Bloodstream Stage Research Articles

Selection of reference genes for mRNA quantification in Trypanosoma brucei

Internal normalization is an established procedure that is necessary for accurate and reliable quantification of differentially regulated mRNAs. The profound changes of gene expression in parasitic life cycles pose a particular challenge on selection of appropriate reference genes for normalization, most importantly when using quantitative real time PCR (qPCR). Here we use the ranking algorithm implemented in the geNorm application to identify suitable Trypanosoma brucei reference genes for comparisons between the bloodstream and procyclic developmental stages and for analysis of mRNA induction by environmental conditions. For these conditions, the TERT gene is a good choice for valid normalization of qPCR and is clearly superior to some other reference genes reported in the literature. For comparison of other conditions, the ranking algorithm is recommended to verify a reliable and valid normalization that is instrumental to quantitative analysis of gene expression.

Evidence that low endocytic activity is not directly responsible for human serum resistance in the insect form of African trypanosomes

BackgroundIn Trypanosoma brucei, the African trypanosome, endocytosis is developmentally regulated and substantially more active in all known mammalian infective stages. In both mammalian and insect stages endocytic activity is likely required for nutrient acquisition, but in bloodstream forms increased endocytosis is involved in recycling the variant surface glycoprotein and removing host immune factors from the surface. However, a rationale for low endocytic activity in insect stages has not been explored. Here we asked if endocytic down-regulation in the procyclic form was associated with resistance to innate trypanolytic immune factors in the blood meal or tsetse fly midgut.FindingsUsing a well-characterized procyclic parasite with augmented endocytic flux mediated via TbRab5A overexpression, we found that insect stage parasites were able to grow both in the presence of trypanosome lytic factor (TLF) provided in human serum, and also in tsetse flies. Additionally, by placing blood stage parasites in restricted glucose medium, we observed that enlargement of the flagellar pocket, a key morphology associated with defective endocytosis, manifests in parallel with loss of cellular ATP levels.ConclusionsThese observations suggest that a high rate of endocytosis per se is insufficient to render insect form parasites sensitive to TLF or tsetse-derived trypanocidal factors. However, the data do suggest that endocytosis is energetically burdensome, as endocytic activity is rapidly compromised on energy depletion in bloodstream stages. Hence an important aspect of endocytic modulation in the nutrient-poor tsetse midgut is likely energetic conservation.

A Trk/HKT-Type K + Transporter from Trypanosoma brucei

The molecular mechanisms of K(+) homeostasis are only poorly understood for protozoan parasites. Trypanosoma brucei subsp. parasites, the causative agents of human sleeping sickness and nagana, are strictly extracellular and need to actively concentrate K(+) from their hosts' body fluids. The T. brucei genome contains two putative K(+) channel genes, yet the trypanosomes are insensitive to K(+) antagonists and K(+) channel-blocking agents, and they do not spontaneously depolarize in response to high extracellular K(+) concentrations. However, the trypanosomes are extremely sensitive to K(+) ionophores such as valinomycin. Surprisingly, T. brucei possesses a member of the Trk/HKT superfamily of monovalent cation permeases which so far had only been known from bacteria, archaea, fungi, and plants. The protein was named TbHKT1 and functions as a Na(+)-independent K(+) transporter when expressed in Escherichia coli, Saccharomyces cerevisiae, or Xenopus laevis oocytes. In trypanosomes, TbHKT1 is expressed in both the mammalian bloodstream stage and the Tsetse fly midgut stage; however, RNA interference (RNAi)-mediated silencing of TbHKT1 expression did not produce a growth phenotype in either stage. The presence of HKT genes in trypanosomatids adds a further piece to the enigmatic phylogeny of the Trk/HKT superfamily of K(+) transporters. Parsimonial analysis suggests that the transporters were present in the first eukaryotes but subsequently lost in several of the major eukaryotic lineages, in at least four independent events.

Distinct donor and acceptor specificities of Trypanosoma brucei oligosaccharyltransferases

Asparagine-linked glycosylation is catalysed by oligosaccharyltransferase (OTase). In Trypanosoma brucei OTase activity is catalysed by single-subunit enzymes encoded by three paralogous genes of which TbSTT3B and TbSTT3C can complement a yeast Δstt3 mutant. The two enzymes have overlapping but distinct peptide acceptor specificities, with TbSTT3C displaying an enhanced ability to glycosylate sites flanked by acidic residues. TbSTT3A and TbSTT3B, but not TbSTT3C, are transcribed in the bloodstream and procyclic life cycle stages of T. brucei. Selective knockdown and analysis of parasite protein N-glycosylation showed that TbSTT3A selectively transfers biantennary Man5GlcNAc2 to specific glycosylation sites whereas TbSTT3B selectively transfers triantennary Man9GlcNAc2 to others. Analysis of T. brucei glycosylation site occupancy showed that TbSTT3A and TbSTT3B glycosylate sites in acidic to neutral and neutral to basic regions of polypeptide, respectively. This embodiment of distinct specificities in single-subunit OTases may have implications for recombinant glycoprotein engineering. TbSTT3A and TbSTT3B could be knocked down individually, but not collectively, in tissue culture. However, both were independently essential for parasite growth in mice, suggesting that inhibiting protein N-glycosylation could have therapeutic potential against trypanosomiasis.

Analysis of expressed sequence tags from the four main developmental stages of Trypanosoma congolense

Trypanosoma congolense is one of the most economically important pathogens of livestock in Africa. Culture-derived parasites of each of the three main insect stages of the T. congolense life cycle, i.e., the procyclic, epimastigote and metacyclic stages, and bloodstream stage parasites isolated from infected mice, were used to construct stage-specific cDNA libraries and expressed sequence tags (ESTs or cDNA clones) in each library were sequenced. Thirteen EST clusters encoding different variant surface glycoproteins (VSGs) were detected in the metacyclic library and 26 VSG EST clusters were found in the bloodstream library, 6 of which are shared by the metacyclic library. Rare VSG ESTs are present in the epimastigote library, and none were detected in the procyclic library. ESTs encoding enzymes that catalyze oxidative phosphorylation and amino acid metabolism are about twice as abundant in the procyclic and epimastigote stages as in the metacyclic and bloodstream stages. In contrast, ESTs encoding enzymes involved in glycolysis, the citric acid cycle and nucleotide metabolism are about the same in all four developmental stages. Cysteine proteases, kinases and phosphatases are the most abundant enzyme groups represented by the ESTs. All four libraries contain T. congolense-specific expressed sequences not present in the Trypanosoma brucei and Trypanosoma cruzi genomes. Normalized cDNA libraries were constructed from the metacyclic and bloodstream stages, and found to be further enriched for T. congolense-specific ESTs. Given that cultured T. congolense offers an experimental advantage over other African trypanosome species, these ESTs provide a basis for further investigation of the molecular properties of these four developmental stages, especially the epimastigote and metacyclic stages for which it is difficult to obtain large quantities of organisms. The T. congolense EST databases are available at: http://www.sanger.ac.uk/Projects/T_congolense/EST_index.shtml.

Identification of Methylated Loci in the Trypanosoma brucei Genome

Previous mass spectrometry and blotting experiments in our laboratory have shown that Trypanosoma brucei, the causative agent of African Sleeping Sickness, contains low levels of 5‐methylcytosine in its genomic DNA. In order to determine the location of 5‐methylcytosine in the genome, an immunoprecipitation strategy was utilized. T. brucei DNA was cut with DpnII, modified by linker addition, and immunoprecipitated with an antibody against 5‐methylcytosine. Immunoprecipitated DNAs were amplified by PCR, inserted into the pGEM‐T Easy plasmid, and analyzed by DNA sequencing. In total, greater than 100 clones have been analyzed representing DNA from the insect stage and bloodstream stage of the parasite. To date, all methylated loci are in the nuclear genome. Several loci were found in the immunoprecipitate multiple times, including variant surface glycoprotein genes and retrotransposon genes. Thus, DNA methylation may regulate expression of these genes. However, housekeeping genes were also found in the immunoprecipitate indicating that DNA methylation is not restricted to a single locus. To pinpoint the position of 5‐methylcytosine within specific genes, sodium bisulfite sequencing of retrotransposon genes was initiated. In total, our data demonstrate that 5‐methylcytosine is present in the nuclear genome and may be overrepresented in multi‐gene families and repeated sequences. This work is supported by National Institutes of Health award AI074035‐01 to K.T.M.

Kinetoplastid guide RNA biogenesis is dependent on subunits of the mitochondrial RNA binding complex 1 and mitochondrial RNA polymerase.

The mitochondrial RNA binding complex 1 (MRB1) is a recently discovered complex of proteins associated with the TbRGG1 and TbRGG2 proteins in Trypanosoma brucei. Based on the phenotype caused by down-regulation of these two proteins, it was proposed to play an unspecified role in RNA editing. RNAi silencing of three newly characterized protein subunits, guide RNA associated proteins (GAPs) 1 and 2 as well as a predicted DExD/H-box RNA helicase, show they are essential for cell growth in the procyclic stage. Furthermore, their down-regulation leads to inhibition of editing in only those mRNAs for which minicircle-encoded guide (g) RNAs are required. However, editing remains unaffected when the maxicircle-encoded cis-acting gRNA is employed. Interestingly, all three proteins are necessary for the expression of the minicircle-encoded gRNAs. Moreover, down-regulation of a fourth assayed putative MRB1 subunit, Nudix hydrolase, does not appear to destabilize gRNAs, and down-regulation of this protein has a general impact on the stability of maxicircle-encoded RNAs. GAP1 and 2 are also essential for the survival of the bloodstream stage, in which the gRNAs become eliminated upon depletion of either protein. Immunolocalization revealed that GAP1 and 2 are concentrated into discrete spots along the mitochondrion, usually localized in the proximity of the kinetoplast. Finally, we demonstrate that the same mtRNA polymerase known to transcribe the maxicircle mRNAs may also have a role in expression of the minicircle-encoded gRNAs.

Hydroxyurea-induced synchronisation of bloodstream stage Trypanosoma brucei

Synchronisation of the Trypanosoma brucei cell cycle proved elusive for many years. A recent report demonstrated that synchronisation of procyclic form cells was possible following treatment with hydroxyurea. Here, that work is extended to the disease-relevant, mammalian-infective bloodstream stage trypanosome. Treatment of bloodstream stage Lister 427 T. brucei cells growing in vitro with 10 μg ml −1 hydroxyurea for 6 h led to an enrichment of cells in S phase. Following removal of the drug, cells proceeded uniformly through one round of the cell cycle, providing a much needed tool to enrich for specific cell cycle stages, in a manner similar to hydroxyurea treatment of procyclic form T. brucei.

Turnover of glycosomes during life-cycle differentiation of Trypanosoma brucei

Protozoan Kinetoplastida, a group that comprises the pathogenic Trypanosoma brucei, compartmentalize several metabolic systems such as the major part of the glycolytic pathway, in multiple peroxisome-like organelles, designated glycosomes. Trypanosomes have a complicated life cycle, involving two major, distinct stages living in the mammalian bloodstream and several stages inhabiting different body parts of the tsetse fly. Previous studies on non-differentiating trypanosomes have shown that the metabolism and enzymatic contents of glycosomes in bloodstream-form and cultured procyclic cells, representative of the stage living in the insect’s midgut, differ considerably. In this study, the morphology of glycosomes and their position relative to the lysosome were followed, as were the levels of some glycosomal enzymes and markers for other subcellular compartments, during the differentiation from bloodstream-form to procyclic trypanosomes. Our studies revealed a small tendency of glycosomes to associate with the lysosome when a population of long-slender bloodstream forms differentiated into short-stumpy forms which are pre-adapted to live in the fly. The same phenomenon was observed during the short-stumpy to procyclic transformation, but then the process was fast and many more glycosomes were associated with the dramatically enlarged degradation organelle. The observations suggested an efficient glycosome turnover involving autophagy. Changes observed in the levels of marker enzymes are consistent with the notion that, during differentiation, glycosomes with enzymatic contents specific for the old life-cycle stage are degraded and new glycosomes with different contents are synthesized, causing that the metabolic repertoire of trypanosomes is, at each stage, optimally adapted to the environmental conditions encountered.

Adaptations of Trypanosoma brucei to gradual loss of kinetoplast DNA: Trypanosoma equiperdum and Trypanosoma evansi are petite mutants of T. brucei

Trypanosoma brucei is a kinetoplastid flagellate, the agent of human sleeping sickness and ruminant nagana in Africa. Kinetoplastid flagellates contain their eponym kinetoplast DNA (kDNA), consisting of two types of interlocked circular DNA molecules: scores of maxicircles and thousands of minicircles. Maxicircles have typical mitochondrial genes, most of which are translatable only after RNA editing. Minicircles encode guide RNAs, required for decrypting the maxicircle transcripts. The life cycle of T. brucei involves a bloodstream stage (BS) in vertebrates and a procyclic stage (PS) in the tsetse fly vector. Partial [dyskinetoplastidy (Dk)] or total [akinetoplastidy (Ak)] loss of kDNA locks the trypanosome in the BS form. Transmission between vertebrates becomes mechanical without PS and tsetse mediation, allowing the parasite to spread outside the African tsetse belt. Trypanosoma equiperdum and Trypanosoma evansi are agents of dourine and surra, diseases of horses, camels, and water buffaloes. We have characterized representative strains of T. equiperdum and T. evansi by numerous molecular and classical parasitological approaches. We show that both species are actually strains of T. brucei, which lost part (Dk) or all (Ak) of their kDNA. These trypanosomes are not monophyletic clades and do not qualify for species status. They should be considered two subspecies, respectively T. brucei equiperdum and T. brucei evansi, which spontaneously arose recently. Dk/Ak trypanosomes may potentially emerge repeatedly from T. brucei.

Acetylation-dependent ADP-ribosylation by Trypanosoma brucei Sir2

Sirtuins are a highly conserved family of proteins implicated in diverse cellular processes such as gene silencing, aging, and metabolic regulation. Although many sirtuins catalyze a well characterized protein/histone deacetylation reaction, there are a number of reports that suggest protein ADP-ribosyltransferase activity. Here we explored the mechanisms of ADP-ribosylation using the Trypanosoma brucei Sir2 homologue TbSIR2rp1 as a model for sirtuins that reportedly display both activities. Steady-state kinetic analysis revealed a highly active histone deacetylase (k cat = 0.1 s(-1), with Km values of 42 microm and for NAD+ and 65 microm for acetylated substrate). A series of biochemical assays revealed that TbSIR2rp1 ADP-ribosylation of protein/histone requires an acetylated substrate. The data are consistent with two distinct ADP-ribosylation pathways that involve an acetylated substrate, NAD+ and TbSIR2rp1 as follows: 1) a noncatalytic reaction between the deacetylation product O-acetyl-ADP-ribose (or its hydrolysis product ADP-ribose) and histones, and 2) a more efficient mechanism involving interception of an ADP-ribose-acetylpeptide-enzyme intermediate by a side-chain nucleophile from bound histone. However, the sum of both ADP-ribosylation reactions was approximately 5 orders of magnitude slower than histone deacetylation under identical conditions. The biological implications of these results are discussed.

Activation of endocytosis as an adaptation to the mammalian host by trypanosomes.

Immune evasion in African trypanosomes is principally mediated by antigenic variation, but rapid internalization of surface-bound immune factors may contribute to survival. Endocytosis is upregulated approximately 10-fold in bloodstream compared to procyclic forms, and surface coat remodeling accompanies transition between these life stages. Here we examined expression of endocytosis markers in tsetse fly stages in vivo and monitored modulation during transition from bloodstream to procyclic forms in vitro. Among bloodstream stages nonproliferative stumpy forms have endocytic activity similar to that seen with rapidly dividing slender forms, while differentiation of stumpy forms to procyclic forms is accompanied by rapid down-regulation of Rab11 and clathrin, suggesting that modulation of endocytic and recycling systems accompanies this differentiation event. Significantly, rapid down-regulation of endocytic markers occurs upon entering the insect midgut and expression of Rab11 and clathrin remains low throughout subsequent development, which suggests that high endocytic activity is not required for remodeling the parasite surface or for survival within the fly. However, salivary gland metacyclic forms dramatically increase expression of clathrin and Rab11, indicating that emergence of mammalian infective forms is coupled to reacquisition of a high-activity endocytic-recycling system. These data suggest that high-level endocytosis in Trypanosoma brucei is an adaptation required for viability in the mammalian host.

Dileucine signal-dependent and AP-1-independent targeting of a lysosomal glycoprotein in Trypanosoma brucei

Sorting of trans-membrane proteins destined for the lysosome is achieved by selective inclusion into post-Golgi transport vesicles. In higher eukaryotes sorting may be mediated by a peptidic motif, principally acidic clusters and tyrosine- or dileucine-based cytoplasmic signals or by inclusion of mannose-6-phosphate (M6P) into the N-glycans of lysosomal proteins. In African trypanosomes a major lysosomal trans-membrane protein is CB-1/p67. The cytoplasmic domain of p67 lacks tyrosine and lysine, but does contain a canonical dileucine sequence embedded within an acidic region. AP-1, -3 and -4 adaptin complexes, which recognise tyrosine- and dileucine-sorting signals, are encoded by the trypanosome genome, but the genes for M6P-receptors or activities required to produce M6P are absent, suggesting that lysosomal delivery of p67 is most likely adaptin-mediated. By construction of p67 reporter constructs we show that the dileucine signal is necessary and sufficient for efficient lysosomal delivery of a trans-membrane protein in bloodstream stage trypanosomes. However, this targeting does not require AP-1, as knockdown of the trypanosome γ-adaptin subunit by RNAi has no detectable effect on the location or maturation of p67. These data suggest that p67 is targeted to the lysosome by dileucine-dependent but AP-1-independent mechanisms.

Targeted depletion of a mitochondrial nucleotidyltransferase suggests the presence of multiple enzymes that polymerize mRNA 3′ tails in Trypanosoma brucei mitochondria

Polyadenylation plays an important role in regulating RNA stability in Trypanosoma brucei mitochondria. To date, little is known about the enzymes responsible for the addition of mRNA 3′ tails in this system. In this study, we characterize a trypanosome homolog of the human mitochondrial poly(A) polymerase, which we term kPAP2. kPAP2 is mitochondrially localized and expressed in both bloodstream and procyclic form trypanosomes. Targeted gene depletion using RNAi showed that kPAP2 is not required for T. brucei growth in either bloodstream or procyclic life stages, nor is it essential for differentiation from bloodstream to procyclic form. We also demonstrate that steady state abundance of several mitochondrial RNAs was largely unaffected upon kPAP2 down-regulation. Interestingly, mRNA 3′ tail analysis of several mRNAs from both life cycle stages in uninduced kPAP2 RNAi cells demonstrated that tail length and uridine content are both regulated in a transcript-specific manner. mRNA-specific tail lengths were maintained upon kPAP2 depletion. However, the percentage of uridine residues in 3′ tails was increased, and conversely the percentage of adenosine residues was decreased, in a distinct subset of mRNAs when kPAP2 levels were down-regulated. Thus, kPAP2 apparently contributes to the incorporation of adenosine residues in 3′ tails of some, but not all, mitochondrial mRNAs. Together, these data suggest that multiple nucleotidyltransferases act on mitochondrial mRNA 3′ ends, and that these enzymes are somewhat redundant and subject to complex regulation.

Mitochondrial Fatty Acid Synthesis in Trypanosoma brucei

Whereas other organisms utilize type I or type II synthases to make fatty acids, trypanosomatid parasites such as Trypanosoma brucei are unique in their use of a microsomal elongase pathway (ELO) for de novo fatty acid synthesis (FAS). Because of the unusual lipid metabolism of the trypanosome, it was important to study a second FAS pathway predicted by the genome to be a type II synthase. We localized this pathway to the mitochondrion, and RNA interference (RNAi) or genomic deletion of acyl carrier protein (ACP) and beta-ketoacyl-ACP synthase indicated that this pathway is likely essential for bloodstream and procyclic life cycle stages of the parasite. In vitro assays show that the largest major fatty acid product of the pathway is C16, whereas the ELO pathway, utilizing ELOs 1, 2, and 3, synthesizes up to C18. To demonstrate mitochondrial FAS in vivo, we radio-labeled fatty acids in cultured procyclic parasites with [(14)C]pyruvate or [(14)C]threonine, either of which is catabolized to [(14)C]acetyl-CoA in the mitochondrion. Although some of the [(14)C]acetyl-CoA may be utilized by the ELO pathway, a striking reduction in radiolabeled fatty acids following ACP RNAi confirmed that it is also consumed by mitochondrial FAS. ACP depletion by RNAi or gene knockout also reduces lipoic acid levels and drastically decreases protein lipoylation. Thus, octanoate (C8), the precursor for lipoic acid synthesis, must also be a product of mitochondrial FAS. Trypanosomes employ two FAS systems: the unconventional ELO pathway that synthesizes bulk fatty acids and a mitochondrial pathway that synthesizes specialized fatty acids that are likely utilized intramitochondrially.

Intense Expression of GFP Gene in Gonads of Chicken Embryos by Transfecting Circulating Primordial Germ Cells <I>in vitro</I> and <I>in vivo</I>

The present study was carried out to develop techniques for introducing exogenous DNA into primordial germ cells (PGCs) and expressing the introduced DNA efficiently in the gonads of developing chicken embryos. PGCs circulating in the bloodstream (stages 14-15) were transfected with GFP gene in vitro or in vivo by lipofection or nucleofection. The manipulated PGCs successfully migrated to the germinal ridges and expressed GFP gene efficiently in the gonads of developing embryos. Intense GFP gene expression was observed in the gonads during the first 8 days following the transfection, during which period the sexual differentiation of gonads and germ cells (GCs) takes place. The GFP gene expression then gradually declined during subsequent embryonic development until hatching. When PGCs were transfected in vivo by lipofection with linearised plasmid DNA, GFP gene was detected in the gonads of 4.3% (19/442) of embryos examined at 20.5 days of culture, whereas less than 1% of embryos detected GFP gene in the gonads of embryos in which PGCs were transfected in vitro or with circular form plasmid DNA. In two of the embryos in which PGCs were transfected in vivo by lipofection with linearised plasmid DNA, GFP gene was expressed clearly in limited areas of the gonads of 20.5-day cultured embryos. The results obtained in this study suggest that the present in vitro and in vivo techniques for PGC manipulation provide a useful experimental system for studying gene functions in the sexual differentiation of gonads and GCs in early chicken embryos.

Knock-downs of Iron-Sulfur Cluster Assembly Proteins IscS and IscU Down-regulate the Active Mitochondrion of Procyclic Trypanosoma brucei

Transformation of the metabolically down-regulated mitochondrion of the mammalian bloodstream stage of Trypanosoma brucei to the ATP-producing mitochondrion of the insect procyclic stage is accompanied by the de novo synthesis of citric acid cycle enzymes and components of the respiratory chain. Because these metabolic pathways contain multiple iron-sulfur (FeS) proteins, their synthesis, including the formation of FeS clusters, is required. However, nothing is known about FeS cluster biogenesis in trypanosomes, organisms that are evolutionarily distant from yeast and humans. Here we demonstrate that two mitochondrial proteins, the cysteine desulfurase TbiscS and the metallochaperone TbiscU, are functionally conserved in trypanosomes and essential for this parasite. Knock-downs of TbiscS and TbiscU in the procyclic stage by means of RNA interference resulted in reduced activity of the marker FeS enzyme aconitase in both the mitochondrion and cytosol because of the lack of FeS clusters. Moreover, down-regulation of TbiscS and TbiscU affected the metabolism of procyclic T. brucei so that their mitochondria resembled the organelle of the bloodstream stage; mitochondrial ATP production was impaired, the activity of the respiratory chain protein complex ubiquinol-cytochrome-c reductase was reduced, and the production of pyruvate as an end product of glucose metabolism was enhanced. These results indicate that mitochondrial FeS cluster assembly is indispensable for completion of the T. brucei life cycle.

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.

An Aurora Kinase Homologue Is Involved in Regulating Both Mitosis and Cytokinesis in Trypanosoma brucei

The chromosomal passenger protein aurora kinases have been implicated in regulating chromosome segregation and cell division. Three aurora kinase homologues were identified (TbAUK1, -2 and -3) in the Trypanosome Genomic Data Base, and their expressions in the procyclic form of Trypanosoma brucei were knocked down individually by using the RNA interference technique. Only a knockdown of TbAUK1 arrested the cells in G(2)/M phase with each cell showing an extended posterior end, two kinetoplasts, and an enlarged nucleus, apparently the result of an inhibited kinetoplast multiplication and a failed mitosis. There is no mitotic spindle structure in the TbAUK1-depleted cell. The two kinetoplasts moved apart from each other but stopped just before cytokinesis, suggesting that cytokinesis was blocked in its early phase. Overexpression of TbAUK1 in the cells resulted in little change in cell growth. By immunofluorescence, TbAUK1 was primarily localized to the nucleus in interphase and to the mitotic spindle during apparent metaphase and anaphase. Thus, differing from other eukaryotes, TbAUK1 has an apparent triple function in coupling mitosis and kinetoplast replication with cytokinesis in T. brucei. T. brucei polo-like kinase, previously identified as the initiator of cytokinesis without apparent involvement in mitosis in the trypanosome, was either depleted or overexpressed in the TbAUK1-deficient cells. A dominant TbAUK1-depleted phenotype was demonstrated in both cases, suggesting that TbAUK1 plays an essential role in cytokinesis that cannot be affected by changes in the level of T. brucei polo-like kinase. To our knowledge, this is the first time that the function of an aurora B-like kinase is a prerequisite for polo-like kinase action in initiating cytokinesis. TbAUK1 is also the first identified protein that couples both mitosis and kinetoplast replication with cytokinesis in the trypanosome.

The F1-ATP synthase complex in bloodstream stage trypanosomes has an unusual and essential function

The F1-ATP synthase complex in bloodstream stage trypanosomes has an unusual and essential function