Flagellum Attachment Research Articles

A putative cAMP-binding protein in Trypanosoma brucei cooperates with FLAM3 to promote flagellar connection and cell morphogenesis

Trypanosoma brucei is a flagellated parasitic protozoan, and within the insect vector the parasite transitions from the trypomastigote form to the epimastigote form by re-positioning its mitochondrial genome and re-locating the flagellum. The mechanisms underlying such morphology changes are still poorly understood, but several flagellum-localized proteins are involved in this process by modulating the flagellum attachment zone (FAZ) that adheres the flagellum to the cell membrane. We report here a putative cAMP-binding protein named cAMP-BP1, which promotes flagellar connection and morphology transition. cAMP-BP1 contains two cyclic nucleotide-binding domains and five calcium-binding C2 domains and localizes to the flagella connector and the new FAZ tip. Depletion of cAMP-BP1 in the trypomastigote form of T. brucei causes major morphology changes, generating epimastigote-like cells with re-positioned kinetoplast and re-located flagellum. At the flagella connector and the new FAZ tip, cAMP-BP1 associates with FLAM3, a regulator of morphology transition, depends on the latter for localization, and is required for FLAM3 localization to the flagella connector. Knockdown of cAMP-BP1 inhibits FAZ elongation and disrupts flagellar connection by impairing flagella connector structural integrity. These results identify a flagella connector- and new FAZ tip-localized protein as a regulator of morphology transition and flagellar connection in trypanosomes and uncover its functional interplay with FLAM3 to promote FAZ elongation for maintaining trypomastigote morphology.

Disruption of Leishmania flagellum attachment zone architecture causes flagellum loss.

Leishmania are flagellated eukaryotic parasites that cause leishmaniasis and are closely related to the other kinetoplastid parasites such as Trypanosoma brucei. In all these parasites there is a cell membrane invagination at the base of the flagellum called the flagellar pocket, which is tightly associated with and sculpted by cytoskeletal structures including the flagellum attachment zone (FAZ). The FAZ is a complex interconnected structure linking the flagellum to the cell body and has critical roles in cell morphogenesis, function and pathogenicity. However, this structure varies dramatically in size and organisation between these different parasites, suggesting changes in protein localisation and function. Here, we screened the localisation and function of the Leishmania orthologues of T. brucei FAZ proteins identified in the genome-wide protein tagging project TrypTag. We identified 27 FAZ proteins and our deletion analysis showed that deletion of two FAZ proteins in the flagellum, FAZ27 and FAZ34 resulted in a reduction in cell body size, and flagellum loss in some cells. Furthermore, after null mutant generation, we observed distinct and reproducible changes to cell shape, demonstrating the ability of the parasite to adapt to morphological perturbations resulting from gene deletion. This process of adaptation has important implications for the study of Leishmania mutants.

Implications of Flagellar Attachment Zone Proteins TcGP72 and TcFLA-1BP in Morphology, Proliferation, and Intracellular Dynamics in Trypanosoma cruzi.

The highly adaptable parasite Trypanosoma cruzi undergoes complex developmental stages to exploit host organisms effectively. Each stage involves the expression of specific proteins and precise intracellular structural organization. These morphological changes depend on key structures that control intracellular components' growth and redistribution. In trypanosomatids, the flagellar attachment zone (FAZ) connects the flagellum to the cell body and plays a pivotal role in cell expansion and structural rearrangement. While FAZ proteins are well-studied in other trypanosomatids, there is limited knowledge about specific components, organization, and function in T. cruzi. This study employed the CRISPR/Cas9 system to label endogenous genes and conduct deletions to characterize FAZ-specific proteins during epimastigote cell division and metacyclogenesis. In T. cruzi, these proteins exhibited distinct organization compared to their counterparts in T. brucei. TcGP72 is anchored to the flagellar membrane, while TcFLA-1BP is anchored to the membrane lining the cell body. We identified unique features in the organization and function of the FAZ in T. cruzi compared to other trypanosomatids. Deleting these proteins had varying effects on intracellular structures, cytokinesis, and metacyclogenesis. This study reveals specific variations that directly impact the success of cell division and differentiation of this parasite.

The microtubule quartet protein SNAP1 in Trypanosoma brucei facilitates flagellum and cell division plane positioning by promoting basal body segregation

The unicellular protozoan Trypanosoma brucei has a single flagellum that is involved in cell motility, cell morphogenesis, and cell division. Inheritance of the newly assembled flagellum during the cell cycle requires its correct positioning, which depends on the faithful duplication or segregation of multiple flagellum-associated cytoskeletal structures, including the basal body, the flagellum attachment zone, and the hook complex. Along the flagellum attachment zone sites a set of four microtubules termed the microtubule quartet (MtQ), whose molecular function remains enigmatic. We recently reported that the MtQ-localized protein NHL1 interacts with the microtubule-binding protein TbSpef1 and regulates flagellum inheritance by promoting basal body rotation and segregation. Here, we identified a TbSpef1- and NHL1-associated protein named SNAP1, which co-localizes with NHL1 and TbSpef1 at the proximal portion of the MtQ, depends on TbSpef1 for localization and is required for NHL1 localization to the MtQ. Knockdown of SNAP1 impairs the rotation and segregation of the basal body, the elongation of the flagellum attachment zone filament, and the positioning of the newly assembled flagellum, thereby causing mis-placement of the cell division plane, a halt in cleavage furrow ingression, and an inhibition of cytokinesis completion. Together, these findings uncover a coordinating role of SNAP1 with TbSpef1 and NHL1 in facilitating flagellum positioning and cell division plane placement for the completion of cytokinesis.

FAZ assembly in bloodstream form Trypanosoma brucei requires kinesin KIN-E.

Trypanosoma brucei, the causative agent of African sleeping sickness, uses its flagellum for movement, cell division, and signaling. The flagellum is anchored to the cell body membrane via the flagellum attachment zone (FAZ), a complex of proteins, filaments, and microtubules that spans two membranes with elements on both flagellum and cell body sides. How FAZ components are carried into place to form this complex is poorly understood. Here, we show that the trypanosome-specific kinesin KIN-E is required for building the FAZ in bloodstream-form parasites. KIN-E is localized along the flagellum with a concentration at its distal tip. Depletion of KIN-E by RNAi rapidly inhibits flagellum attachment and leads to cell death. A detailed analysis reveals that KIN-E depletion phenotypes include failure in cytokinesis completion, kinetoplast DNA missegregation, and transport vesicle accumulation. Together with previously published results in procyclic form parasites, these data suggest KIN-E plays a critical role in FAZ assembly in T. brucei.

Abstract 15752: Differential IgG Repertoire in Individuals With Chagas Cardiomyopathy

Chagas cardiomyopathy (CC) is caused by the infection of Trypanosoma cruzi ( T. cruzi ) and represents a major etiology for heart failure in Latin America. Trying fill gaps related to humoral response in CC, we used a PhIP-Seq technology to describe differential IgG profile between individuals with CC and individuals infected by T. cruzi , but without symptoms (Indeterminate - IND). We built a library comprising all T. cruzi proteome (12766 proteins) and incubated against 1293 serum samples: 396 T. cruzi seronegative samples (98 controls and 298 individuals with cardiomyopathy caused by other causes than Chagas disease) and 897 T. cruzi seropositive samples (684 CC and 213 IND). 1870 peptides were increased in T. cruzi seropositive IgG compared to T. cruzi seronegative samples. Peptides from Nucleoporin NUP149, a flagellar attachment zone protein, basal body protein, surface protein TolT and cytoskeleton associated protein were the peptides with highest IgG response. Using as little as 5 peptides, I derived a classifier model for T. cruzi infection with 97% accuracy. Then, I compared CC and IND samples using logistic regression models after splitting our sample into 2 cohort groups. From these, 316 peptides were significantly different between CC and IND: 289 peptides were increased in CC (27% of them belong to the trans-sialidase class) and 27 peptides were increased in IND (FDR>0.01). Aiming to develop a classifier to separate CCC from IND, I analyzed our data using machine learning approaches. LASSO returned the best model using 48 peptides, with 70% of accuracy, 74% of sensitivity and 69% of specificity. The peptides selected for the model belong to 38 proteins, 21% being unknow function proteins, 18% trans-sialidases, 8% flagellar associated proteins, and 60S ribosomal protein, Nucleoporins and R27-2 proteins classes representing 5% each. Ninety (90%) of selected peptides in LASSO were identified in the previous results using the logistic regression model. I showed for the first time that Chagas Cardiomyopathy individuals have a differential IgG profile against T. cruzi proteins compared to IND individuals. I described a differential IgG response against 84 sequences (62 increased in CCC and 22 increased in IND) that can be related to disease development.

Light chain 2 is a Tctex-type related axonemal dynein light chain that regulates directional ciliary motility in Trypanosoma brucei.

Flagellar motility is essential for the cell morphology, viability, and virulence of pathogenic kinetoplastids. Trypanosoma brucei flagella beat with a bending wave that propagates from the flagellum's tip to its base, rather than base-to-tip as in other eukaryotes. Thousands of dynein motor proteins coordinate their activity to drive ciliary bending wave propagation. Dynein-associated light and intermediate chains regulate the biophysical mechanisms of axonemal dynein. Tctex-type outer arm dynein light chain 2 (LC2) regulates flagellar bending wave propagation direction, amplitude, and frequency in Chlamydomonas reinhardtii. However, the role of Tctex-type light chains in regulating T. brucei motility is unknown. Here, we used a combination of bioinformatics, in-situ molecular tagging, and immunofluorescence microscopy to identify a Tctex-type light chain in the procyclic form of T. brucei (TbLC2). We knocked down TbLC2 expression using RNAi in both wild-type and FLAM3, a flagellar attachment zone protein, knockdown cells and quantified TbLC2's effects on trypanosome cell biology and biophysics. We found that TbLC2 knockdown reduced the directional persistence of trypanosome cell swimming, induced an asymmetric ciliary bending waveform, modulated the bias between the base-to-tip and tip-to-base beating modes, and increased the beating frequency. Together, our findings are consistent with a model of TbLC2 as a down-regulator of axonemal dynein activity that stabilizes the forward tip-to-base beating ciliary waveform characteristic of trypanosome cells. Our work sheds light on axonemal dynein regulation mechanisms that contribute to pathogenic kinetoplastids' unique tip-to-base ciliary beating nature and how those mechanisms underlie dynein-driven ciliary motility more generally.

Use of recombinant calflagin protein as a potential candidate for diagnosis of Trypanosoma evansi infection

Serodiagnosis of surra, caused by Trypanosoma evansi, is still based on native antigens purified from bloodstream form of T. evansi grown in rodents. In order to investigate prospective diagnostic possibilities as an alternative for native antigens, we cloned, expressed 26 kDa calflagin protein containing 218 amino acids from T. evansi (Indian Strain) in Escherichia coli. The potential of recombinant calflagin (rCLF) protein as diagnostic antigen was evaluated in immunoblot and indirect ELISA using experimentally infected equine serum samples from 0 to 84 days post infection. The antibodies against T. evansi were detected with rCLF antigen in serum samples of experimentally infected equines as early as 10 days and 14 days post infection, using immunoblot and ELISA respectively. No cross-reactivity was observed with rCLF antigen in ELISA with different serum samples of equines positive for Equine herpesvirus 1, Burkholderia mallei, and Theileria equi infections. Several immunoreactive regions ranging from 10 to 28 kDa were detected using distinct T. evansi isolates (pony, cattle, donkey and camel origin) indicating presence of multiple calflagin family members in a single trypanosome. Indirect immunofluorescence antibody test with anti-CLF rabbit hyperimmune serum showed localisation of native immunogenic protein near attachment of flagellum. The rCLF protein was found to be a potential diagnostic candidate for distinguishing T. evansi positive and negative equine serum sample, suggesting that it could be used for serological surveys in animals for surra. In addition, it could be used with other potential diagnostic candidates to improve the diagnostic efficiency.

Identification of Prazosin as a Potential Flagellum Attachment Zone 1(FAZ1) Inhibitor for the Treatment of Human African Trypanosomiasis.

Human African trypanosomiasis (HAT) remains a health threat to sub-Saharan Africa. The current treatments suffer from drug resistance and life-threatening side effects, making drug discovery for HAT still important. A high-throughput screening of the library of pharmaceutically active compounds identified prazosin, an α-adrenoceptor antagonist, that showed selective activity toward Trypanosoma brucei brucei. Furthermore, a series of prazosin analogues were examined, and overall, the new analogues had improved activity and selectivity. To elucidate the binding partner, a biotin-conjugated probe was synthesized, and a protein pulldown assay combined with a proteomic analysis identified the flagellum attachment zone 1 (FAZ1) filament as an interacting partner. Additionally, prazosin treatment resulted in dysfunction of the flagellum of trypanosome cells, which is indicative of a FAZ1 irregularity. We also examined the drug distribution by utilizing immunofluorescence with a designed fluorescent analogue that showed partial colocalization with FAZ1. With the activity of the prazosin analogues, a structure-activity relationship (SAR) was summarized for future lead optimization. Our findings provide a new group of FAZ1 inhibitors as novel antitrypanosomal agents.

A Spef1-interacting microtubule quartet protein in Trypanosoma brucei promotes flagellar inheritance by regulating basal body segregation

The human parasite Trypanosoma brucei contains a motile flagellum that determines the plane of cell division, controls cell morphology, and mediates cell–cell communication. During the cell cycle, inheritance of the newly formed flagellum requires its correct positioning toward the posterior of the cell, which depends on the faithful segregation of multiple flagellum-associated cytoskeletal structures including the basal body, the flagellar pocket collar, the flagellum attachment zone, and the hook complex. A specialized group of four microtubules termed the microtubule quartet (MtQ) originates from the basal body and runs through the flagellar pocket collar and the hook complex to extend, along the flagellum attachment zone, toward the anterior of the cell. However, the physiological function of the MtQ is poorly understood, and few MtQ-associated proteins have been identified and functionally characterized. We report here that an MtQ-localized protein named NHL1 interacts with the microtubule-binding protein TbSpef1 and depends on TbSpef1 for its localization to the MtQ. We show that RNAi-mediated knockdown of NHL1 impairs the segregation of flagellum-associated cytoskeletal structures, resulting in mispositioning of the new flagellum. Furthermore, knockdown of NHL1 also causes misplacement of the cell division plane in dividing trypanosome cells, halts cleavage furrow ingression, and inhibits completion of cytokinesis. These findings uncover a crucial role for the MtQ-associated protein NHL1 in regulating basal body segregation to promote flagellar inheritance in T. brucei.

Candidates for Balancing Selection in Leishmania donovani Complex Parasites.

The Leishmania donovani species complex is the causative agent of visceral leishmaniasis, which cause 20–40,000 fatalities a year. Here, we conduct a screen for balancing selection in this species complex. We used 384 publicly available L. donovani and L. infantum genomes, and sequence 93 isolates of L. infantum from Brazil to describe the global diversity of this species complex. We identify five genetically distinct populations that are sufficiently represented by genomic data to search for signatures of selection. We find that signals of balancing selection are generally not shared between populations, consistent with transient adaptive events, rather than long-term balancing selection. We then apply multiple diversity metrics to identify candidate genes with robust signatures of balancing selection, identifying a curated set of 24 genes with robust signatures. These include zeta toxin, nodulin-like, and flagellum attachment proteins. This study highlights the extent of genetic divergence between L. donovani complex parasites and provides genes for further study.

The kinesin of the flagellum attachment zone in Leishmania is required for cell morphogenesis, cell division and virulence in the mammalian host

Leishmania parasites possess a unique and complex cytoskeletal structure termed flagellum attachment zone (FAZ) connecting the base of the flagellum to one side of the flagellar pocket (FP), an invagination of the cell body membrane and the sole site for endocytosis and exocytosis. This structure is involved in FP architecture and cell morphogenesis, but its precise role and molecular composition remain enigmatic. Here, we characterized Leishmania FAZ7, the only known FAZ protein containing a kinesin motor domain, and part of a clade of trypanosomatid-specific kinesins with unknown functions. The two paralogs of FAZ7, FAZ7A and FAZ7B, display different localizations and functions. FAZ7A localizes at the basal body, while FAZ7B localizes at the distal part of the FP, where the FAZ structure is present in Leishmania. While null mutants of FAZ7A displayed normal growth rates, the deletion of FAZ7B impaired cell growth in both promastigotes and amastigotes of Leishmania. The kinesin activity is crucial for its function. Deletion of FAZ7B resulted in altered cell division, cell morphogenesis (including flagellum length), and FP structure and function. Furthermore, knocking out FAZ7B induced a mis-localization of two of the FAZ proteins, and disrupted the molecular organization of the FP collar, affecting the localization of its components. Loss of the kinesin FAZ7B has important consequences in the insect vector and mammalian host by reducing proliferation in the sand fly and pathogenicity in mice. Our findings reveal the pivotal role of the only FAZ kinesin as part of the factors important for a successful life cycle of Leishmania.

Role for the flagellum attachment zone in Leishmania anterior cell tip morphogenesis.

The shape and form of the flagellated eukaryotic parasite Leishmania is sculpted to its ecological niches and needs to be transmitted to each generation with great fidelity. The shape of the Leishmania cell is defined by the sub-pellicular microtubule array and the positioning of the nucleus, kinetoplast and the flagellum within this array. The flagellum emerges from the anterior end of the cell body through an invagination of the cell body membrane called the flagellar pocket. Within the flagellar pocket the flagellum is laterally attached to the side of the flagellar pocket by a cytoskeletal structure called the flagellum attachment zone (FAZ). During the cell cycle single copy organelles duplicate with a new flagellum assembling alongside the old flagellum. These are then segregated between the two daughter cells by cytokinesis, which initiates at the anterior cell tip. Here, we have investigated the role of the FAZ in the morphogenesis of the anterior cell tip. We have deleted the FAZ filament protein, FAZ2 and investigated its function using light and electron microscopy and infection studies. The loss of FAZ2 caused a disruption to the membrane organisation at the anterior cell tip, resulting in cells that were connected to each other by a membranous bridge structure between their flagella. Moreover, the FAZ2 null mutant was unable to develop and proliferate in sand flies and had a reduced parasite burden in mice. Our study provides a deeper understanding of membrane-cytoskeletal interactions that define the shape and form of an individual cell and the remodelling of that form during cell division.

Maintenance of hook complex integrity and centrin arm assembly facilitates flagellum inheritance in Trypanosoma brucei

Inheritance of the newly assembled flagellum in the human parasite Trypanosoma brucei depends on the faithful duplication and segregation of multiple flagellum-associated cytoskeletal structures, including the hook complex and its associated centrin arm. The biological functions of this unique hook complex-centrin arm assembly remain poorly understood. Here, we report a hook complex-associated protein named BOH2 that plays an essential role in promoting flagellum inheritance. BOH2 localizes to the hooked part of the hook complex by bridging the hook complex, the centrin arm, and the flagellum attachment zone filament. Depletion of BOH2 caused the loss of the shank part of the hook complex and its associated protein TbSmee1, disrupted the assembly of the centrin arm and the recruitment of centrin arm-associated protein CAAP1, inhibited the assembly of the flagellum attachment zone, and caused flagellum mispositioning and detachment. These results demonstrate crucial roles of BOH2 in maintaining hook complex integrity and promoting centrin arm formation and suggest that proper assembly of the hook complex-centrin arm structure facilitates flagellum inheritance.

FAZ27 cooperates with FLAM3 and ClpGM6 to maintain cell morphology in Trypanosoma brucei.

The human parasite Trypanosoma brucei transitions from the trypomastigote form to the epimastigote form in the insect vector by repositioning its mitochondrial genome and flagellum-associated cytoskeleton. The molecular mechanisms underlying such changes in cell morphology remain elusive, but recent works demonstrated the involvement of three flagellar proteins, FLAM3, ClpGM6 and KIN-E, in this process by controlling the elongation of the flagellum attachment zone (FAZ). In this report, we identified a FAZ flagellum domain-localizing protein named FAZ27 and characterized its role in cell morphogenesis. Depletion of FAZ27 in the trypomastigote form caused major morphological changes and repositioning of the mitochondrial genome and flagellum-associated cytoskeleton, generating epimastigote-like cells. Furthermore, proximity biotinylation and co-immunoprecipitation identified FLAM3 and ClpGM6 as FAZ27-interacting proteins, and analyses of their functional interplay revealed an interdependency for assembly into the FAZ flagellum domain. Finally, we showed that assembly of FAZ27 occurred proximally, identical to the assembly pattern of other FAZ sub-domain proteins. Taken together, these results demonstrate a crucial role for the FAZ flagellum domain in controlling cell morphogenesis and suggest a coordinated assembly of all the FAZ sub-domains at the proximal end of the FAZ.

In situ structural analysis of the flagellum attachment zone in Trypanosoma brucei using cryo-scanning transmission electron tomography

The flagellum of Trypanosoma brucei is a 20 µm-long organelle responsible for locomotion and cell morphogenesis. The flagellum attachment zone (FAZ) is a multi-protein complex whose function is to attach the flagellum to the cell body but also to guide cytokinesis. Cryo-transmission electron microscopy is a tool of choice to access the structure of the FAZ in a close-to-native state. However, because of the large dimension of the cell body, the whole FAZ cannot be structurally studied in situ at the nanometre scale in 3D using classical transmission electron microscopy approaches. In the present work, cryo-scanning transmission electron tomography, a new method capable of investigating cryo-fixed thick biological samples, has been used to study whole T. brucei cells at the bloodstream stage. The method has been used to visualise and characterise the structure and organisation of the FAZ filament. It is composed of an array of cytoplasmic stick-like structures. These sticks are heterogeneously distributed between the posterior part and the anterior tip of the cell. This cryo-STET investigation provides new insights into the structure of the FAZ filament. In combination with protein structure predictions, this work proposes a new model for the elongation of the FAZ.

Trypanosomes have divergent kinesin-2 proteins that function differentially in flagellum biosynthesis and cell viability.

Trypanosoma brucei, the causative agent of African sleeping sickness, has a flagellum that is crucial for motility, pathogenicity, and viability. In most eukaryotes, the intraflagellar transport (IFT) machinery drives flagellum biogenesis, and anterograde IFT requires kinesin-2 motor proteins. In this study, we investigated the function of the two T. brucei kinesin-2 proteins, TbKin2a and TbKin2b, in bloodstream form trypanosomes. We found that, compared to kinesin-2 proteins across other phyla, TbKin2a and TbKin2b show greater variation in neck, stalk and tail domain sequences. Both kinesins contributed additively to flagellar lengthening. Silencing TbKin2a inhibited cell proliferation, cytokinesis and motility, whereas silencing TbKin2b did not. TbKin2a was localized on the flagellum and colocalized with IFT components near the basal body, consistent with it performing a role in IFT. TbKin2a was also detected on the flagellar attachment zone, a specialized structure that connects the flagellum to the cell body. Our results indicate that kinesin-2 proteins in trypanosomes play conserved roles in flagellar biosynthesis and exhibit a specialized localization, emphasizing the evolutionary flexibility of motor protein function in an organism with a large complement of kinesins.

Modulation of flagellar rotation in surface-attached bacteria: A pathway for rapid surface-sensing after flagellar attachment.

Attachment is a necessary first step in bacterial commitment to surface-associated behaviors that include colonization, biofilm formation, and host-directed virulence. The Gram-negative opportunistic pathogen Pseudomonas aeruginosa can initially attach to surfaces via its single polar flagellum. Although many bacteria quickly detach, some become irreversibly attached and express surface-associated structures, such as Type IV pili, and behaviors, including twitching motility and biofilm initiation. P. aeruginosa that lack the GTPase FlhF assemble a randomly placed flagellum that is motile; however, we observed that these mutant bacteria show defects in biofilm formation comparable to those seen for non-motile, aflagellate bacteria. This phenotype was associated with altered behavior of ΔflhF bacteria immediately following surface-attachment. Forward and reverse genetic screens led to the discovery that FlhF interacts with FimV to control flagellar rotation at a surface, and implicated cAMP signaling in this pathway. Although cAMP controls many transcriptional programs in P. aeruginosa, known targets of this second messenger were not required to modulate flagellar rotation in surface-attached bacteria. Instead, alterations in switching behavior of the motor appeared to result from direct or indirect effects of cAMP on switch complex proteins and/or the stators associated with them.

The trypanosome-specific proteins FPRC and CIF4 regulate cytokinesis initiation by recruiting CIF1 to the cytokinesis initiation site

The evolutionarily early divergent human parasite Trypanosoma brucei proliferates through binary cell fission in both its tsetse fly vector and mammalian host. The parasite divides unidirectionally along the longitudinal cell axis from the anterior cell tip toward the posterior cell tip through a mechanism distinct from that in the cells of its human host. Initiation of cytokinesis in T. brucei is regulated by two evolutionarily conserved protein kinases, the Polo-like kinase TbPLK and the Aurora B kinase TbAUK1, and a cohort of trypanosome-specific proteins, including the three cytokinesis initiation factors CIF1, CIF2, and CIF3. Here, using RNAi, in situ epitope tagging of proteins, GST pulldown, and coimmunoprecipitation assays, and immunofluorescence and scanning electron microscopy analyses, we report the identification and functional characterization of two trypanosome-specific proteins, flagellum attachment zone tip-localizing protein required for cytokinesis (FPRC) and CIF4. We found that the two proteins colocalize to the distal tips of the new and the old flagellum attachment zones and are required for cytokinesis initiation. Knockdown of FPRC or CIF4 disrupted the localization of CIF1, suggesting that they function upstream of CIF1. Moreover, depletion of CIF4 abolished FPRC localization, indicating that CIF4 acts upstream of FPRC. Together, these results identify two new cytokinesis regulators in T. brucei and integrate them into the CIF1-mediated cytokinesis regulatory pathway. These findings highlight the existence of a cytokinesis pathway in T. brucei that is different from that of its mammalian host and therefore suggest that cytokinesis in T. brucei could potentially be exploited as a new drug target.

Flagellum Removal by a Nectar Metabolite Inhibits Infectivity of a Bumblebee Parasite.

Plant phytochemicals can act as natural "medicines" for animals against parasites [1-3]. Some nectar metabolites, for example, reduce parasite infections in bees [4-7]. Declining plant diversity through anthropogenic landscape change [8-11] could reduce the availability of medicinal nectar plants for pollinators, exacerbating their decline [12]. Existing studies are, however, limited by (1) a lack of mechanistic insights into how phytochemicals affect pollinator diseases and (2) the restriction to few, commercially available chemicals, thereby potentially neglecting plants with the biggest antiparasitic effects. To rapidly identify plants with the greatest potential as natural bee medicines, we developed a bioactivity-directed fractionation assay for nectar metabolites. We evaluated 17 important nectar plants against the bumblebee pathogen Crithidia bombi (Trypanosomatidae) [13-17]. The most bioactive species washeather (Calluna vulgaris), the second most productive UK nectar plant [10]. We identified 4-(3-oxobut-1-enylidene)-3,5,5-trimethylcyclohex-2-en-1-one (callunene) from heather nectar as a potent inhibitor of C.bombi. Wild bumblebees (Bombus terrestris) foraging on heather ingest callunene at concentrations causing complete C.bombi inhibition. Feeding on callunene was prophylactic against infections. We show that C.bombi establishes infections by flagellar anchoring to the ileum epithelium. Short-term callunene exposure induced flagellum loss in C.bombi choanomastigotes, resulting in a loss of infectivity. We conclude that plant secondary metabolites can disrupt parasite flagellum attachment, revealing a mechanism behind their prophylactic effects. The decline of heathlands [18-21] reduces the availability of natural bee "medicine" and could exacerbate the contribution of diseases to pollinator declines. VIDEO ABSTRACT.