Kent L. Hill

Double-stranded RNA interference in Trypanosoma brucei using head-to-head promoters

The discovery of double-stranded RNA interference (dsRNAi) in Trypanosoma brucei provides a convenient method to generate knockout phenotypes in this protozoan parasite [Ngo H, Tschudi C, Gull K, Ullu E. Double-stranded RNA induces mRNA degradation in Trypanosoma brucei. Proc Natl Acad Sci USA 1998;95:14687-14692]. The presence of double-stranded RNA (dsRNA) dominantly silences gene expression in a sequence-specific manner by causing the corresponding endogenous RNA to be degraded. To simplify the generation of knockout phenotypes in T. brucei via dsRNAi, we used two promoters arranged as an inverted repeat on a plasmid. This promoter arrangement generates transcripts of both strands of DNA inserted between the promoters, which then form dsRNA. We have used plasmids encoding either two T. brucei ribosomal RNA promoters or two bacteriophage T7 promoters to interfere with expression of alpha-tubulin (TUB), green fluorescent protein (GFP), paraflagellar rod protein A (PFRA), flagellum-adhesion glycoprotein 1 (FLA1), and histone 2B (H2B) in T. brucei. We show here that FLA1 is required for flagellar attachment in T. brucei and that H2B is required for parasite growth. Thus, the two-promoter approach efficiently generates dsRNAi in T. brucei and can be used to produce both specific and random knockout phenotypes in T. brucei. This approach should be useful in generating knockout phenotypes in other kinetoplastid parasites including Trypanosoma cruzi and Leishmania.

CCDC39 is required for assembly of inner dynein arms and the dynein regulatory complex and for normal ciliary motility in humans and dogs

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 asymmetry. 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.

Flagellar Motility Contributes to Cytokinesis in Trypanosoma brucei and Is Modulated by an Evolutionarily Conserved Dynein Regulatory System

ABSTRACT 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.

Multiple mechanisms of immune evasion by African trypanosomes

During infection of a mammalian host, African trypanosomes are in constant contact with the host’s immune system. These protozoan parasites are infamous for their ability to evade the immune responses by periodically switching their major variant surface glycoprotein (VSG), a phenomenon called antigenic variation. Antigenic variation, however, is likely to be only one of several mechanisms enabling these organisms to thrive in the face of the immune defenses. The ability to grow in high levels of interferon-gamma (IFN-γ) and to avoid complement-mediated destruction may also facilitate the parasite’s survival. In this review we summarize (i) the activation of trypanosome genes for three different VSGs during antigenic variation, (ii) the secretion of a trypanosome protein that induces host CD8+ T cells to produce IFN-γ, and (iii) the evidence for trypansome protein similar to a surface protease of Leishmania that plays a role in resistance to complement-mediated lysis.

The dynein regulatory complex is required for ciliary motility and otolith biogenesis in the inner ear

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). 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.

The Trypanosoma brucei Flagellum: Moving Parasites in New Directions

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.

Independent Analysis of the Flagellum Surface and Matrix Proteomes Provides Insight into Flagellum Signaling in Mammalian-infectious Trypanosoma brucei

The flagellum of African trypanosomes is an essential and multifunctional organelle that functions in motility, cell morphogenesis, and host-parasite interaction. Previous studies of the trypanosome flagellum have been limited by the inability to purify flagella without first removing the flagellar membrane. This limitation is particularly relevant in the context of studying flagellum signaling, as signaling requires surface-exposed proteins in the flagellar membrane and soluble signaling proteins in the flagellar matrix. Here we employ a combination of genetic and mechanical approaches to purify intact flagella from the African trypanosome, Trypanosoma brucei, in its mammalian-infectious stage. We combined flagellum purification with affinity-purification of surface-exposed proteins to conduct independent proteomic analyses of the flagellum surface and matrix fractions. The proteins identified encompass a broad range of molecular functionalities, including many predicted to function in signaling. Immunofluorescence and RNA interference studies demonstrate flagellum localization and function for proteins identified and provide insight into mechanisms of flagellum attachment and motility. The flagellum surface proteome includes many T. brucei-specific proteins and is enriched for proteins up-regulated in the mammalian-infectious stage of the parasite life-cycle. The combined results indicate that the flagellum surface presents a diverse and dynamic host-parasite interface that is well-suited for host-parasite signaling.

Functional genomics in Trypanosoma brucei identifies evolutionarily conserved components of motile flagella.

Cilia and flagella are highly conserved, complex organelles involved in a variety of important functions. Flagella are required for motility of several human pathogens and ciliary defects lead to a variety of fatal and debilitating human diseases. Many of the major structural components of cilia and flagella are known, but little is known about regulation of flagellar beat. Trypanosoma brucei, the causative agent of African sleeping sickness, provides an excellent model for studying flagellar motility. We have used comparative genomics to identify a core group of 50 genes unique to organisms with motile flagella. These genes, referred to as T. brucei components of motile flagella (TbCMF) include 30 novel genes, and human homologues of many of the TbCMF genes map to loci associated with human ciliary diseases. To characterize TbCMF protein function we used RNA interference to target 41 TbCMF genes. Sedimentation assays and direct observation demonstrated clear motility defects in a majority of these knockdown mutants. Epitope tagging, fluorescence localization and biochemical fractionation demonstrated flagellar localization for several TbCMF proteins. Finally, ultrastructural analysis identified a family of novel TbCMF proteins that function to maintain connections between outer doublet microtubules, suggesting that they are the first identified components of nexin links. Overall, our results provide insights into the workings of the eukaryotic flagellum, identify several novel human disease gene candidates, reveal unique aspects of the trypanosome flagellum and underscore the value of T. brucei as an experimental system for studying flagellar biology.

Trypanin is a cytoskeletal linker protein and is required for cell motility in African trypanosomes

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.

Biology and mechanism of trypanosome cell motility.

Movement of microbial pathogens plays an important role in pathogen biology, host-pathogen interaction, and disease pathogenesis. Microbial pathogens employ a variety of mechanisms for cell locomotion, including passive movement within their hosts circulation, cooptation of host cytoskeletal and