Radial spoke protein 9 is necessary for axoneme assembly in Plasmodium but not in trypanosomatid parasites.

Flagella are important for eukaryote cell motility, including in sperm, and are vital for life cycle progression of many unicellular eukaryotic pathogens. The '9+2' axoneme in most motile flagella comprises nine outer doublet and two central-pair singlet microtubules. T-shaped radial spokes protrude from the outer doublets towards the central pair and are necessary for effective beating. We asked whether there were radial spoke adaptations associated with parasite lineage-specific properties in apicomplexans and trypanosomatids. Following an orthologue search for experimentally uncharacterised radial spoke proteins (RSPs), we identified and analysed RSP9. Trypanosoma brucei and Leishmania mexicana have an extensive RSP complement, including two divergent RSP9 orthologues, necessary for flagellar beating and swimming. Detailed structural analysis showed that neither orthologue is needed for axoneme assembly in Leishmania. In contrast, Plasmodium has a reduced set of RSPs including a single RSP9 orthologue, deletion of which in Plasmodium berghei leads to failure of axoneme formation, failed male gamete release, greatly reduced fertilisation and inefficient life cycle progression in the mosquito. This indicates contrasting selection pressures on axoneme complexity, likely linked to the different mode of assembly of trypanosomatid versus Plasmodium flagella.

Unbalanced Arginine pathway and altered maturation of pleural macrophages in Th2-deficient mice during Litomosoides sigmodontis filarial infection.

Filarial parasites are tissue dwelling worms transmitted by hematophagous vectors. Understanding the mechanisms regulating microfilariae (the parasite offspring) development is a prerequisite for controlling transmission in filarial infections. Th2 immune responses are key for building efficient anti-parasite responses but have been shown to also lead to detrimental tissue damage in the presence of microfilariae. Litomosoides sigmodontis, a rodent filaria residing in the pleural cavity was therefore used to characterize pleuropulmonary pathology and associated immune responses in wild-type and Th2 deficient mice. Wild-type and Th2-deficient mice (Il-4rα-/-/Il-5-/- ) were infected with L. sigmodontis and parasite outcome was analyzed during the patent phase (when microfilariae are in the general circulation). Pleuropulmonary manifestations were investigated and pleural and bronchoalveolar cells were characterized by RNA analysis, imaging and/or flow cytometry focusing on macrophages. Il-4rα-/-/Il-5-/- mice were hypermicrofilaremic and showed an enhanced filarial survival but also displayed a drastic reduction of microfilaria-driven pleural cavity pathologies. In parallel, pleural macrophages from Il-4rα-/-/Il-5-/- mice lacked expression of prototypical alternative activation markers RELMα and Chil3 and showed an altered balance of some markers of the arginine metabolic pathway. In addition, monocytes-derived F4/80intermediate macrophages from infected Il-4rα-/-/Il-5-/- mice failed to mature into resident F4/80high large macrophages. Altogether these data emphasize that the presence of both microfilariae and IL-4R/IL-5 signaling are critical in the development of the pathology and in the phenotype of macrophages. In Il-4rα-/-/Il-5-/- mice, the balance is in favor of parasite development while limiting the pathology associated with the host immune response.

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.

Vital role for Plasmodium berghei Kinesin8B in axoneme assembly during male gamete formation and mosquito transmission.

Sexual development is an essential phase in the Plasmodium life cycle, where male gametogenesis is an unusual and extraordinarily rapid process. It produces 8 haploid motile microgametes, from a microgametocyte within 15 minutes. Its unique achievement lies in linking the assembly of 8 axonemes in the cytoplasm to the three rounds of intranuclear genome replication, forming motile microgametes, which are expelled in a process called exflagellation. Surprisingly little is known about the actors involved in these processes. We are interested in kinesins, molecular motors that could play potential roles in male gametogenesis. We have undertaken a functional characterization in Plasmodium berghei of kinesin-8B (PbKIN8B) expressed specifically in male gametocytes and gametes. By generating Pbkin8B-gfp parasites, we show that PbKIN8B is specifically expressed during male gametogenesis and is associated with the axoneme. We created a ΔPbkin8B knockout cell line and analysed the consequences of the absence of PbKIN8B on male gametogenesis. We show that the ability to produce sexually differentiated gametocytes is not affected in ΔPbkin8B parasites and that the 3 rounds of genome replication occur normally. Nevertheless, the development to free motile microgametes is halted and the life cycle is interrupted in vivo. Ultrastructural analysis revealed that intranuclear mitoses are unaffected whereas cytoplasmic microtubules, although assembled in doublets and elongated, fail to assemble in the normal axonemal '9+2' structure and become motile. Absence of a functional axoneme prevented microgamete assembly and release from the microgametocyte, severely reducing infection of the mosquito vector. This is the first functional study of a kinesin involved in male gametogenesis. These results reveal a previously unknown role for PbKIN8B in male gametogenesis, providing new insights into Plasmodium flagellar formation.

Intraflagellar transport is required for the maintenance of the trypanosome flagellum composition but not its length.

Intraflagellar transport (IFT) is required for construction of most cilia and flagella. Here, we used electron microscopy, immunofluorescence and live video microscopy to show that IFT is absent or arrested in the mature flagellum of Trypanosoma brucei upon RNA interference (RNAi)-mediated knockdown of IFT88 and IFT140, respectively. Flagella assembled prior to RNAi did not shorten, showing that IFT is not essential for the maintenance of flagella length. Although the ultrastructure of the axoneme was not visibly affected, flagellar beating was strongly reduced and the distribution of several flagellar components was drastically modified. The R subunit of the protein kinase A was no longer concentrated in the flagellum but was largely found in the cell body whereas the kinesin 9B motor was accumulating at the distal tip of the flagellum. In contrast, the distal tip protein FLAM8 was dispersed along the flagellum. This reveals that IFT also functions in maintaining the distribution of some flagellar proteins after construction of the organelle is completed.

The more we know, the more we have to discover: an exciting future for understanding cilia and ciliopathies.

The Cilia 2014 conference was organised by four European networks: the Ciliopathy Alliance, the Groupement de Recherche CIL, the Nordic Cilia and Centrosome Network and the EU FP7 programme SYSCILIA. More than 400 delegates from 27 countries gathered at the Institut Pasteur conference centre in Paris, including 30 patients and patient representatives. The meeting offered a unique opportunity for exchange between different scientific and medical communities. Major highlights included new discoveries about the roles of motile and immotile cilia during development and homeostasis, the mechanism of cilium construction, as well as progress in diagnosis and possible treatment of ciliopathies. The contributions to the cilia field of flagellated infectious eukaryotes and of systems biology were also presented.

A Protein Phosphatase 1 involved in correct nucleus positioning in trypanosomes.

Reversible protein phosphorylation is a key regulator in intracellular functions. In the African trypanosome, Trypanosoma brucei, the serine-threonine phosphatase PP1-3, is localised in the cytoplasm. RNAi mediated knockdown of PP1-3 leads to a coordinated rearrangement of cellular organelles and compartments in the procyclic trypanosome. These parasites display their nucleus at the very posterior end of the cell. The kinetoplast is very close to the nucleus, and often located in a more anterior position. The lysosomal compartment, which in a normal procyclic cell is situated between nucleus and kinetoplast, is now positioned towards the anterior end of the cell. The Flagellum Attachment Zone, essential for cytokinesis, is still constructed, allowing initiation of the cleavage furrow and cell division. These adaptations allow dividing cells to distribute their organelles among the daughter cells and to proliferate normally. PP1-3 is therefore essential in conserving the intracellular organisation of the procyclic trypanosome cell.

Probiotics for the control of parasites: an overview.

Probiotics are defined as live organisms, which confer benefits to the host. Their efficiency was demonstrated for the treatment of gastrointestinal disorders, respiratory infections, and allergic symptoms, but their use is mostly limited to bacterial and viral diseases. During the last decade, probiotics as means for the control of parasite infections were reported covering mainly intestinal diseases but also some nongut infections, that are all of human and veterinary importance. In most cases, evidence for a beneficial effect was obtained by studies using animal models. In a few cases, cellular interactions between probiotics and pathogens or relevant host cells were also investigated using in vitro culture systems. However, molecular mechanisms mediating the beneficial effects are as yet poorly understood. These studies indicate that probiotics might indeed provide a strain-specific protection against parasites, probably through multiple mechanisms. But more unravelling studies are needed to justify probiotic utilisation in therapeutics.

Flagellar kinesins in protists.

Cilia and flagella are organelles of the cell body present in many eukaryotic cells. Although their basic structure is well conserved from unicellular organisms to mammals, they show amazing diversity in number, structure, molecular composition, disposition and function. These complex organelles are generally assembled by the action of intraflagellar transport, which is powered by kinesin and dynein motor proteins. Several types of kinesins can function in flagella. They all have a well-conserved motor domain with characteristic signatures, but display exhaustive diversification of some domains. This diversity can be explained by the multitude of functions fulfilled by these proteins (transport of cargoes along microtubules, polymerization and depolymerization of microtubules). Functional and phylogenetic analyses reveal that at least seven kinesin families are involved in flagellum assembly and function. In protists, where cilia and flagella fulfill many essential roles, this diversity of function is also observed.

Evaluation of nucleoside hydrolase inhibitors for treatment of African trypanosomiasis.

In this paper, we present the biochemical and biological evaluation of N-arylmethyl-substituted iminoribitol derivatives as potential chemotherapeutic agents against trypanosomiasis. Previously, a library of 52 compounds was designed and synthesized as potent and selective inhibitors of Trypanosoma vivax inosine-adenosine-guanosine nucleoside hydrolase (IAG-NH). However, when the compounds were tested against bloodstream-form Trypanosoma brucei brucei, only one inhibitor, N-(9-deaza-adenin-9-yl)methyl-1,4-dideoxy-1,4-imino-d-ribitol (UAMC-00363), displayed significant activity (mean 50% inhibitory concentration [IC(50)] +/- standard error, 0.49 +/- 0.31 microM). Validation in an in vivo model of African trypanosomiasis showed promising results for this compound. Several experiments were performed to investigate why only UAMC-00363 showed antiparasitic activity. First, the compound library was screened against T. b. brucei IAG-NH and inosine-guanosine nucleoside hydrolase (IG-NH) to confirm the previously demonstrated inhibitory effects of the compounds on T. vivax IAG-NH. Second, to verify the uptake of these compounds by T. b. brucei, their affinities for the nucleoside P1 and nucleoside/nucleobase P2 transporters of T. b. brucei were tested. Only UAMC-00363 displayed significant affinity for the P2 transporter. It was also shown that UAMC-00363 is concentrated in the cell via at least one additional transporter, since P2 knockout mutants of T. b. brucei displayed no resistance to the compound. Consequently, no cross-resistance to the diamidine or the melaminophenyl arsenical classes of trypanocides is expected. Third, three enzymes of the purine salvage pathway of procyclic T. b. brucei (IAG-NH, IG-NH, and methylthioadenosine phosphorylase [MTAP]) were investigated using RNA interference. The findings from all these studies showed that it is probably not sufficient to target only the nucleoside hydrolase activity to block the purine salvage pathway of T. b. brucei and that, therefore, it is possible that UAMC-00363 acts on an additional target.

Kinesin 9 family members perform separate functions in the trypanosome flagellum.

Numerous eukaryote genome projects have uncovered a variety of kinesins of unknown function. The kinesin 9 family is limited to flagellated species. Our phylogenetic experiments revealed two subfamilies: KIF9A (including Chlamydomonas reinhardtii KLP1) and KIF9B (including human KIF6). The function of KIF9A and KIF9B was investigated in the protist Trypanosoma brucei that possesses a single motile flagellum. KIF9A and KIF9B are strongly associated with the cytoskeleton and are required for motility. KIF9A is localized exclusively in the axoneme, and its depletion leads to altered motility without visible structural modifications. KIF9B is found in both the axoneme and the basal body, and is essential for the assembly of the paraflagellar rod (PFR), a large extra-axonemal structure. In the absence of KIF9B, cells grow abnormal flagella with excessively large blocks of PFR-like material that alternate with regions where only the axoneme is present. The functional diversity of the kinesin 9 family illustrates the capacity for adaptation of organisms to suit specific cytoskeletal requirements.

Flagellum elongation is required for correct structure, orientation and function of the flagellar pocket in Trypanosoma brucei.

In trypanosomes, the flagellum is rooted in the flagellar pocket, a surface micro-domain that is the sole site for endocytosis and exocytosis. By analysis of anterograde or retrograde intraflagellar transport in IFT88RNAi or IFT140RNAi mutant cells, we show that elongation of the new flagellum is not required for flagellar pocket formation but is essential for its organisation, orientation and function. Transmission electron microscopy revealed that the flagellar pocket exhibited a modified shape (smaller, distorted and/or deeper) in cells with abnormally short or no flagella. Scanning electron microscopy analysis of intact and detergent-extracted cells demonstrated that the orientation of the flagellar pocket collar was more variable in trypanosomes with short flagella. The structural protein BILBO1 was present but its localisation and abundance was altered. The membrane flagellar pocket protein CRAM leaked out of the pocket and reached the short flagella. CRAM also accumulated in intracellular compartments, indicating defects in routing of resident flagellar pocket proteins. Perturbations of vesicular trafficking were obvious; vesicles were observed in the lumen of the flagellar pocket or in the short flagella, and fluid-phase endocytosis was drastically diminished in non-flagellated cells. We propose a model to explain the role of flagellum elongation in correct flagellar pocket organisation and function.

Intraflagellar transport and functional analysis of genes required for flagellum formation in trypanosomes.

Intraflagellar transport (IFT) is the bidirectional movement of protein complexes required for cilia and flagella formation. We investigated IFT by analyzing nine conventional IFT genes and five novel putative IFT genes (PIFT) in Trypanosoma brucei that maintain its existing flagellum while assembling a new flagellum. Immunostaining against IFT172 or expression of tagged IFT20 or green fluorescent protein GFP::IFT52 revealed the presence of IFT proteins along the axoneme and at the basal body and probasal body regions of both old and new flagella. IFT particles were detected by electron microscopy and exhibited a strict localization to axonemal microtubules 3-4 and 7-8, suggesting the existence of specific IFT tracks. Rapid (>3 microm/s) bidirectional intraflagellar movement of GFP::IFT52 was observed in old and new flagella. RNA interference silencing demonstrated that all individual IFT and PIFT genes are essential for new flagellum construction but the old flagellum remained present. Inhibition of IFTB proteins completely blocked axoneme construction. Absence of IFTA proteins (IFT122 and IFT140) led to formation of short flagella filled with IFT172, indicative of defects in retrograde transport. Two PIFT proteins turned out to be required for retrograde transport and three for anterograde transport. Finally, flagellum membrane elongation continues despite the absence of axonemal microtubules in all IFT/PIFT mutant.

A repetitive protein essential for the flagellum attachment zone filament structure and function in Trypanosoma brucei.

The flagellum is attached along the length of the cell body in the protozoan parasite Trypanosoma brucei and is a defining morphological feature of this parasite. The flagellum attachment zone (FAZ) is a complex structure and has been characterised morphologically as comprising a FAZ filament structure and the specialised microtubule quartet (MtQ) plus the specialised areas of flagellum: plasma membrane attachment. Unfortunately, we have no information as to the molecular identity of the FAZ filament components. Here, by screening an expression library with the monoclonal antibody L3B2 which identifies the FAZ filament we identify a novel repeat containing protein FAZ1. It is kinetoplastid-specific and provides the first molecular component of the FAZ filament. Knockdown of FAZ1 by RNA interference (RNAi) results in the assembly of a compromised FAZ and defects in flagellum attachment and cytokinesis in procyclic trypanosomes. The complexity of FAZ structure and assembly is revealed by the use of other monoclonal antibody markers illustrating that FAZ1 is only one protein of a complex structure. The cytokinesis defects provide further evidence for the role of an attached flagellum in cellular morphogenesis in these trypanosomes.

Basal body positioning is controlled by flagellum formation in Trypanosoma brucei.

To perform their multiple functions, cilia and flagella are precisely positioned at the cell surface by mechanisms that remain poorly understood. The protist Trypanosoma brucei possesses a single flagellum that adheres to the cell body where a specific cytoskeletal structure is localised, the flagellum attachment zone (FAZ). Trypanosomes build a new flagellum whose distal tip is connected to the side of the old flagellum by a discrete structure, the flagella connector. During this process, the basal body of the new flagellum migrates towards the posterior end of the cell. We show that separate inhibition of flagellum assembly, base-to-tip motility or flagella connection leads to reduced basal body migration, demonstrating that the flagellum contributes to its own positioning. We propose a model where pressure applied by movements of the growing new flagellum on the flagella connector leads to a reacting force that in turn contributes to migration of the basal body at the proximal end of the flagellum.

Conserved and specific functions of axoneme components in trypanosome motility.

The Trypanosoma brucei flagellum is unusual as it is attached along the cell body and contains, in addition to an apparently conventional axoneme, a structure called the paraflagellar rod, which is essential for cell motility. Here, we investigated flagellum behaviour in normal and mutant trypanosome cell lines where expression of genes encoding various axoneme proteins (PF16, PF20, DNAI1, LC2) had been silenced by RNAi. First, we show that the propulsive wave (normally used for forward motility) is abolished in the absence of outer dynein arms, whereas the reverse wave (normally used for changing direction) still occurs. Second, in contrast to Chlamydomonas--but like metazoa, the central pair adopts a fixed orientation during flagellum beating. This orientation becomes highly variable in central-pair- and outer-dynein-arm-mutants. Third, the paraflagellar rod contributes to motility by facilitating three-dimensional wave propagation and controlling cell shape. Fourth, motility is required to complete the last stage of cell division in both insect and bloodstream stages of the parasite. Finally, our study also reveals the conservation of molecular components of the trypanosome flagellum. Coupled to the ease of reverse genetics, it raises the interest of trypanosomes as model organisms to study cilia and flagella.

The flagellum of trypanosomes.

Eukaryotic cilia and flagella are cytoskeletal organelles that are remarkably conserved from protists to mammals. Their basic unit is the axoneme, a well-defined cylindrical structure composed of microtubules and up to 250 associated proteins. These complex organelles are assembled by a dynamic process called intraflagellar transport. Flagella and cilia perform diverse motility and sensitivity functions in many different organisms. Trypanosomes are flagellated protozoa, responsible for various tropical diseases such as sleeping sickness and Chagas disease. In this review, we first describe general knowledge on the flagellum: its occurrence in the living world, its molecular composition, and its mode of assembly, with special emphasis on the exciting developments that followed the discovery of intraflagellar transport. We then present recent progress regarding the characteristics of the trypanosome flagellum, highlighting the original contributions brought by this organism. The most striking phenomenon is the involvement of the flagellum in several aspects of the trypanosome cell cycle, including cell morphogenesis, basal body migration, and cytokinesis.

Novel roles for the flagellum in cell morphogenesis and cytokinesis of trypanosomes.

Flagella and cilia are elaborate cytoskeletal structures conserved from protists to mammals, where they fulfil functions related to motility or sensitivity. Here we demonstrate novel roles for the flagellum in the control of cell size, shape, polarity and division of the protozoan Trypanosoma brucei. To investigate the function of the flagellum, its formation was perturbed by inducible RNA interference silencing of com ponents required for intraflagellar transport, a dynamic process necessary for flagellum assembly. First, we show that down-regulation of intraflagellar transport leads to assembly of a shorter flagellum. Strikingly, cells with a shorter flagellum are smaller, with a direct correlation between flagellum length and cell size. Detailed morphogenetic analysis reveals that the tip of the new flagellum defines the point where cytokinesis is initiated. Secondly, when new flagellum formation is completely blocked, non-flagellated cells are very short, lose their normal shape and polarity, and fail to undergo cytokinesis. We show that flagellum elongation controls formation of cytoskeletal structures (present in the cell body) that act as molecular organizers of the cell.