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1.  "With a Little Help from My Friends"—Social Motility in Trypanosoma brucei 
PLoS Pathogens  2015;11(12):e1005272.
PMCID: PMC4683075  PMID: 26679190
2.  Insect Stage-Specific Adenylate Cyclases Regulate Social Motility in African Trypanosomes 
Eukaryotic Cell  2014;14(1):104-112.
Sophisticated systems for cell-cell communication enable unicellular microbes to act as multicellular entities capable of group-level behaviors that are not evident in individuals. These group behaviors influence microbe physiology, and the underlying signaling pathways are considered potential drug targets in microbial pathogens. Trypanosoma brucei is a protozoan parasite that causes substantial human suffering and economic hardship in some of the most impoverished regions of the world. T. brucei lives on host tissue surfaces during transmission through its tsetse fly vector, and cultivation on surfaces causes the parasites to assemble into multicellular communities in which individual cells coordinate their movements in response to external signals. This behavior is termed “social motility,” based on its similarities with surface-induced social motility in bacteria, and it demonstrates that trypanosomes are capable of group-level behavior. Mechanisms governing T. brucei social motility are unknown. Here we report that a subset of receptor-type adenylate cyclases (ACs) in the trypanosome flagellum regulate social motility. RNA interference-mediated knockdown of adenylate cyclase 6 (AC6), or dual knockdown of AC1 and AC2, causes a hypersocial phenotype but has no discernible effect on individual cells in suspension culture. Mutation of the AC6 catalytic domain phenocopies AC6 knockdown, demonstrating that loss of adenylate cyclase activity is responsible for the phenotype. Notably, knockdown of other ACs did not affect social motility, indicating segregation of AC functions. These studies reveal interesting parallels in systems that control social behavior in trypanosomes and bacteria and provide insight into a feature of parasite biology that may be exploited for novel intervention strategies.
PMCID: PMC4279026  PMID: 25416239
3.  Mouse infection and pathogenesis by Trypanosoma brucei motility mutants 
Cellular microbiology  2014;16(6):912-924.
The flagellum of Trypanosoma brucei is an essential and multifunctional organelle that drives parasite motility and is receiving increased attention as a potential drug target. In the mammalian host, parasite motility is suspected to contribute to infection and disease pathogenesis. However, it has not been possible to test this hypothesis owing to lack of motility mutants that are viable in the bloodstream life cycle stage that infects the mammalian host. We recently identified a bloodstream-form motility mutant in 427-derived T. brucei in which point mutations in the LC1 dynein subunit disrupt propulsive motility but do not affect viability. These mutants have an actively beating flagellum, but cannot translocate. Here we demonstrate that the LC1 point mutant fails to show enhanced cell motility upon increasing viscosity of the surrounding medium, which is a hallmark of wild type T. brucei, thus indicating that motility of the mutant is fundamentally altered compared to wild type cells. We next used the LC1 point mutant to assess the influence of trypanosome motility on infection in mice. We surprisingly found that disrupting parasite motility has no discernible effect on T. brucei bloodstream infection. Infection time-course, maximum parasitemia, number of waves of parasitemia, clinical features and disease outcome are indistinguishable between motility mutant and control parasites. Our studies provide an important step toward understanding the contribution of parasite motility to infection and a foundation for future investigations of T. brucei interaction with the mammalian host.
PMCID: PMC4264834  PMID: 24286532
Trypanosome; flagellum; dynein; motility; pathogenesis
4.  Cyclic AMP Regulates Social Behavior in African Trypanosomes 
mBio  2015;6(3):e01954-14.
The protozoan parasite Trypanosoma brucei engages in surface-induced social behavior, termed social motility, characterized by single cells assembling into multicellular groups that coordinate their movements in response to extracellular signals. Social motility requires sensing and responding to extracellular signals, but the underlying mechanisms are unknown. Here we report that T. brucei social motility depends on cyclic AMP (cAMP) signaling systems in the parasite’s flagellum (synonymous with cilium). Pharmacological inhibition of cAMP-specific phosphodiesterase (PDE) completely blocks social motility without impacting the viability or motility of individual cells. Using a fluorescence resonance energy transfer (FRET)-based sensor to monitor cAMP dynamics in live cells, we demonstrate that this block in social motility correlates with an increase in intracellular cAMP levels. RNA interference (RNAi) knockdown of the flagellar PDEB1 phenocopies pharmacological PDE inhibition, demonstrating that PDEB1 is required for social motility. Using parasites expressing distinct fluorescent proteins to monitor individuals in a genetically heterogeneous community, we found that the social motility defect of PDEB1 knockdowns is complemented by wild-type parasites in trans. Therefore, PDEB1 knockdown cells are competent for social motility but appear to lack a necessary factor that can be provided by wild-type cells. The combined data demonstrate that the role of cyclic nucleotides in regulating microbial social behavior extends to African trypanosomes and provide an example of transcomplementation in parasitic protozoa.
In bacteria, studies of cell-cell communication and social behavior have profoundly influenced our understanding of microbial physiology, signaling, and pathogenesis. In contrast, mechanisms underlying social behavior in protozoan parasites are mostly unknown. Here we show that social behavior in the protozoan parasite Trypanosoma brucei is governed by cyclic-AMP signaling systems in the flagellum, with intriguing parallels to signaling systems that control bacterial social behavior. We also generated a T. brucei social behavior mutant and found that the mutant phenotype is complemented by wild-type cells grown in the same culture. Our findings open new avenues for dissecting social behavior and signaling in protozoan parasites and illustrate the capacity of these organisms to influence each other’s behavior in mixed communities.
PMCID: PMC4436052  PMID: 25922395
5.  Insect Stage-Specific Receptor Adenylate Cyclases Are Localized to Distinct Subdomains of the Trypanosoma brucei Flagellar Membrane 
Eukaryotic Cell  2014;13(8):1064-1076.
Increasing evidence indicates that the Trypanosoma brucei flagellum (synonymous with cilium) plays important roles in host-parasite interactions. Several studies have identified virulence factors and signaling proteins in the flagellar membrane of bloodstream-stage T. brucei, but less is known about flagellar membrane proteins in procyclic, insect-stage parasites. Here we report on the identification of several receptor-type flagellar adenylate cyclases (ACs) that are specifically upregulated in procyclic T. brucei parasites. Identification of insect stage-specific ACs is novel, as previously studied ACs were constitutively expressed or confined to bloodstream-stage parasites. We show that procyclic stage-specific ACs are glycosylated, surface-exposed proteins that dimerize and possess catalytic activity. We used gene-specific tags to examine the distribution of individual AC isoforms. All ACs examined localized to the flagellum. Notably, however, while some ACs were distributed along the length of the flagellum, others specifically localized to the flagellum tip. These are the first transmembrane domain proteins to be localized specifically at the flagellum tip in T. brucei, emphasizing that the flagellum membrane is organized into specific subdomains. Deletion analysis reveals that C-terminal sequences are critical for targeting ACs to the flagellum, and sequence comparisons suggest that differential subflagellar localization might be specified by isoform-specific C termini. Our combined results suggest insect stage-specific roles for a subset of flagellar adenylate cyclases and support a microdomain model for flagellar cyclic AMP (cAMP) signaling in T. brucei. In this model, cAMP production is compartmentalized through differential localization of individual ACs, thereby allowing diverse cellular responses to be controlled by a common signaling molecule.
PMCID: PMC4135804  PMID: 24879126
6.  Trypanosoma brucei Translation Initiation Factor Homolog EIF4E6 Forms a Tripartite Cytosolic Complex with EIF4G5 and a Capping Enzyme Homolog 
Eukaryotic Cell  2014;13(7):896-908.
Trypanosomes lack the transcriptional control characteristic of the majority of eukaryotes that is mediated by gene-specific promoters in a one-gene–one-promoter arrangement. Rather, their genomes are transcribed in large polycistrons with no obvious functional linkage. Posttranscriptional regulation of gene expression must thus play a larger role in these organisms. The eIF4E homolog TbEIF4E6 binds mRNA cap analogs in vitro and is part of a complex in vivo that may fulfill such a role. Knockdown of TbEIF4E6 tagged with protein A-tobacco etch virus protease cleavage site-protein C to approximately 15% of the normal expression level resulted in viable cells that displayed a set of phenotypes linked to detachment of the flagellum from the length of the cell body, if not outright flagellum loss. While these cells appeared and behaved as normal under stationary liquid culture conditions, standard centrifugation resulted in a marked increase in flagellar detachment. Furthermore, the ability of TbEIF4E6-depleted cells to engage in social motility was reduced. The TbEIF4E6 protein forms a cytosolic complex containing a triad of proteins, including the eIF4G homolog TbEIF4G5 and a hypothetical protein of 70.3 kDa, referred to as TbG5-IP. The TbG5-IP analysis revealed two domains with predicted secondary structures conserved in mRNA capping enzymes: nucleoside triphosphate hydrolase and guanylyltransferase. These complex members have the potential for RNA interaction, either via the 5′ cap structure for TbEIF4E6 and TbG5-IP or through RNA-binding domains in TbEIF4G5. The associated proteins provide a signpost for future studies to determine how this complex affects capped RNA molecules.
PMCID: PMC4135740  PMID: 24839125
7.  Motility and more: the flagellum of Trypanosoma brucei 
Nature reviews. Microbiology  2014;12(7):505-518.
A central feature of trypanosome cell biology and life cycle is the parasite’s single flagellum, which is an essential and multifunctional organelle involved in cell propulsion, morphogenesis and cytokinesis. The flagellar membrane is also a specialized subdomain of the cell surface that harbors multiple parasite virulence factors with roles in signaling and host-parasite interactions. In this review, we discuss the structure, assembly and function of the trypanosome flagellum, including canonical roles in cell motility as well as novel and emerging roles in cell morphogenesis and host-parasite interaction.
PMCID: PMC4278896  PMID: 24931043
8.  CMF22 Is a Broadly Conserved Axonemal Protein and Is Required for Propulsive Motility in Trypanosoma brucei 
Eukaryotic Cell  2013;12(9):1202-1213.
The eukaryotic flagellum (or cilium) is a broadly conserved organelle that provides motility for many pathogenic protozoa and is critical for normal development and physiology in humans. Therefore, defining core components of motile axonemes enhances understanding of eukaryotic biology and provides insight into mechanisms of inherited and infectious diseases in humans. In this study, we show that component of motile flagella 22 (CMF22) is tightly associated with the flagellar axoneme and is likely to have been present in the last eukaryotic common ancestor. The CMF22 amino acid sequence contains predicted IQ and ATPase associated with a variety of cellular activities (AAA) motifs that are conserved among CMF22 orthologues in diverse organisms, hinting at the importance of these domains in CMF22 function. Knockdown by RNA interference (RNAi) and rescue with an RNAi-immune mRNA demonstrated that CMF22 is required for propulsive cell motility in Trypanosoma brucei. Loss of propulsive motility in CMF22-knockdown cells was due to altered flagellar beating patterns, rather than flagellar paralysis, indicating that CMF22 is essential for motility regulation and likely functions as a fundamental regulatory component of motile axonemes. CMF22 association with the axoneme is weakened in mutants that disrupt the nexin-dynein regulatory complex, suggesting potential interaction with this complex. Our results provide insight into the core machinery required for motility of eukaryotic flagella.
PMCID: PMC3811564  PMID: 23851336
9.  Approaches for functional analysis of flagellar proteins in African trypanosomes 
Methods in cell biology  2009;93:10.1016/S0091-679X(08)93002-8.
The eukaryotic flagellum is a highly conserved organelle serving motility, sensory and transport functions. Although genetic, genomic and proteomic studies have led to the identification of hundreds of flagellar and putative flagellar proteins, precisely how these proteins function individually and collectively to drive flagellum motility and other functions remains to be determined. In this chapter we provide an overview of tools and approaches available for studying flagellum protein function in the protozoan parasite Trypanosoma brucei. We begin by outlining techniques for in vitro cultivation of both T. brucei lifecycle stages, as well as transfection protocols for the delivery of DNA constructs. We then describe specific assays used to assess flagellum function including flagellum preparation and quantitative motility assays. We conclude the chapter with a description of molecular genetic approaches for manipulating gene function. In summary, the availability of potent molecular tools, as well as the health and economic relevance of T. brucei as a pathogen, combine to make the parasite an attractive and integral experimental system for the functional analysis of flagellar proteins.
PMCID: PMC3821762  PMID: 20409810
10.  The Trypanosoma brucei Flagellum: Moving Parasites in New Directions 
Annual review of microbiology  2009;63:10.1146/annurev.micro.091208.073353.
African trypanosomes are devastating human and animal pathogens. Trypanosoma brucei rhodesiense and T. b. gambiense subspecies cause the fatal human disease known as African sleeping sickness. It is estimated that several hundred thousand new infections occur annually and the disease is fatal if untreated. T. brucei is transmitted by the tsetse fly and alternates between bloodstream-form and insect-form life cycle stages that are adapted to survive in the mammalian host and the insect vector, respectively. The importance of the flagellum for parasite motility and attachment to the tsetse fly salivary gland epithelium has been appreciated for many years. Recent studies have revealed both conserved and novel features of T. brucei flagellum structure and composition, as well as surprising new functions that are outlined here. These discoveries are important from the standpoint of understanding trypanosome biology and identifying novel drug targets, as well as for advancing our understanding of fundamental aspects of eukaryotic flagellum structure and function.
PMCID: PMC3821760  PMID: 19575562
flagellum; cilium; motility; axoneme; trypanosome; dynein; cytokinesis
11.  The dynein regulatory complex is required for ciliary motility and otolith biogenesis in the inner ear 
Nature  2008;457(7226):10.1038/nature07520.
In teleosts, proper balance and hearing depend on mechanical sensors in the inner ear. These sensors include actin-based microvilli and microtubule-based cilia that extend from the surface of sensory hair cells and attach to biomineralized ‘ear stones’ (or otoliths)1. Otolith number, size and placement are under strict developmental control, but the mechanisms that ensure otolith assembly atop specific cells of the sensory epithelium are unclear. Here we demonstrate that cilia motility is required for normal otolith assembly and localization. Using in vivo video microscopy, we show that motile tether cilia at opposite poles of the otic vesicle create fluid vortices that attract otolith precursor particles, thereby biasing an otherwise random distribution to direct localized otolith seeding on tether cilia. Independent knockdown of subunits for the dynein regulatory complex and outer-arm dynein disrupt cilia motility, leading to defective otolith biogenesis. These results demonstrate a requirement for the dynein regulatory complex in vertebrates and show that cilia-driven flow is a key epigenetic factor in controlling otolith biomineralization.
PMCID: PMC3821763  PMID: 19043402
12.  Social Parasites 
Current opinion in microbiology  2011;14(6):642-648.
Summary of recent advances
Protozoan parasites cause tremendous human suffering worldwide, but strategies for therapeutic intervention are limited. Recent studies illustrate that the paradigm of microbes as social organisms can be brought to bear on questions about parasite biology, transmission and pathogenesis. This review discusses recent work demonstrating adaptation of social behaviors by parasitic protozoa that cause African sleeping sickness and malaria. The recognition of social behavior and cell-cell communication as a ubiquitous property of bacteria has transformed our view of microbiology, but protozoan parasites have not generally been considered in this context. Works discussed illustrate the potential for concepts of sociomicrobiology to provide insight into parasite biology and should stimulate new approaches for thinking about parasites and parasite-host interactions.
PMCID: PMC3230740  PMID: 22020108
Cell-cell communication; social behavior; Trypanosoma; Plasmodium
13.  CCDC39 is required for assembly of inner dynein arms and the dynein regulatory complex and for normal ciliary motility in humans and dogs 
Nature genetics  2010;43(1):72-78.
Primary ciliary dyskinesia (PCD) is an inherited disorder characterized by recurrent infections of the upper and lower respiratory tract, reduced fertility in males and situs inversus in about 50% of affected individuals (Kartagener syndrome). It is caused by motility defects in the respiratory cilia that are responsible for airway clearance, the flagella that propel sperm cells and the nodal monocilia that determine left-right asymmetry1. Recessive mutations that cause PCD have been identified in genes encoding components of the outer dynein arms, radial spokes and cytoplasmic pre-assembly factors of axonemal dyneins, but these mutations account for only about 50% of cases of PCD. We exploited the unique properties of dog populations to positionally clone a new PCD gene, CCDC39. We found that loss-of-function mutations in the human ortholog underlie a substantial fraction of PCD cases with axonemal disorganization and abnormal ciliary beating. Functional analyses indicated that CCDC39 localizes to ciliary axonemes and is essential for assembly of inner dynein arms and the dynein regulatory complex.
PMCID: PMC3509786  PMID: 21131972
14.  Three-Dimensional Structure of the Trypanosome Flagellum Suggests that the Paraflagellar Rod Functions as a Biomechanical Spring 
PLoS ONE  2012;7(1):e25700.
Flagellum motility is critical for normal human development and for transmission of pathogenic protozoa that cause tremendous human suffering worldwide. Biophysical principles underlying motility of eukaryotic flagella are conserved from protists to vertebrates. However, individual cells exhibit diverse waveforms that depend on cell-specific elaborations on basic flagellum architecture. Trypanosoma brucei is a uniflagellated protozoan parasite that causes African sleeping sickness. The T. brucei flagellum is comprised of a 9+2 axoneme and an extra-axonemal paraflagellar rod (PFR), but the three-dimensional (3D) arrangement of the underlying structural units is poorly defined. Here, we use dual-axis electron tomography to determine the 3D architecture of the T. brucei flagellum. We define the T. brucei axonemal repeating unit. We observe direct connections between the PFR and axonemal dyneins, suggesting a mechanism by which mechanochemical signals may be transmitted from the PFR to axonemal dyneins. We find that the PFR itself is comprised of overlapping laths organized into distinct zones that are connected through twisting elements at the zonal interfaces. The overall structure has an underlying 57nm repeating unit. Biomechanical properties inferred from PFR structure lead us to propose that the PFR functions as a biomechanical spring that may store and transmit energy derived from axonemal beating. These findings provide insight into the structural foundations that underlie the distinctive flagellar waveform that is a hallmark of T. brucei cell motility.
PMCID: PMC3250385  PMID: 22235240
15.  Structure-Function Analysis of Dynein Light Chain 1 Identifies Viable Motility Mutants in Bloodstream-Form Trypanosoma brucei ▿ † 
Eukaryotic Cell  2011;10(7):884-894.
The flagellum of Trypanosoma brucei is an essential and multifunctional organelle that is receiving increasing attention as a potential drug target and as a system for studying flagellum biology. RNA interference (RNAi) knockdown is widely used to test the requirement for a protein in flagellar motility and has suggested that normal flagellar motility is essential for viability in bloodstream-form trypanosomes. However, RNAi knockdown alone provides limited functional information because the consequence is often loss of a multiprotein complex. We therefore developed an inducible system that allows functional analysis of point mutations in flagellar proteins in T. brucei. Using this system, we identified point mutations in the outer dynein light chain 1 (LC1) that allow stable assembly of outer dynein motors but do not support propulsive motility. In procyclic-form trypanosomes, the phenotype of LC1 mutants with point mutations differs from the motility and structural defects of LC1 knockdowns, which lack the outer-arm dynein motor. Thus, our results distinguish LC1-specific functions from broader functions of outer-arm dynein. In bloodstream-form trypanosomes, LC1 knockdown blocks cell division and is lethal. In contrast, LC1 point mutations cause severe motility defects without affecting viability, indicating that the lethal phenotype of LC1 RNAi knockdown is not due to defective motility. Our results demonstrate for the first time that normal motility is not essential in bloodstream-form T. brucei and that the presumed connection between motility and viability is more complex than might be interpreted from knockdown studies alone. These findings open new avenues for dissecting mechanisms of flagellar protein function and provide an important step in efforts to exploit the potential of the flagellum as a therapeutic target in African sleeping sickness.
PMCID: PMC3147417  PMID: 21378260
16.  Parasites in motion: flagellum-driven cell motility in African trypanosomes 
Current opinion in microbiology  2010;13(4):459-465.
Motility of the sleeping sickness parasite, Trypanosoma brucei, impacts disease transmission and pathogenesis. Trypanosome motility is driven by a flagellum that harbors a canonical 9 + 2 axoneme, together with trypanosome-specific elaborations. Trypanosome flagellum biology and motility have been the object of intense research over the last two years. These studies have led to the discovery of a novel form of motility, termed social motility, and provided revision of long-standing models for cell propulsion. Recent work has also uncovered novel structural features and motor proteins associated with the flagellar apparatus and has identified candidate signaling molecules that are predicted to regulate flagellar motility. Together with earlier inventories of flagellar proteins from proteomic and genomic studies, the stage is now set to move forward with functional studies to elucidate molecular mechanisms and investigate parasite motility in the context of host-parasite interactions.
PMCID: PMC3225338  PMID: 20591724
Flagellum; Cilium; Motility; Axoneme; Trypanosome; Social Behavior
17.  The flagellum of Trypanosoma brucei: new tricks from an old dog 
African trypanosomes, i.e. Trypanosoma brucei and related sub-species, are devastating human and animal pathogens that cause significant human mortality and limit sustained economic development in sub-Saharan Africa. Trypanosoma brucei is a highly motile protozoan parasite and coordinated motility is central to both disease pathogenesis in the mammalian host and parasite development in the tsetse fly vector. Since motility is critical for parasite development and pathogenesis, understanding unique aspects of the T. brucei flagellum may uncover novel targets for therapeutic intervention in African sleeping sickness. Moreover, studies of conserved features of the T. brucei flagellum are directly relevant to understanding fundamental aspects of flagellum and cilium function in other eukaryotes, making T. brucei an important model system. The T. brucei flagellum contains a canonical 9 + 2 axoneme, together with additional features that are unique to kinetoplastids and a few closely-related organisms. Until recently, much of our knowledge of the structure and function of the trypanosome flagellum was based on analogy and inference from other organisms. There has been an explosion in functional studies in T. brucei in recent years, revealing conserved as well as novel and unexpected structural and functional features of the flagellum. Most notably, the flagellum has been found to be an essential organelle, with critical roles in parasite motility, morphogenesis, cell division and immune evasion. This review highlights recent discoveries on the T. brucei flagellum.
PMCID: PMC3210513  PMID: 18472102
Flagellum; Cilium; Motility; Axoneme; Trypanosome; Dynein; Cytokinesis
18.  Social Motility in African Trypanosomes 
PLoS Pathogens  2010;6(1):e1000739.
African trypanosomes are devastating human and animal pathogens that cause significant human mortality and limit economic development in sub-Saharan Africa. Studies of trypanosome biology generally consider these protozoan parasites as individual cells in suspension cultures or in animal models of infection. Here we report that the procyclic form of the African trypanosome Trypanosoma brucei engages in social behavior when cultivated on semisolid agarose surfaces. This behavior is characterized by trypanosomes assembling into multicellular communities that engage in polarized migrations across the agarose surface and cooperate to divert their movements in response to external signals. These cooperative movements are flagellum-mediated, since they do not occur in trypanin knockdown parasites that lack normal flagellum motility. We term this behavior social motility based on features shared with social motility and other types of surface-induced social behavior in bacteria. Social motility represents a novel and unexpected aspect of trypanosome biology and offers new paradigms for considering host-parasite interactions.
Author Summary
African trypanosomes, e.g. Trypanosoma brucei, and related kinetoplastid parasites cause morbidity and mortality in several million people worldwide. Trypanosomes are protists and are thus generally considered to behave as single-celled microorganisms. In other microorganisms, social interactions among individuals lead to development of multicellular communities with specialized and advantageous capabilities versus single cells. The concept of bacteria acting as groups of cells communicating and cooperating with one another has had a major impact on our understanding of bacterial physiology and pathogenesis, but this paradigm has not been applied to parasitic protozoa. Here we report that T. brucei is capable of social behavior when exposed to semisolid surfaces. This behavior, termed social motility, is characterized by the assembly of parasites into multicellular communities with emergent properties that are not evident in single cells. Parasites within communities exhibit polarized movements and cooperate to coordinate their movements in response to an external stimulus. Social motility offers many potential advantages, such as facilitating colonization and navigation through host tissues. The identification of social behavior in T. brucei reveals a novel and unexpected aspect of parasite biology and provides new concepts for considering host-parasite interactions.
PMCID: PMC2813273  PMID: 20126443
19.  Trypanin, a Component of the Flagellar Dynein Regulatory Complex, Is Essential in Bloodstream Form African Trypanosomes 
PLoS Pathogens  2006;2(9):e101.
The Trypanosoma brucei flagellum is a multifunctional organelle with critical roles in motility, cellular morphogenesis, and cell division. Although motility is thought to be important throughout the trypanosome lifecycle, most studies of flagellum structure and function have been restricted to the procyclic lifecycle stage, and our knowledge of the bloodstream form flagellum is limited. We have previously shown that trypanin functions as part of a flagellar dynein regulatory system that transmits regulatory signals from the central pair apparatus and radial spokes to axonemal dyneins. Here we investigate the requirement for this dynein regulatory system in bloodstream form trypanosomes. We demonstrate that trypanin is localized to the flagellum of bloodstream form trypanosomes, in a pattern identical to that seen in procyclic cells. Surprisingly, trypanin RNA interference is lethal in the bloodstream form. These knockdown mutants fail to initiate cytokinesis, but undergo multiple rounds of organelle replication, accumulating multiple flagella, nuclei, kinetoplasts, mitochondria, and flagellum attachment zone structures. These findings suggest that normal flagellar beat is essential in bloodstream form trypanosomes and underscore the emerging concept that there is a dichotomy between trypanosome lifecycle stages with respect to factors that contribute to cell division and cell morphogenesis. This is the first time that a defined dynein regulatory complex has been shown to be essential in any organism and implicates the dynein regulatory complex and other enzymatic regulators of flagellar motility as candidate drug targets for the treatment of African sleeping sickness.
African trypanosomes are protozoan parasites that cause African sleeping sickness, a fatal disease with devastating health and economic consequences. These parasites are indigenous to a 9 million-km2 area of sub-Saharan Africa where 60 million people live at risk of infection every day. In addition to the tremendous human health burden posed by trypanosomes, their infection of wild and domestic animals presents a barrier to sustained economic development of vast regions of otherwise productive land. Current drugs used for treatment of sleeping sickness are antiquated, toxic, and often ineffective; thus, there is a dire need for the development of innovative approaches for therapeutic intervention. Trypanosomes are highly motile and this motility requires coordinated regulation of axonemal dynein, a molecular motor that drives beating of the parasite's flagellum. In the present work, the authors demonstrate that the protein trypanin, which is part of a signaling system that regulates the flagellar dynein motor, is essential in bloodstream stage African trypanosomes. This surprising finding raises the possibility that numerous enzymes and regulatory proteins that are necessary for flagellar motility may represent novel targets for chemotherapeutic intervention in African sleeping sickness.
PMCID: PMC1579245  PMID: 17009870
20.  Flagellar Motility Contributes to Cytokinesis in Trypanosoma brucei and Is Modulated by an Evolutionarily Conserved Dynein Regulatory System†  
Eukaryotic Cell  2006;5(4):696-711.
The flagellum of Trypanosoma brucei is a multifunctional organelle with critical roles in motility and other aspects of the trypanosome life cycle. Trypanin is a flagellar protein required for directional cell motility, but its molecular function is unknown. Recently, a trypanin homologue in Chlamydomonas reinhardtii was reported to be part of a dynein regulatory complex (DRC) that transmits regulatory signals from central pair microtubules and radial spokes to axonemal dynein. DRC genes were identified as extragenic suppressors of central pair and/or radial spoke mutations. We used RNA interference to ablate expression of radial spoke (RSP3) and central pair (PF16) components individually or in combination with trypanin. Both rsp3 and pf16 single knockdown mutants are immotile, with severely defective flagellar beat. In the case of rsp3, this loss of motility is correlated with the loss of radial spokes, while in the case of pf16 the loss of motility correlates with an aberrant orientation of the central pair microtubules within the axoneme. Genetic interaction between trypanin and PF16 is demonstrated by the finding that loss of trypanin suppresses the pf16 beat defect, indicating that the DRC represents an evolutionarily conserved strategy for dynein regulation. Surprisingly, we discovered that four independent mutants with an impaired flagellar beat all fail in the final stage of cytokinesis, indicating that flagellar motility is necessary for normal cell division in T. brucei. These findings present the first evidence that flagellar beating is important for cell division and open the opportunity to exploit enzymatic activities that drive flagellar beat as drug targets for the treatment of African sleeping sickness.
PMCID: PMC1459671  PMID: 16607017
21.  Biology and Mechanism of Trypanosome Cell Motility 
Eukaryotic Cell  2003;2(2):200-208.
PMCID: PMC154846  PMID: 12684369
22.  Trypanin is a cytoskeletal linker protein and is required for cell motility in African trypanosomes 
The Journal of Cell Biology  2002;156(5):867-877.
The cytoskeleton of eukaryotic cells is comprised of a complex network of distinct but interconnected filament systems that function in cell division, cell motility, and subcellular trafficking of proteins and organelles. A gap in our understanding of this dynamic network is the identification of proteins that connect subsets of cytoskeletal structures. We previously discovered a family of cytoskeleton-associated proteins that includes GAS11, a candidate human tumor suppressor upregulated in growth-arrested cells, and trypanin, a component of the flagellar cytoskeleton of African trypanosomes. Although these proteins are intimately associated with the cytoskeleton, their function has yet to be determined. Here we use double-stranded RNA interference to block trypanin expression in Trypanosoma brucei, and demonstrate that this protein is required for directional cell motility. Trypanin(−) mutants have an active flagellum, but are unable to coordinate flagellar beat. As a consequence, they spin and tumble uncontrollably, occasionally moving backward. Immunofluorescence experiments demonstrate that trypanin is located along the flagellum/flagellum attachment zone and electron microscopic analysis revealed that cytoskeletal connections between the flagellar apparatus and subpellicular cytoskeleton are destabilized in trypanin(−) mutants. These results indicate that trypanin functions as a cytoskeletal linker protein and offer insights into the mechanisms of flagellum-based cell motility.
PMCID: PMC2173309  PMID: 11864997
trypanin; microtubule-associated protein; cytoskeleton; cell motility; dsRNA interference
23.  The Gene for a T Lymphocyte Triggering Factor from African Trypanosomes  
An early and essential event in the protective immune response against most viruses and protozoa is the production of interferon-γ (IFN-γ). In contrast, during infection with African trypanosomes, protozoan parasites that cause human sleeping sickness, the increased levels of IFN-γ do not correlate with a protective response. We showed previously that African trypanosomes express a protein called T lymphocyte triggering factor (TLTF), which triggers CD8+ T lymphocytes to proliferate and to secrete IFN-γ. Here, we isolate the gene for TLTF and demonstrate that the recombinant version of TLTF specifically induces CD8+, but not CD4+, T cells to secrete IFN-γ. Studies with TLTF fused to the green fluorescent protein show that TLTF is localized to small vesicles that are found primarily at or near the flagellar pocket, the site of secretion in trypanosomes. TLTF is likely to be only the first example of a class of proteins that we designate as trypanokines, i.e., factors secreted by trypanosomes that modulate the cytokine network of the host immune system for the benefit of the parasite.
PMCID: PMC2199003  PMID: 9236195

Results 1-23 (23)