Trypanosoma brucei glycosomal glyceraldehyde-3-phosphate dehydrogenase genes are stage-regulated at the transcriptional level.
ABSTRACT: Regions 5' of the glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) gene from Trypanosoma brucei were tested for their ability to promote chloramphenicol acetyl-transferase (CAT) expression on reintroduction by electroporation into the parasite. Deletion analysis mapped the gGAPDH promoter to within 403 nts of the start of translation. A transcription initiation site was mapped at around -190 nts from the ATG start codon by RNase protection and by primer extension. The higher expression of gGAPDH in bloodstream T. brucei, compared to procyclic (insect) forms, was largely attributed to differences in promoter activity. The gGAPDH promoter gave rise to relatively high CAT signals upon transfection into bloodstream T. brucei and relatively low signals in procyclic T. brucei, compared with levels resulting from transfection with the procyclic acidic repetitive protein (PARP) promoter. In addition, RNase protection data showed a higher level of gGAPDH primary transcripts in bloodstream. T. brucei. The PARP mini-exon addition region abolished transient CAT expression directed by either the gGAPDH or PARP promoters in bloodstream T. brucei implying that transplicing can be a point of stage-specific gene regulation.
Project description:The bloodstream lifecycle stage of the kinetoplastid parasite Trypanosoma brucei relies solely on glucose metabolism for ATP production, which occurs in peroxisome-like organelles (glycosomes). Many studies have been conducted on glucose uptake and metabolism, but none thus far have been able to monitor changes in cellular and organellar glucose concentration in live parasites. We have developed a non-destructive technique for monitoring changes in cytosolic and glycosomal glucose levels in T. brucei using a fluorescent protein biosensor (FLII12Pglu-700??6) in combination with flow cytometry. T. brucei parasites harboring the biosensor allowed for observation of cytosolic glucose levels. Appending a type 1 peroxisomal targeting sequence caused biosensors to localize to glycosomes, which enabled observation of glycosomal glucose levels. Using this approach, we investigated cytosolic and glycosomal glucose levels in response to changes in external glucose or 2-deoxyglucose concentration. These data show that procyclic form and bloodstream form parasites maintain different glucose concentrations in their cytosol and glycosomes. In procyclic form parasites, the cytosol and glycosomes maintain indistinguishable glucose levels (3.4 ± 0.4mM and 3.4 ± 0.5mM glucose respectively) at a 6.25mM external glucose concentration. In contrast, bloodstream form parasites maintain glycosomal glucose levels that are ~1.8-fold higher than the surrounding cytosol, equating to 1.9 ± 0.6mM in cytosol and 3.5 ± 0.5mM in glycosomes. While the mechanisms of glucose transport operating in the glycosomes of bloodstream form T. brucei remain unresolved, the methods described here will provide a means to begin to dissect the cellular machinery required for subcellular distribution of this critical hexose.
Project description:?-Ketoglutarate decarboxylase (?-KDE1) is a Krebs cycle enzyme found in the mitochondrion of the procyclic form (PF) of Trypanosoma brucei. The bloodstream form (BF) of T. brucei lacks a functional Krebs cycle and relies exclusively on glycolysis for ATP production. Despite the lack of a functional Krebs cycle, ?-KDE1 was expressed in BF T. brucei and RNA interference knockdown of ?-KDE1 mRNA resulted in rapid growth arrest and killing. Cell death was preceded by progressive swelling of the flagellar pocket as a consequence of recruitment of both flagellar and plasma membranes into the pocket. BF T. brucei expressing an epitope-tagged copy of ?-KDE1 showed localization to glycosomes and not the mitochondrion. We used a cell line transfected with a reporter construct containing the N-terminal sequence of ?-KDE1 fused to green fluorescent protein to examine the requirements for glycosome targeting. We found that the N-terminal 18 amino acids of ?-KDE1 contain overlapping mitochondrion- and peroxisome-targeting sequences and are sufficient to direct localization to the glycosome in BF T. brucei. These results suggest that ?-KDE1 has a novel moonlighting function outside the mitochondrion in BF T. brucei.
Project description:The actin genes of Trypanosoma brucei are transcribed constitutively during the parasite life-cycle, by a polymerase sensitive to alpha-amanitin. The start region of the actin gene transcription unit was mapped by virtue of the accumulation of promoter-proximal transcripts which occurs following moderate UV irradiation. This region, located about 4 kilobases upstream from the genes, was able to direct transient expression of the bacterial Chloramphenicol Acetyl Transferase (CAT) gene in both bloodstream and procyclic forms of the parasite. The essential region of the promoter was defined by deletion, and appeared to be within 600 bp upstream from the putative transcription start site. It does not share significant homology with the other trypanosome promoters described so far (VSG, procyclin, rDNA), which all direct alpha-amanitin resistant transcription.
Project description:KREPB5 is an essential component of ∼ 20S editosomes in Trypanosoma brucei which contains a degenerate, noncatalytic RNase III domain. To explore the function of this protein, we used a novel approach to make and screen numerous conditional null T. brucei bloodstream form cell lines that express randomly mutagenized KREPB5 alleles. We identified nine single amino acid substitutions that could not complement the conditional loss of wild-type KREPB5. Seven of these were within the RNase III domain, and two were in the C-terminal region that has no homology to known motifs. Exclusive expression of these mutated KREPB5 alleles in the absence of wild-type allele expression resulted in growth inhibition, the loss of ∼ 20S editosomes, and inhibition of RNA editing in BF cells. Eight of these mutations were lethal in bloodstream form parasites but not in procyclic-form parasites, showing that multiple domains function in a life cycle-dependent manner. Amino acid changes at a substantial number of positions, including up to 7 per allele, allowed complementation and thus did not block KREPB5 function. Hence, the degenerate RNase III domain and a newly identified domain are critical for KREPB5 function and have differential effects between the life cycle stages of T. brucei that differentially edit mRNAs.
Project description:The polycistronic procylcin PARP (for procyclic acidic repetitive protein) A transcription unit of Trypanosoma brucei was completely characterized by the mapping of the termination region. In addition to the tandem of procyclin genes and GRESAG 2.1, this 7.5- to 9.5-kb unit contained another gene for a putative surface protein, termed PAG (for procyclin-associated gene) 3. The terminal 3-kb sequence did not contain significant open reading frames and cross-hybridized with the beginning of one or several transcription units specific to the bloodstream form. At least three separate fragments from the terminal region were able to inhibit chloramphenicol acetyltransferase expression when inserted between either the PARP, the ribosomal, or the variable surface glycoprotein promoter and a chloramphenicol acetyltransferase reporter gene. This inhibition was due to an orientation-dependent transcription termination caused by the combination of several attenuator elements with no obvious sequence conservation. The procyclin transcription terminator appeared unable to inhibit transcription by polymerase II.
Project description:Mitochondrial protein translocation machinery in the kinetoplastid parasites, like Trypanosoma brucei, has been characterized poorly. In T. brucei genome database, one homolog for a protein translocator of mitochondrial inner membrane (Tim) has been found, which is closely related to Tim17 from other species. The T. brucei Tim17 (TbTim17) has a molecular mass 16.2kDa and it possesses four characteristic transmembrane domains. The protein is localized in the mitochondrial inner membrane. The level of TbTim17 protein is 6-7-fold higher in the procyclic form that has a fully active mitochondrion, than in the mammalian bloodstream form of T. brucei, where many of the mitochondrial activities are suppressed. Knockdown of TbTim17 expression by RNAi caused a cessation of cell growth in the procyclic form and reduced growth rate in the bloodstream form. Depletion of TbTim17 decreased mitochondrial membrane potential more in the procyclic than bloodstream form. However, TbTim17 knockdown reduced the expression level of several nuclear encoded mitochondrial proteins in both the forms. Furthermore, import of presequence containing nuclear encoded mitochondrial proteins was significantly reduced in TbTim17 depleted mitochondria of the procyclic as well as the bloodstream form, confirming that TbTim17 is critical for mitochondrial protein import in both developmental forms. Together, these show that TbTim17 is the translocator of nuclear encoded mitochondrial proteins and its expression is regulated according to mitochondrial activities in T. brucei.
Project description:The variant surface glycoprotein (VSG) and procyclin are the major surface proteins of the bloodstream and procyclic stages, respectively, of Trypanosoma brucei. The promoter regions of the VSG and procyclin gene transcription units could be mapped thanks to the specific enrichment of initial transcripts that occurs following UV irradiation. Whereas the VSG gene is 45 kb distant from its promoter, procyclin genes are located immediately downstream. We show, by run-on assays on isolated nuclei and by cDNA analysis, that transcription occurs from both promoters in bloodstream as well as in procyclic forms. It is inferred that the control of the stage-specific expression of VSG and procyclin genes is not effected at the level of transcription initiation, but most probably by interfering with the elongation and stability of the specific transcripts.
Project description:In nearly all eukaryotes, cellular differentiation is governed by changes in transcription, and stabilized by chromatin and DNA modification. Gene expression control in the pathogen Trypanosoma brucei, in contrast, relies almost exclusively on post-transcriptional mechanisms, so RNA binding proteins must assume the burden that is usually borne by transcription factors. T. brucei multiply in the blood of mammals as bloodstream forms, and in the midgut of Tsetse flies as procyclic forms. We show here that a single RNA-binding protein, RBP10, promotes the bloodstream-form trypanosome differentiation state. Depletion of RBP10 from bloodstream-form trypanosomes gives cells that can grow only as procyclic forms; conversely, expression of RBP10 in procyclic forms converts them to bloodstream forms. RBP10 binds to procyclic-specific mRNAs containing an UAUUUUUU motif, targeting them for translation repression and destruction. Products of RBP10 target mRNAs include not only the major procyclic surface protein and enzymes of energy metabolism, but also protein kinases and stage-specific RNA-binding proteins: this suggests that alterations in RBP10 trigger a regulatory cascade.
Project description:Nowadays, most reverse genetics approaches in Trypanosoma brucei, a protozoan parasite of medical and veterinary importance, rely on pre-established cell lines. Consequently, inducible experimentation is reduced to a few laboratory strains. Here we described a new transgene expression system based exclusively on endogenous transcription activities and a minimum set of regulatory components that can easily been adapted to different strains. The pTbFIX vectors are designed to contain the sequence of interest under the control of an inducible rRNA promoter along with a constitutive dicistronic unit encoding a nucleus targeted tetracycline repressor and puromycin resistance genes in a tandem "head-to-tail" configuration. Upon doxycycline induction, the system supports regulatable GFP expression (170 to 400 fold) in both bloodstream and procyclic T. brucei forms. Furthermore we have adapted the pTbFIX plasmid to perform RNAi experimentation. Lethal phenotypes, including ?-tubulin and those corresponding to the enolase and clathrin heavy chain genes, were successfully recapitulated in procyclic and bloodstream parasites thus showing the versatility of this new tool.
Project description:The genes which encode glycosomal glyceraldehyde-phosphate dehydrogenase (gGAPDH) of Trypanosoma cruzi are arranged as a tandemly repeated pair on a single chromosome and are identical at the level of nucleotide sequence. They are separated by an intergenic region which contains a 317 base pair sequence with the properties of a retroposon. The genes express a 1.5 kb mRNA and a 38 kd protein. The amino acid sequence contains features characteristic of glycosomal enzymes such as peptide insertions and a C-terminal extension. However, T. cruzi gGAPDH lacks one of the positively charged 'hotspot' motifs which have been proposed as topogenic signals for import into the glycosome, a unique microbody-like organelle. Molecular modelling of the T. cruzi and T. brucei enzymes suggests that neither structure would fulfil the requirements of the 'hotspot' glycosomal import model.