mediated protein depletion, biochemical in vitro assays and a mouse model of infection to study the single mitotic Kinesin-13 in T. brucei. Subcellular localisation of all five T. brucei Kinesin-13s revealed distinct distributions, indicating that the expansion of this kinesin family in kinetoplastids is accompanied by functional diversification. Only a single kinesin (TbKif13-1) has a nuclear localisation. Using active, recombinant TbKif13-1 in in vitro assays we experimentally confirm the depolymerising properties of this kinesin. We analyse the biological function of TbKif13-1 by RNAi-mediated protein depletion and show its central role in regulating spindle assembly during mitosis. Absence of the protein leads to abnormally long and bent mitotic spindles, causing chromosome mis-segregation and cell death. RNAi-depletion in a mouse model of infection completely prevents infection with the parasite. Given its essential role in mitosis, proliferation and survival of the parasite and the availability of a simple in vitro activity assay, TbKif13-1 has been identified as an excellent potential drug target.

The role of the Kinesin-13 family protein TbKif13-2 in flagellar length control of Trypanosoma brucei.

TbKif13-2, a member of the microtubule-depolymerising Kinesin-13 family was localised at the tip of the flagellum in Trypanosoma brucei. Its predicted activity suggested a role in the regulation of axonemal length. However, using gene deletion and overexpression of TbKif13-2 we show that, in procyclic T. brucei, this kinesin has only a very limited effect on flagellar length. Gene deletion resulted in no significant elongation of the flagellum and overexpression only slightly decreased flagellar length and the rate of growth of a new flagellum during cell division. This is in contrast to studies in Leishmania major, where overexpression of the TbKif13-2 homologue resulted in a significant length reduction of the flagellum. Knockout of TbKif13-2 has, however, an effect on the initial growth of the emerging new flagellum. In conclusion, we show that TbKif13-2 has only a marginal impact on flagellar length in T. brucei.

Expression and cellular localisation of calpain-like proteins in Trypanosoma brucei.

Calpains are a ubiquitous family of calcium-dependent cysteine proteases involved in a wide range of cell regulatory and differentiation processes. In many protozoan organisms, atypical calpains have been discovered that lack the characteristic calcium-binding penta-EF-hand motif of typical vertebrate calpains and most of these novel calpain-like proteins are non-enzymatic homologues of typical calpains. The gene family is particularly expanded in ciliates and kinetoplastids, comprising 25 members in the parasite Trypanosoma brucei. Unique to kinetoplastids, some calpain-like proteins contain N-terminal dual myristoylation/palmitoylation signals, a protein modification involved in protein-membrane associations. We analyzed the expression of calpain-like proteins in the insect (procyclic) and bloodstream-stage of T. brucei using quantitative real time PCR and identified the differential expression of some of the calpain genes. We also present a comprehensive analysis of the subcellular localisation of selected members of this protein family in trypanosomes. Here, of particular interest is the role of protein acylation for targeting to the flagellum. We show that, although acylation is important for flagellar targeting, additional signals are required to specify the precise subcellular localisation.

Distinct and overlapping roles for two Dicer-like proteins in the RNA interference pathways of the ancient eukaryote Trypanosoma brucei.

Trypanosoma brucei is one of the most ancient eukaryotes where RNA interference (RNAi) is operational and is the only single-cell pathogen where RNAi has been extensively studied and used as a tool for functional analyses. Here, we report that the T. brucei RNAi pathway, although relying on a single Argonaute protein (AGO1), is initiated by the activities of two distinct Dicer-like enzymes. Both TbDCL1, a mostly cytoplasmic protein, and the previously undescribed nuclear enzyme TbDCL2 contribute to the biogenesis of siRNAs from retroposons. However, TbDCL2 has a predominant role in generating siRNAs from chromosomal internal repeat transcripts that accumulate at the nucleolus in RNAi-deficient cells and in initiating the endogenous RNAi response against retroposons and repeats alike. Moreover, siRNAs generated by both TbDCL1 and TbDCL2 carry a 5'-monophosphate and a blocked 3' terminus, suggesting that 3' end modification is an ancient trait of siRNAs. We thus propose a model whereby TbDCL2 fuels the T. brucei nuclear RNAi pathway and TbDCL1 patrols the cytoplasm, posttranscriptionally silencing potentially harmful nucleic acid parasites that may access the cytoplasm. Nevertheless, we also provide evidence for cross-talk between the two Dicer-like enzymes, because TbDCL2 is implicated in the generation of 35- to 65-nucleotide intermediate transcripts that appear to be substrates for TbDCL1. Our finding that dcl2KO cells are more sensitive to RNAi triggers than wild-type cells has significant implications for reverse genetic analyses in this important human pathogen.

Functional characterization of cohesin SMC3 and separase and their roles in the segregation of large and minichromosomes in Trypanosoma brucei.

Minichromosomes in the nuclear genome of Trypanosoma brucei exhibit unusual patterns of mitotic segregation. To address whether differences in their mode of segregation in relation to large chromosomes are reflected at a molecular level, we characterized two different proteins that have highly conserved functions in eukaryotic chromosomes segregation: the SMC3 protein, a component of the chromatid cohesion apparatus, and the protease separase that resolves the cohesin complex at the onset of anaphase and has, in other organisms, additional functions during mitosis. Using in situ hybridization we show that RNA interference-mediated depletion of SMC3 has no visible effect on the segregation of the minichromosomal population but interferes with the faithful mitotic separation of large chromosomes. In contrast, separase depletion causes missegregation of both mini- and large chromosomes. We also show that SMC3 persists as a soluble protein throughout the cell cycle and only associates with chromatin between G1 and metaphase. Separase is present in the cell during the entire cell cycle, but is excluded from the nucleus until the metaphase-anaphase transition, thereby providing a potential control mechanism to prevent the untimely cleavage of the cohesin complex.

The Argonaute protein TbAGO1 contributes to large and mini-chromosome segregation and is required for control of RIME retroposons and RHS pseudogene-associated transcripts.

The protist Trypanosoma brucei possesses a single Argonaute gene called TbAGO1 that is necessary for RNAi silencing. We previously showed that in strain 427, TbAGO1 knock-out leads to a slow growth phenotype and to chromosome segregation defects. Here we report that the slow growth phenotype is linked to defects in segregation of both large and mini-chromosome populations, with large chromosomes being the most affected. These phenotypes are completely reversed upon inducible re-expression of TbAGO1 fused to GFP, demonstrating their link with TbAGO1. Trypanosomes that do not express TbAGO1 show a general increase in the abundance of transcripts derived from the short retroposon RIME (Ribosomal Interspersed Mobile Element). Supplementary large RIME transcripts emerge in the absence of RNAi, a phenomenon coupled to the disappearance of short transcripts. These fluctuations are reversed by inducible expression of GFP::TbAGO1. Furthermore, we use a combination of Northern blots, RT-PCR and sequencing to reveal that RNAi controls expression of transcripts derived from RHS (Retrotransposon Hot Spot) pseudogenes (RHS genes with retro-element(s) integrated within their coding sequence). Absence of RNAi also leads to an increase of steady-state transcripts from regular RHS genes (those without retro-element), indicating a role for pseudogene in control of gene expression. However, analysis of retroposon abundance and arrangement in the genome of multiple clonal cell lines of TbAGO1-/- failed to reveal movement of mobile elements despite the increased amounts of retroposon transcripts.