The protozoan parasite Trypanosoma brucei lives in the bloodstream of vertebrates or in a tsetse fly. Expression of a GPI-phospholipase C polypeptide (GPI-PLCp) in the parasite is restricted to the bloodstream form. Events controlling the amount of GPI-PLCp expressed during differentiation are not completely understood. Our metabolic “pulse-chase” analysis reveals that GPI-PLCp is stable in bloodstream form. However, during differentiation of bloodstream to insect stage (procyclic) T. brucei, translation GPI-PLC mRNA ceases within 8 h of initiating transformation. GPI-PLCp is not lost precipitously from newly-transformed procyclic trypanosomes. Nascent procyclics contain 400-fold more GPI-PLCp than established insect stage T. brucei. Reduction of GPI-PLCp in early-stage procyclics is linked to parasite replication. Sixteen cell divisions are required to reduce the amount of GPI-PLCp in newly-differentiated procyclics to levels present in the established procyclic. GPI-PLCp is retained in strains of T. brucei that fail to replicate after differentiation of the bloodstream to the procyclic form. Thus, at least two factors control levels of GPI-PLCp during differentiation of bloodstream T. brucei; (i) repression of GPI-PLC mRNA translation, and (ii) sustained replication of newly-transformed procyclic T. brucei. These studies illustrate the importance of repeated cell divisions in controlling the steady-state amount of GPI-PLCp during differentiation of the African trypanosome.
The protozoan parasite Trypanosoma brucei has a complex digenetic lifecycle between a mammalian host and an insect vector, and adaption of its proteome between lifecycle stages is essential to its survival and virulence. We have optimized a procedure for growing Trypanosoma brucei procyclic form cells in conditions suitable for stable isotope labeling by amino acids in culture (SILAC) and report a comparative proteomic analysis of cultured procyclic form and bloodstream form T. brucei cells. In total we were able to identify 3959 proteins and quantify SILAC ratios for 3553 proteins with a false discovery rate of 0.01. A large number of proteins (10.6%) are differentially regulated by more the 5-fold between lifecycle stages, including those involved in the parasite surface coat, and in mitochondrial and glycosomal energy metabolism. Our proteomic data is broadly in agreement with transcriptomic studies, but with significantly larger fold changes observed at the protein level than at the mRNA level.
Trypanosomes are protozoan parasites that cycle between a mammalian host (bloodstream form) and an insect host, the Tsetse fly (procyclic stage). In trypanosomes, all mRNAs are trans-spliced as part of their maturation. Genome-wide analysis of trans-splicing indicates the existence of alternative trans-splicing, but little is known regarding RNA-binding proteins that participate in such regulation. In this study, we performed functional analysis of the Trypanosoma brucei heterogeneous nuclear ribonucleoproteins (hnRNP) F/H homologue, a protein known to regulate alternative splicing in metazoa. The hnRNP F/H is highly expressed in the bloodstream form of the parasite, but is also functional in the procyclic form. Transcriptome analyses of RNAi-silenced cells were used to deduce the RNA motif recognized by this protein. A purine rich motif, AAGAA, was enriched in both the regulatory regions flanking the 3′ splice site and poly (A) sites of the regulated genes. The motif was further validated using mini-genes carrying wild-type and mutated sequences in the 3′ and 5′ UTRs, demonstrating the role of hnRNP F/H in mRNA stability and splicing. Biochemical studies confirmed the binding of the protein to this proposed site. The differential expression of the protein and its inverse effects on mRNA level in the two lifecycle stages demonstrate the role of hnRNP F/H in developmental regulation.
Porin is the most abundant outer membrane (OM) protein of mitochondria. It forms the aqueous channel on the mitochondrial OM and mediates major metabolite flux between mitochondria and cytosol. Mitochondrial porin in Trypanosoma brucei, a unicellular parasitic protozoan and the causative agent of African trypanosomiasis, possesses a β-barrel structure similar to the bacterial OM porin OmpA. T. brucei porin (TbPorin) is present as a monomer as well as an oligomer on the mitochondrial OM, and its expression is developmentally regulated. In spite of its distinct structure, the TbPorin function is similar to those of other eukaryotic porins. TbPorin RNA interference (RNAi) reduced cell growth in both procyclic and bloodstream forms. The depletion of TbPorin decreased ATP production by inhibiting metabolite flux through the OM. Additionally, the level of trypanosome alternative oxidase (TAO) decreased, whereas the levels of cytochrome-dependent respiratory complexes III and IV increased in TbPorin-depleted mitochondria. Furthermore, the depletion of TbPorin reduced cellular respiration via TAO, which is not coupled with oxidative phosphorylation, but increased the capacity for cyanide-sensitive respiration. Together, these data reveal that TbPorin knockdown reduced the mitochondrial ATP level, which in turn increased the capacity of the cytochrome-dependent respiratory pathway (CP), in an attempt to compensate for the mitochondrial energy crisis. However, a simultaneous decrease in the substrate-level phosphorylation due to TbPorin RNAi caused growth inhibition in the procyclic form. We also found that the expressions of TAO and CP proteins are coordinately regulated in T. brucei according to mitochondrial energy demand.
Trypanosoma brucei is the causative agent of human African sleeping sickness and Nagana in cattle. In addition to being an important pathogen T. brucei has developed into a model system in cell biology.
Using Stable Isotope Labelling of Amino acids in Cell culture (SILAC) in combination with mass spectrometry we determined the abundance of >1600 proteins in the long slender (LS), short stumpy (SS) mammalian bloodstream form stages relative to the procyclic (PC) insect-form stage. In total we identified 2645 proteins, corresponding to ~30% of the total proteome and for the first time present a comprehensive overview of relative protein levels in three life stages of the parasite.
We can show the extent of pre-adaptation in the SS cells, especially at the level of the mitochondrial proteome. The comparison to a previously published report on monomorphic in vitro grown bloodstream and procyclic T. brucei indicates a loss of stringent regulation particularly of mitochondrial proteins in these cells when compared to the pleomorphic in vivo situation. In order to better understand the different levels of gene expression regulation in this organism we compared mRNA steady state abundance with the relative protein abundance-changes and detected moderate but significant correlation indicating that trypanosomes possess a significant repertoire of translational and posttranslational mechanisms to regulate protein abundance.
In 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.
Using 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.
These 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.
The cytoskeleton of Trypanosoma brucei, a unicellular eukaryote and a parasitic protozoan, is defined by the subpellicular microtubule corset that is arranged underneath the plasma membrane. We recently identified two orphan kinesins, TbKIN-C and TbKIN-D, that cooperate to regulate the organization of the subpellicular microtubule corset and thereby maintain cell morphology in the procyclic form of T. brucei. In this report, we characterize the function of TbKIN-C and TbKIN-D in the bloodstream form of T. brucei and investigate their functional cooperation in both the bloodstream and procyclic forms. TbKIN-C and TbKIN-D form a tight complex in vivo in the bloodstream form. TbKIN-C is strongly enriched at the posterior tip of the cell, whereas TbKIN-D is distributed throughout the cell body at all cell cycle stages. RNAi of TbKIN-C or TbKIN-D in the bloodstream form inhibits cell proliferation and leads to cell death, due to cytokinesis defects. RNAi of TbKIN-C and TbKIN-D also results in defects in basal body segregation, but does not affect the synthesis and segregation of the flagellum and the flagellum attachment zone (FAZ) filament. Knockdown of TbKIN-C and TbKIN-D does not disrupt the organization of the subpellicular microtubule corset, but produces multinucleated cells with an enlarged flagellar pocket and misplaced flagella. Interestingly, depletion of TbKIN-C results in rapid degradation of TbKIN-D and, similarly, knockdown of TbKIN-C destabilizes TbKIN-D, suggesting that formation of TbKIN-C/TbKIN-D complex stabilizes both kinesins and is required for the two kinesins to execute their essential cellular functions. Altogether, our results demonstrate the essential role of the two kinesins in cell morphogenesis and cytokinesis in the bloodstream form and the requirement of heteromeric complex formation for maintaining the stability of the two kinesins.
Trypanosoma brucei brucei has two distinct developmental stages, the procyclic stage in the insect and the bloodstream stage in the mammalian host. The significance of each developmental stage is punctuated by specific changes in metabolism. In the insect, T. b. brucei is strictly dependent on mitochondrial function and thus respiration to generate the bulk of its ATP, whereas in the mammalian host it relies heavily on glycolysis. These observations have raised questions about the importance of mitochondrial function in the bloodstream. Peculiarly, akinetoplastic strains of Trypanosoma brucei evansi that lack mitochondrial DNA do exist in the wild and are developmentally locked in the glycolysis-dependent bloodstream stage. Using RNAi we show that two mitochondrion-imported proteins, mitochondrial RNA polymerase and guide RNA associated protein 1, are still imported into the nucleic acids-lacking organelle of T. b. evansi, making the need for these proteins futile. We also show that, like in the T. b. brucei procyclic stage, the mitochondria of both bloodstream stage of T. b. brucei and T. b. evansi import various tRNAs, including those that undergo thiolation. However, we were unable to detect mitochondrial thiolation in the akinetoplastic organelle. Taken together, these data suggest a lack of connection between nuclear and mitochondrial communication in strains of T. b. evansi that lost mitochondrial genome and that do not required an insect vector for survival.
Trypanosoma; tRNA; protein import; mitochondrion; kinetoplast
Studies on the cell-cycle of Trypanosoma brucei have revealed several unusual characteristics that differ from the model eukaryotic organisms. However, the inability to isolate homogenous populations of parasites in distinct cell-cycle stages has limited the analysis of trypanosome cell division and complicated the understanding of mutant phenotypes with possible impact on cell-cycle related events. Although hydroxyurea-induced cell-cycle arrest in procyclic and bloodstream forms has been applied recently with success, such block-release protocols can complicate the analysis of cell-cycle regulated events and have the potential to disrupt important cell-cycle checkpoints. An alternative approach based on flow cytometry of parasites stained with Vybrant DyeCycle Orange circumvents this problem, but is restricted to procyclic form parasites. Here, we apply Vybrant Dyecycle Violet staining coupled with flow cytometry to effectively select different cell-cycle stages of bloodstream form trypanosomes. Moreover, the sorted parasites remain viable, although synchrony is rapidly lost. This method enables cell-cycle enrichment of populations of trypanosomes in their mammal infective stage, particularly at the G1 phase.
Trypanosoma brucei; Cell-cycle; Vybrant DyeCycle Violet; Flow cytometry; Synchronisation
The compartmentalization of glycolytic enzymes into specialized organelles, the glycosomes, allows the bloodstream form of Trypanosoma brucei to rely solely on glycolysis for its energy production. The biogenesis of glycosomes in these parasites has been studied intensively as a potential target for chemotherapy. We have adapted the recently developed methods for stable transformation of T. brucei to the in vivo analysis of glycosomal protein import. Firefly luciferase, a peroxisomal protein in the lantern of the insect, was expressed in stable transformants of the procyclic form of T. brucei, where it was found to accumulate inside the glycosomes. Mutational analysis of the peroxisomal targeting signal serine-lysine-leucine (SKL) located at the C-terminus of luciferase showed that replacement of the serine residue (Serine548) with a small neutral amino acid (A, C, G, H, N, P, T) still resulted in an import efficiency of 50-100% of the wild-type luciferase. Lysine549 could be substituted with an amino acid capable of hydrogen bonding (H, M, N, Q, R, S), whereas the C-terminal leucine550 could be replaced with a subset of hydrophobic amino acids (I, M, Y). Thus, a peroxisome-like C-terminal SKL-dependent targeting mechanism may function in T. brucei to import luciferase into the glycosomes. However, a few significant differences exist between the glycosomal targeting signals identified here and the tripeptide sequences that direct proteins to mammalian or yeast peroxisomes.
Most mitochondrial mRNAs in Trypanosoma brucei require RNA editing for maturation and translation. The edited RNAs primarily encode proteins of the oxidative phosphorylation system. These parasites undergo extensive changes in energy metabolism between the insect and bloodstream stages which are mirrored by alterations in RNA editing. Two U-specific exonucleases, KREX1 and KREX2, are both present in protein complexes (editosomes) that catalyze RNA editing but the relative roles of each protein are not known.
The requirement for KREX2 for RNA editing in vivo was assessed in both procyclic (insect) and bloodstream form parasites by methods that use homologous recombination for gene elimination. These studies resulted in null mutant cells in which both alleles were eliminated. The viability of these cells demonstrates that KREX2 is not essential in either life cycle stage, despite certain defects in RNA editing in vivo. Furthermore, editosomes isolated from KREX2 null cells require KREX1 for in vitro U-specific exonuclease activity.
KREX2 is a U-specific exonuclease that is dispensable for RNA editing in vivo in T. brucei BFs and PFs. This result suggests that the U deletion activity, which is required for RNA editing, is primarily mediated in vivo by KREX1 which is normally found associated with only one type of editosome. The retention of the KREX2 gene implies a non-essential role or a role that is essential in other life cycle stages or conditions.
The coat of Trypanosoma brucei consists mainly of glycosylphosphatidylinositol-anchored proteins that are present in several million copies and are characteristic of defined stages of the life cycle. While these major components of the coats of bloodstream forms and procyclic (insect midgut) forms are well characterised, very little is known about less abundant stage-regulated surface proteins and their roles in infection and transmission. By creating epitope-tagged versions of procyclic-specific surface antigen 2 (PSSA-2) we demonstrated that it is a membrane-spanning protein that is expressed by several different life cycle stages in tsetse flies, but not by parasites in the mammalian bloodstream. In common with other membrane-spanning proteins in T. brucei, PSSA-2 requires its cytoplasmic domain in order to exit the endoplasmic reticulum. Correct localisation of PSSA-2 requires phosphorylation of a cytoplasmic threonine residue (T305), a modification that depends on the presence of TbMAPK4. Mutation of T305 to alanine (T305A) has no effect on the localisation of the protein in cells that express wild type PSSA-2. In contrast, this protein is largely intracellular when expressed in a null mutant background. A variant with a T305D mutation gives strong surface expression in both the wild type and null mutant, but slows growth of the cells, suggesting that it may function as a dominant negative mutant. The PSSA-2 null mutant exhibits no perceptible phenotype in culture and is fully competent at establishing midgut infections in tsetse, but is defective in colonising the salivary glands and the production of infectious metacyclic forms. Given the protein's structure and the effects of mutation of T305 on proliferation and localisation, we postulate that PSSA-2 might sense and transmit signals that contribute to the parasite's decision to divide, differentiate or migrate.
PUF proteins are a conserved family of RNA binding proteins found in all eukaryotes examined so far. This study focussed on PUF5, one of 11 PUF family members encoded in the Trypanosoma brucei genome. Native PUF5 is present at less than 50000 molecules per cell in both bloodstream and procyclic form trypanosomes. C-terminally myc-tagged PUF5 was mainly found in the cytoplasm and could be cross-linked to RNA. PUF5 knockdown by RNA interference had no effect on the growth of bloodstream forms. Procyclic forms lacking PUF5 grew normally, but expression of PUF5 bearing a 21 kDa tandem affinity purification tag inhibited growth. Knockdown of PUF5 did not have any effect on the ability of trypanosomes to differentiate from the mammalian to the insect form of the parasite.
The cell surface glycoconjugates of trypanosomatid parasites are intimately involved in parasite survival, infectivity, and virulence in their insect vectors and mammalian hosts. Although there is a considerable body of work describing their structure, biosynthesis, and function, little is known about the sugar nucleotide pools that fuel their biosynthesis. In order to identify and quantify parasite sugar nucleotides, we developed an analytical method based on liquid chromatography-electrospray ionization-tandem mass spectrometry using multiple reaction monitoring. This method was applied to the bloodstream and procyclic forms of Trypanosoma brucei, the epimastigote form of T. cruzi, and the promastigote form of Leishmania major. Five sugar nucleotides, GDP-α-d-mannose, UDP-α-d-N-acetylglucosamine, UDP-α-d-glucose, UDP-α-galactopyranose, and GDP-β-l-fucose, were common to all three species; one, UDP-α-d-galactofuranose, was common to T. cruzi and L. major; three, UDP-β-l-rhamnopyranose, UDP-α-d-xylose, and UDP-α-d-glucuronic acid, were found only in T. cruzi; and one, GDP-α-d-arabinopyranose, was found only in L. major. The estimated demands for each monosaccharide suggest that sugar nucleotide pools are turned over at very different rates, from seconds to hours. The sugar nucleotide survey, together with a review of the literature, was used to define the routes to these important metabolites and to annotate relevant genes in the trypanosomatid genomes.
We examined the expression of a nucleus-encoded mitochondrial protein, cytochrome c, during the life cycle of Trypanosoma brucei. The bloodstream forms of T. brucei, the long slender and short stumpy trypanosomes, have inactive mitochondria with no detectable cytochrome-mediated respiration. The insect form of T. brucei, the procyclic trypanosomes, has fully functional mitochondria. Cytochrome c is spectrally undetectable in the bloodstream forms of trypanosomes, but during differentiation to the procyclic form, spectrally detected holo-cytochrome c accumulates rapidly. We have purified T. brucei cytochrome c and raised antibodies that react to both holo- and apo-cytochrome c. In addition, we isolated a partial cDNA to trypanosome cytochrome c. An examination of protein expression and steady-state mRNA levels in T. brucei indicated that bloodstream trypanosomes did not express cytochrome c but maintained significant steady-state levels of cytochrome c mRNA. The results suggest that in T. brucei, cytochrome c is developmentally regulated by a posttranscriptional mechanism which prevents either translation or accumulation of cytochrome c in the bloodstream trypanosomes.
Trypanosome alternative oxidase (TAO) and the cytochrome oxidase (COX) are two developmentally regulated terminal oxidases of the mitochondrial electron transport chain in Trypanosoma brucei. Here, we have compared the import of TAO and cytochrome oxidase subunit IV (COIV), two stage specific nuclear encoded mitochondrial proteins, into the bloodstream and procyclic form mitochondria of T. brucei to understand the import processes in two different developmental stages. Under in vitro conditions TAO and COIV were imported and processed into isolated mitochondria from both the bloodstream and procyclic forms. With mitochondria isolated from the procyclic form, the import of TAO and COIV was dependent on the mitochondrial inner membrane potential (Δψ) and required protein(s) on the outer membrane. Import of these proteins also depended on the presence of both internal and external ATP. However, import of TAO and COIV into isolated mitochondria from the bloodstream form was not inhibited after the mitochondrial Δψ was dissipated by valinomycin, CCCP, or valinomycin and oligomycin in combination. In contrast, import of these proteins into bloodstream mitochondria was abolished after the hydrolysis of ATP by apyrase or removal of the ATP and ATP-generating system, suggesting that import is dependent on the presence of external ATP. Together, these data suggest that nuclear encoded proteins such as TAO and COIV are imported in the mitochondria of the bloodstream and the procyclic forms via different mechanism. Differential import conditions of nuclear encoded mitochondrial proteins of T. brucei possibly help it to adapt to different life forms.
Trypanosoma brucei; Mitochondrial protein import; Membrane potential; Trypanosome alternative oxidase
Trypanosomes are protozoan agents of major parasitic diseases such as Chagas' disease in South America and sleeping sickness of humans and nagana disease of cattle in Africa. They are transmitted to mammalian hosts by specific insect vectors. Their life cycle consists of a succession of differentiation and growth phases requiring regulated gene expression to adapt to the changing extracellular environment. Typical of such stage-specific expression is that of the major surface antigens of Trypanosoma brucei, procyclin in the procyclic (insect) form and the variant surface glycoprotein (VSG) in the bloodstream (mammalian) form. In trypanosomes, the regulation of gene expression is effected mainly at posttranscriptional levels, since primary transcription of most of the genes occurs in long polycistronic units and is constitutive. The transcripts are processed by transsplicing and polyadenylation under the influence of intergenic polypyrimidine tracts. These events show some developmental regulation. Untranslated sequences of the mRNAs seem to play a prominent role in the stage-specific control of individual gene expression, through a modulation of mRNA abundance. The VSG and procyclin transcription units exhibit particular features that are probably related to the need for a high level of expression. The promoters and RNA polymerase driving the expression of these units resemble those of the ribosomal genes. Their mutually exclusive expression is ensured by controls operating at several levels, including RNA elongation. Antigenic variation in the bloodstream is achieved through DNA rearrangements or alternative activation of the telomeric VSG gene expression sites. Recent discoveries, such as the existence of a novel nucleotide in telomeric DNA and the generation of point mutations in VSG genes, have shed new light on the mechanisms and consequences of antigenic variation.
A series of azasterol derivatives, designed as potential inhibitors of the Δ24-sterol methyltransferase enzyme (24-SMT), were synthesized and evaluated for their activities against parasitic protozoa. Values in the nanomolar range were obtained for 50% effective dose against the Trypanosoma brucei subsp. rhodesiense bloodstream form cultured in vitro. In order to investigate the mode of action, Trypanosoma brucei subsp. brucei 24-SMT was cloned and overexpressed and compounds were assayed for inhibitory activity. None of the inhibitors tested appeared to be active against the enzyme. Sterol composition analysis showed that only cholestane type sterols are present in membranes of bloodstream forms while ergosterol is a major component of procyclic sterol extracts. Interestingly, Northern blot analysis showed the presence of 24-SMT mRNA in both the procyclic and the bloodstream forms of the parasite, although levels of mRNA were threefold lower in the latter. Likewise, Western blot analysis and activity determinations evidenced the existence of active enzyme in both forms of the parasite. We conclude that the designed compounds act at sites other than 24-SMT in Trypanosoma brucei.
The survival of Trypanosoma brucei, the causative agent of Sleeping Sickness and Nagana, is facilitated by the expression of a dense surface coat of glycosylphosphatidylinositol (GPI)-anchored proteins in both its mammalian and tsetse fly hosts. We have characterized T. brucei GPI8, the gene encoding the catalytic subunit of the GPI:protein transamidase complex that adds preformed GPI anchors onto nascent polypeptides. Deletion of GPI8 (to give Δgpi8) resulted in the absence of GPI-anchored proteins from the cell surface of procyclic form trypanosomes and accumulation of a pool of non–protein-linked GPI molecules, some of which are surface located. Procyclic Δgpi8, while viable in culture, were unable to establish infections in the tsetse midgut, confirming that GPI-anchored proteins are essential for insect-parasite interactions. Applying specific inducible GPI8 RNAi with bloodstream form parasites resulted in accumulation of unanchored variant surface glycoprotein and cell death with a defined multinuclear, multikinetoplast, and multiflagellar phenotype indicative of a block in cytokinesis. These data show that GPI-anchored proteins are essential for the viability of bloodstream form trypanosomes even in the absence of immune challenge and imply that GPI8 is important for proper cell cycle progression.
Trypanosoma brucei, the etiologic agent of African sleeping sickness, divides into insect (procyclic) and bloodstream forms. These two forms are subject to distinct cell cycle regulations, with cytokinesis controlled primarily by basal body/kinetoplast segregation in the procyclic form but by mitosis in the bloodstream form. Polo-like kinases (PLKs), known to play essential roles in regulating both mitosis and cytokinesis among eukaryotes, have a homologue in T. brucei, TbPLK, which regulates only cytokinesis. In our previous study, overexpressed triply hemagglutinin-tagged TbPLK (TbPLK-3HA) in the procyclic form localized to a mid-dorsal point and the anterior tip of the cell along the flagellum attachment zone (FAZ). In our current study, TbPLK-3HA expressed at the endogenous level was identified at the same dorsal location of both procyclic and bloodstream forms, albeit it was no longer detectable at the anterior tip of the cell. Endogenously expressed TbPLK fused with an enhanced yellow fluorescent protein (EYFP) localized to the same dorsal location along the FAZs in living procyclic and bloodstream cells. Fluorescence-activated cell sorter analysis of hydroxyurea-synchronized procyclic cells revealed that TbPLK-EYFP emerges during S phase, persists through G2/M phase, and vanishes in G1 phase. An indicated TbPLK-EYFP association with the FAZs of G2/M cells may thus represent a timely localization to a potential initiation site of cytokinesis, which agrees with the recognized role of TbPLK in cytokinetic initiation.
Ubiquitous among eukaryotes, lipid droplets are organelles that function to coordinate intracellular lipid homeostasis. Their morphology and abundance is affected by numerous genes, many of which are involved in lipid metabolism. In this report we identify a Trypanosoma brucei protein kinase, LDK, and demonstrate its localization to the periphery of lipid droplets. Association with lipid droplets was abrogated when the hydrophobic domain of LDK was deleted, supporting a model in which the hydrophobic domain is associated with or inserted into the membrane monolayer of the organelle. RNA interference knockdown of LDK modestly affected the growth of mammalian bloodstream-stage parasites but did not affect the growth of insect (procyclic)-stage parasites. However, the abundance of lipid droplets dramatically decreased in both cases. This loss was dominant over treatment with myriocin or growth in delipidated serum, both of which induce lipid body biogenesis. Growth in delipidated serum also increased LDK autophosphorylation activity. Thus, LDK is required for the biogenesis or maintenance of lipid droplets and is one of the few protein kinases specifically and predominantly associated with an intracellular organelle.
The protozoan parasite, Trypanosoma brucei, is spread by the tsetse fly and causes trypanosomiasis in humans and animals. Both the life cycle and cell cycle of the parasite are complex. Trypanosomes have eleven cdc2-related kinases (CRKs) and ten cyclins, an unusually large number for a single celled organism. To date, relatively little is known about the function of many of the CRKs and cyclins, and only CRK3 has previously been shown to be cyclin-dependent in vivo. Here we report the identification of a previously uncharacterised CRK:cyclin complex between CRK12 and the putative transcriptional cyclin, CYC9. CRK12:CYC9 interact to form an active protein kinase complex in procyclic and bloodstream T. brucei. Both CRK12 and CYC9 are essential for the proliferation of bloodstream trypanosomes in vitro, and we show that CRK12 is also essential for survival of T. brucei in a mouse model, providing genetic validation of CRK12:CYC9 as a novel drug target for trypanosomiasis. Further, functional characterisation of CRK12 and CYC9 using RNA interference reveals roles for these proteins in endocytosis and cytokinesis, respectively.
The evolutionarily ancient parasite, Trypanosoma brucei, is unusual in that the majority of its genes are regulated post-transcriptionally, leading to the suggestion that transcript abundance of most genes does not vary significantly between different life cycle stages despite the fact that the parasite undergoes substantial cellular remodelling and metabolic changes throughout its complex life cycle. To investigate this in the clinically relevant sub-species, Trypanosoma brucei gambiense, which is the causative agent of the fatal human disease African sleeping sickness, we have compared the transcriptome of two different life cycle stages, the potentially human-infective bloodstream forms with the non-human-infective procyclic stage using digital gene expression (DGE) analysis.
Over eleven million unique tags were generated, producing expression data for 7360 genes, covering 81% of the genes in the genome. Compared to microarray analysis of the related T. b. brucei parasite, approximately 10 times more genes with a 2.5-fold change in expression levels were detected. The transcriptome analysis revealed the existence of several differentially expressed gene clusters within the genome, indicating that contiguous genes, presumably from the same polycistronic unit, are co-regulated either at the level of transcription or transcript stability.
DGE analysis is extremely sensitive for detecting gene expression differences, revealing firstly that a far greater number of genes are stage-regulated than had previously been identified and secondly and more importantly, this analysis has revealed the existence of several differentially expressed clusters of genes present on what appears to be the same polycistronic units, a phenomenon which had not previously been observed in microarray studies. These differentially regulated clusters of genes are in addition to the previously identified RNA polymerase I polycistronic units of variant surface glycoproteins and procyclin expression sites, which encode the major surface proteins of the parasite. This raises a number of questions regarding the function and regulation of the gene clusters that clearly warrant further study.
In Trypanosoma brucei, endocytosis is developmentally regulated and is substantially more active in the mammalian infective stage, where it likely plays a role in immune evasion. The small GTPase TbRAB11 is highly expressed in the mammalian stage and mediates recycling of glycosylphosphatidylinositol-anchored proteins, including the variant surface glycoprotein (VSG) and the transferrin receptor, plus trafficking of internalized anti-VSG antibody and transferrin. No function has been assigned to TbRAB11 in the procyclic (insect) stage trypanosome. The importance of TbRAB11 to both bloodstream and procyclic form viability was assessed by RNA interference (RNAi). Suppression of TbRAB11 in the bloodstream form was rapidly lethal and led to cells with round morphology and an enlarged flagellar pocket. TbRAB11 RNAi was also lethal in procyclic forms, which also became rounded, but progression to cell death was significantly slower and the flagellar pocket remained normal. In bloodstream forms, silencing of TbRAB11 had no effect on exocytosis of newly synthesized VSG, fluid-phase endocytosis, or transferrin uptake, but export of internalized transferrin was inhibited. Lectin endocytosis assays revealed a block to postendosomal transport mediated by suppressing TbRAB11. By contrast, in procyclic forms, depletion of TbRAB11 blocks both fluid-phase endocytosis and internalization of surface proteins. In normal bloodstream forms, most VSG is recycled, but in procyclics, internalized surface proteins accumulated in the lysosome. These data demonstrate that TbRAB11 controls recycling and is essential in both life stages of T. brucei but that its primary role is subject to developmental variation.
Trypanosomatid parasites possess extremely divergent transcription factors whose identification typically relied on biochemical, structural and functional analyses because they could not be identified by standard sequence analysis. For example, subunits of the Trypanosoma brucei mediator and class I transcription factor A (CITFA) have no sequence resemblance to putative counterparts in higher eukaryotes. Therefore, homologous in vitro transcription systems have been crucial in evaluating the transcriptional roles of T. brucei proteins but so far such systems have been restricted to the insect-stage, procyclic form (PF) of the parasite. Here, we report the development of a homologous system for the mammalian-infective, bloodstream form (BF) of T. brucei which supports accurately initiated transcription from three different RNA polymerase (pol) I promoters as well as from the RNA pol II-recruiting spliced leader RNA gene promoter. The system is based on a small scale extract preparation procedure which accommodates the low cell densities obtainable in BF culture. BF and PF systems behave surprisingly similar and we show that the CITFA complex purified from procyclic extract is fully functional in the BF system indicating that the transcriptional machinery in general is equivalent in both life cycle stages. A notable difference, however, was observed with the RNA pol I-recruiting GPEET procyclin promoter whose reduced promoter strength and increased sensitivity to manganese ions in the BF system suggests the specific presence of a transcriptional activator in the PF system.