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Isabel Roditi - One of the best experts on this subject based on the ideXlab platform.
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RNA-Seq analysis validates the use of culture-derived Trypanosoma brucei and provides new markers for mammalian and insect life-cycle stages
BMC Genomics, 2018Co-Authors: Arunasalam Naguleswaran, Nicholas Doiron, Isabel RoditiAbstract:Background Trypanosoma brucei brucei , the parasite causing Nagana in domestic animals, is closely related to the parasites causing sleeping sickness, but does not infect humans. In addition to its importance as a pathogen, the relative ease of genetic manipulation and an innate capacity for RNAi extend its use as a model organism in cell and infection biology. During its development in its mammalian and insect (tsetse fly) hosts, T. b. brucei passes through several different life-cycle stages. There are currently four life-cycle stages that can be cultured: slender Forms and stumpy Forms, which are equivalent to Forms found in the mammal, and early and late Procyclic Forms, which are equivalent to Forms in the tsetse midgut. Early Procyclic Forms show coordinated group movement (social motility) on semi-solid surfaces, whereas late Procyclic Forms do not. Results RNA-Seq was performed on biological replicates of each life-cycle stage. These constitute the first datasets for culture-derived slender and stumpy bloodstream Forms and early and late Procyclic Forms. Expression profiles confirmed that genes known to be stage-regulated in the animal and insect hosts were also regulated in culture. Sequence reads of 100–125 bases provided sufficient precision to uncover differential expression of closely related genes. More than 100 transcripts showed peak expression in stumpy Forms, including adenylate cyclases and several components of inositol metabolism. Early and late Procyclic Forms showed differential expression of 73 transcripts, a number of which encoded proteins that were previously shown to be stage-regulated. Moreover, two adenylate cyclases previously shown to reduce social motility are up-regulated in late Procyclic Forms. Conclusions This study validates the use of cultured bloodstream Forms as alternatives to animal-derived parasites and yields new markers for all four stages. In addition to underpinning recent findings that early and late Procyclic Forms are distinct life-cycle stages, it could provide insights into the reasons for their different biological properties.
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Analysis and growth of T. brucei bloodstream and Procyclic form TbMCP14 (conditional) null mutants.
2015Co-Authors: Juan P. De Macêdo, Isabel Roditi, Gabriela Schumann Burkard, Moritz Niemann, Michael P. Barrett, Henri Vial, Pascal Mäser, André Schneider, Peter BütikoferAbstract:A) Agarose gel of PCR products obtained using primers binding in the UTR regions outside the TbMCP14 ORF. In bloodstream form (bs) parasites, the two endogenous TbMCP14 alleles (∞, 1363 bp) were replaced by hygromycin (*, 2762 bp) and blasticidin (#, 2135 bp) resistance genes, whereas in Procyclic Forms (pc), the endogenous alleles were replaced by blasticidin (#, 2135 bp) and phleomycin (#, 2111 bp) resistance genes. The numbers on the right indicate the migration of base pair markers. B) Growth of T. brucei bloodstream form TbMCP14 null mutants in culture (red circles), in comparison with the parental cell line (black squares, indicated as NY-SM). C) Growth of T. brucei Procyclic form TbMCP14 conditional null mutants in the presence (black squares, +MCP14) or absence (red circles,-MCP14) of tetracycline. Data points in B and C represent mean values ± standard deviations from three independent experiments; for some data points, the error bars are smaller than the symbols. D) SDS-PAGE/immunoblot analysis of lysates from T. brucei Procyclic form TbMCP14 conditional null mutants grown in the presence (+) or absence (-) of tetracycline for indicated times. cMyc-tagged TbMCP14 was visualized with anti-cMyc antibody. Hsp70 was visualized with anti-HSP70 antibody and represents a loading control. Molecular mass markers are indicated.
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ATP production by crude mitochondria from TbMCP14 conditional knock-out Procyclic Forms.
2015Co-Authors: Juan P. De Macêdo, Isabel Roditi, Gabriela Schumann Burkard, Moritz Niemann, Michael P. Barrett, Henri Vial, Pascal Mäser, André Schneider, Peter BütikoferAbstract:A) Growth curves of parasites cultured in SDM80 containing 10% normal FBS in the presence (black squares, +MCP14) or absence (circles,-MCP14) of tetracycline. Data points represent mean values ± standard deviations from three independent experiments. After 6 days of culture, parasites were permeabilized with 0.01% digitonin and crude mitochondria were isolated. The inset shows SDS-PAGE/immunoblots probed with anti-cMyc antibody to detect TbMCP14 (lower band) and anti-HSP70 antibody to demonstrate equal loading. The samples were run on the same blot and exposed for equal times (for depletion of TbMCP14 see also Fig 7D and S5 Fig). B,C,D) ATP production in isolated mitochondria was measured by addition of 67 μM ADP and 5 mM succinate (B), 2-ketoglutarate (C) or proline (D). Antimycin (2.7 μM, panels B, D) or atractyloside (43 μM, panel C) were added to inhibit complex III of the electron transport chain or ADP/ATP translocation, respectively. The bars are mean values ± SD from five independent experiments. The asterisks represent statistical difference relative to the respective controls (**p
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social motility of african trypanosomes is a property of a distinct life cycle stage that occurs early in tsetse fly transmission
PLOS Pathogens, 2014Co-Authors: Simon Imhof, Xuan Lan Vu, Kapila Gunasekera, Sebastian Knusel, Isabel RoditiAbstract:The protozoan pathogen Trypanosoma brucei is transmitted between mammals by tsetse flies. The first compartment colonised by trypanosomes after a blood meal is the fly midgut lumen. Trypanosomes present in the lumen—designated as early Procyclic Forms—express the stage-specific surface glycoproteins EP and GPEET procyclin. When the trypanosomes establish a mature infection and colonise the ectoperitrophic space, GPEET is down-regulated, and EP becomes the major surface protein of late Procyclic Forms. A few years ago, it was discovered that Procyclic form trypanosomes exhibit social motility (SoMo) when inoculated on a semi-solid surface. We demonstrate that SoMo is a feature of early Procyclic Forms, and that late Procyclic Forms are invariably SoMo-negative. In addition, we show that, apart from GPEET, other markers are differentially expressed in these two life-cycle stages, both in culture and in tsetse flies, indicating that they have different biological properties and should be considered distinct stages of the life cycle. Differentially expressed genes include two closely related adenylate cyclases, both hexokinases and calflagins. These findings link the phenomenon of SoMo in vitro to the parasite Forms found during the first 4–7 days of a midgut infection. We postulate that ordered group movement on plates reflects the migration of parasites from the midgut lumen into the ectoperitrophic space within the tsetse fly. Moreover, the process can be uncoupled from colonisation of the salivary glands. Although they are the major surface proteins of Procyclic Forms, EP and GPEET are not essential for SoMo, nor, as shown previously, are they required for near normal colonisation of the fly midgut.
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insights into the regulation of gpeet procyclin during differentiation from early to late Procyclic Forms of trypanosoma brucei
Molecular and Biochemical Parasitology, 2013Co-Authors: Sebastian Knusel, Isabel RoditiAbstract:The Procyclic form of Trypanosoma brucei colonises the gut of its insect vector, the tsetse fly. GPEET and EP procyclins constitute the parasite's surface coat at this stage of the life cycle, and the presence or absence of GPEET distinguishes between early and late Procyclic Forms, respectively. Differentiation from early to late Procyclic Forms in vivo occurs in the fly midgut and can be mimicked in culture. Our analysis of this transition in vitro delivered new insights into the process of GPEET repression. First, we could show that parasites followed a concrete sequence of events upon triggering differentiation: after undergoing an initial growth arrest, cells lost GPEET protein, and finally late Procyclic Forms resumed proliferation. Second, we determined the stability of both GPEET and EP mRNA during differentiation. GPEET mRNA is exceptionally stable in early Procyclic Forms, with a half-life >6h. The GPEET mRNA detected in late Procyclic form cultures is a mixture of transcripts from both bona fide late Procyclic Forms and GPEET-positive 'laggard' parasites present in these cultures. However, its stability was clearly reduced during differentiation and in late Procyclic form cultures. Alternatively processed GPEET transcripts were enriched in samples from late Procyclic Forms, suggesting that altered mRNA processing might contribute to repression of GPEET in this developmental stage. In addition, we detected GPEET transcripts with non-templated oligo(U) tails that were enriched in late Procyclic Forms. To the best of our knowledge, this is the first study reporting a uridylyl-tailed, nuclear-encoded mRNA species in trypanosomatids or any other protozoa.
Christine Clayton - One of the best experts on this subject based on the ideXlab platform.
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the zinc finger proteins zc3h20 and zc3h21 stabilise mrnas encoding membrane proteins and mitochondrial proteins in insect form trypanosoma brucei
Molecular Microbiology, 2020Co-Authors: Bin Liu, Kevin Kamanyi Marucha, Christine ClaytonAbstract:ZC3H20 and ZC3H21 are related trypanosome proteins with two C(x)8 C(x)5 C(x)3 H zinc finger motifs. ZC3H20 is present at a low level in replicating mammalian-infective bloodstream Forms, but becomes more abundant when they undergo growth arrest at high density; ZC3H21 appears only in the Procyclic form of the parasite, which infects Tsetse flies. Each protein binds to several hundred mRNAs, with overlapping but not identical specificities. Both increase expression of bound mRNAs, probably through recruitment of the MKT1-PBP1 complex. At least 28 of the bound mRNAs decrease after depletion of ZC3H20, or of ZC3H20 and ZC3H21 together; their products include Procyclic-specific proteins of the plasma membrane and energy metabolism. Simultaneous depletion of ZC3H20 and ZC3H21 causes Procyclic Forms to shrink and stop growing; in addition to decreases in target mRNAs, there are other changes suggestive of loss of developmental regulation. The bloodstream-form-specific protein RBP10 controls ZC3H20 and ZC3H21 expression. Interestingly, some ZC3H20/21 target mRNAs also bind to and are repressed by RBP10, allowing for dynamic regulation as RBP10 decreases and ZC3H20 and ZC3H21 increase during differentiation.
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the zinc finger proteins zc3h20 and zc3h21 stabilise mrnas encoding membrane proteins and mitochondrial proteins in insect form trypanosoma brucei
bioRxiv, 2019Co-Authors: Bin Liu, Kevin Kamanyi Marucha, Christine ClaytonAbstract:ZC3H20 and ZC3H21 are related trypanosome proteins with two C(x)8C(x)5C(x)3H zinc finger motifs. ZC3H20 is unstable in mammalian-infective bloodstream Forms, but becomes more abundant as they transform to growth-arrested stumpy form, while ZC3H21 appears only in the Procyclic form of the parasite, which infects Tsetse flies. Each protein binds to several hundred mRNAs, with overlapping but not identical specificities. Both increase expression of bound mRNAs, probably through recruitment of the MKT1-PBP1 complex. At least seventy of the bound mRNAs decrease after RNAi targeting ZC3H20 or ZC3H20 and ZC3H21; their products include Procyclic-specific proteins of the plasma membrane and energy metabolism. Simultaneous depletion of ZC3H20 and ZC3H21 causes Procyclic Forms to shrink and stop growing; in addition to decreases in target mRNAs, there are other changes suggestive of loss of developmental regulation. The bloodstream-form specific protein RBP10 controls ZC3H20 and ZC3H21 expression. Interestingly, some ZC3H20/21 target mRNAs also bind to and are repressed by RBP10, allowing for dynamic regulation as RBP10 decreases and ZC3H20 and ZC3H21 increase during differentiation.
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the zinc finger proteins zc3h20 and zc3h21 stabilise mrnas encoding membrane proteins and mitochondrial proteins in insect form trypanosoma brucei
bioRxiv, 2019Co-Authors: Bin Liu, Kevin Kamanyi Marucha, Christine ClaytonAbstract:ZC3H20 and ZC3H21 are related trypanosome proteins with two C(x)8C(x)5C(x)3H zinc finger motifs. ZC3H20 is unstable in mammalian-infective bloodstream Forms, but becomes more abundant as they transform to growth-arrested stumpy form, while ZC3H21 appears only in the Procyclic form of the parasite, which infects Tsetse flies. Each protein binds to several hundred mRNAs, with overlapping but not identical specificities. Both increase expression of bound mRNAs, probably through recruitment of the MKT1-PBP1 complex. At least seventy of the bound mRNAs decrease after RNAi targeting ZC3H20 or ZC3H20 and ZC3H21; their products include Procyclic-specific proteins of the plasma membrane and energy metabolism. Simultaneous depletion of ZC3H20 and ZC3H21 causes Procyclic Forms to shrink and stop growing; in addition to decreases in target mRNAs, there are other changes suggestive of loss of developmental regulation. The bloodstream-form specific protein RBP10 controls ZC3H20 and ZC3H21 expression. Interestingly, some ZC3H20/21 target mRNAs also bind to and are repressed by RBP10, allowing for dynamic regulation as RBP10 decreases and ZC3H20 and ZC3H21 increase during differentiation.
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ZC3H30 is in a granule fraction after heat shock.
2018Co-Authors: Chaitali Chakraborty, Christine ClaytonAbstract:A) In Procyclic trypanosomes grown at 27°C, ZC3H30 is in the cytosolic fraction. Trypanosomes were disrupted using silicon carbide, and fractionated by centrifugation. 5 x 106 cell-equivalents were loaded on each lane of an SDS-PAGE gel and the proteins were detected by Western blotting. Tot: total lysate; C: cytosol: Sup: detergent-soluble organellar proteins; Pel: IGEPAL-insoluble pellet. TR is trypanothione reductase, a cytosolic protein, and XRND is in the nucleus. (B) ZC3H30 moves to the granule fraction after heat shock. Trypanosomes expressing V5-ZC3H30, with or without a 1h heat shock at 41°C, were fractionated into various soluble and granular fractions, using the cytoskeleton as a molecular sieve to trap stress granules. T: Unfractionated total cell lysate; SN1-3: soluble supernatants; SN4: molecules trapped inside cytoskeletons but not sedimented at 20000g (10min); G: sedimentable granules. Proteins were detected from equal proportions of each fraction. DHH1 and SCD6 are stress granule markers and trypanothione reductase (TR) is in the soluble fraction. (C) Loss of GFP-Tb927.8.3820 after 24h RNAi induction in Procyclic Forms (control for panel (D)). The Ponceau protein stain serves as a loading control. (D) ZC3H30 is in the granule fraction in heat-shocked Tb927.8.3820-depleted cells. As in (B), but using cells with RNAi against Tb927.8.3820 (see (C)).
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expression of the rna binding protein rbp10 promotes the bloodstream form differentiation state in trypanosoma brucei
PLOS Pathogens, 2017Co-Authors: Elisha Mugo, Christine ClaytonAbstract: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.
Peter Bütikofer - One of the best experts on this subject based on the ideXlab platform.
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Analysis and growth of T. brucei bloodstream and Procyclic form TbMCP14 (conditional) null mutants.
2015Co-Authors: Juan P. De Macêdo, Isabel Roditi, Gabriela Schumann Burkard, Moritz Niemann, Michael P. Barrett, Henri Vial, Pascal Mäser, André Schneider, Peter BütikoferAbstract:A) Agarose gel of PCR products obtained using primers binding in the UTR regions outside the TbMCP14 ORF. In bloodstream form (bs) parasites, the two endogenous TbMCP14 alleles (∞, 1363 bp) were replaced by hygromycin (*, 2762 bp) and blasticidin (#, 2135 bp) resistance genes, whereas in Procyclic Forms (pc), the endogenous alleles were replaced by blasticidin (#, 2135 bp) and phleomycin (#, 2111 bp) resistance genes. The numbers on the right indicate the migration of base pair markers. B) Growth of T. brucei bloodstream form TbMCP14 null mutants in culture (red circles), in comparison with the parental cell line (black squares, indicated as NY-SM). C) Growth of T. brucei Procyclic form TbMCP14 conditional null mutants in the presence (black squares, +MCP14) or absence (red circles,-MCP14) of tetracycline. Data points in B and C represent mean values ± standard deviations from three independent experiments; for some data points, the error bars are smaller than the symbols. D) SDS-PAGE/immunoblot analysis of lysates from T. brucei Procyclic form TbMCP14 conditional null mutants grown in the presence (+) or absence (-) of tetracycline for indicated times. cMyc-tagged TbMCP14 was visualized with anti-cMyc antibody. Hsp70 was visualized with anti-HSP70 antibody and represents a loading control. Molecular mass markers are indicated.
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ATP production by crude mitochondria from TbMCP14 conditional knock-out Procyclic Forms.
2015Co-Authors: Juan P. De Macêdo, Isabel Roditi, Gabriela Schumann Burkard, Moritz Niemann, Michael P. Barrett, Henri Vial, Pascal Mäser, André Schneider, Peter BütikoferAbstract:A) Growth curves of parasites cultured in SDM80 containing 10% normal FBS in the presence (black squares, +MCP14) or absence (circles,-MCP14) of tetracycline. Data points represent mean values ± standard deviations from three independent experiments. After 6 days of culture, parasites were permeabilized with 0.01% digitonin and crude mitochondria were isolated. The inset shows SDS-PAGE/immunoblots probed with anti-cMyc antibody to detect TbMCP14 (lower band) and anti-HSP70 antibody to demonstrate equal loading. The samples were run on the same blot and exposed for equal times (for depletion of TbMCP14 see also Fig 7D and S5 Fig). B,C,D) ATP production in isolated mitochondria was measured by addition of 67 μM ADP and 5 mM succinate (B), 2-ketoglutarate (C) or proline (D). Antimycin (2.7 μM, panels B, D) or atractyloside (43 μM, panel C) were added to inhibit complex III of the electron transport chain or ADP/ATP translocation, respectively. The bars are mean values ± SD from five independent experiments. The asterisks represent statistical difference relative to the respective controls (**p
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characterization of choline uptake in trypanosoma brucei Procyclic and bloodstream Forms
Molecular and Biochemical Parasitology, 2013Co-Authors: Juan Pereira De Macedo, Henri Vial, Pascal Mäser, Remo S Schmidt, Doris Rentsch, Erwin Sigel, Peter BütikoferAbstract:Choline is an essential nutrient for eukaryotic cells, where it is used as precursor for the synthesis of choline-containing phospholipids, such as phosphatidylcholine (PC). According to published data, Trypanosoma brucei parasites are unable to take up choline from the environment but instead use lyso-phosphatidylcholine as precursor for choline lipid synthesis. We now show that T. brucei Procyclic Forms in culture readily incorporate [3H]-labeled choline into PC, indicating that trypanosomes express a transporter for choline at the plasma membrane. Characterization of the transport system in T. brucei Procyclic and bloodstream Forms shows that uptake of choline is independent of sodium and potassium ions and occurs with a Km in the low micromolar range. In addition, we demonstrate that choline uptake can be blocked by the known choline transport inhibitor, hemicholinium-3, and by synthetic choline analogs that have been established as anti-malarials. Together, our results show that T. brucei parasites express an uptake system for choline and that exogenous choline is used for PC synthesis.
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Trypanosoma congolense Procyclins: Unmasking Cryptic Major Surface Glycoproteins in Procyclic Forms
Eukaryotic cell, 2006Co-Authors: Silvia Utz, Christina Kunz Renggli, Isabel Roditi, Igor C. Almeida, Alvaro Acosta-serrano, Peter BütikoferAbstract:In the tsetse fly, the protozoan parasite Trypanosoma congolense is covered by a dense layer of glycosylphosphatidylinositol (GPI)-anchored molecules. These include a protease-resistant surface molecule (PRS), which is expressed by Procyclic Forms early in infection, and a glutamic acid- and alanine-rich protein (GARP), which appears at later stages. Since neither of these surface antigens is expressed at intermediate stages, we investigated whether a GPI-anchored protein of 50 to 58 kDa, previously detected in Procyclic culture Forms, might constitute the coat of these parasites. We therefore partially purified the protein from T. congolense Kilifi Procyclic Forms, obtained an N-terminal amino acid sequence, and identified its gene. Detailed analyses showed that the mature protein consists almost exclusively of 13 heptapeptide repeats (EPGENGT). The protein is densely N glycosylated, with up to 13 high-mannose oligosaccharides ranging from Man(5)GlcNAc(2) to Man(9)GlcNAc(2) linked to the peptide repeats. The lipid moiety of the glycosylphosphatidylinositol is composed of sn-1-stearoyl-2-lyso-glycerol-3-HPO(4)-1-(2-O-acyl)-d-myo-inositol. Heavily glycosylated proteins with similar repeats were subsequently identified in T. congolense Savannah Procyclic Forms. Collectively, this group of proteins was named T. congolense procyclins to reflect their relationship to the EP and GPEET procyclins of T. brucei. Using an antiserum raised against the EPGENGT repeat, we show that T. congolense procyclins are expressed continuously in the fly midgut and thus form the surface coat of cells that are negative for both PRS and GARP.
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coordinate expression of gpeet procyclin and its membrane associated kinase in trypanosoma brucei Procyclic Forms
Journal of Biological Chemistry, 2003Co-Authors: Annecatherine Schlaeppi, Tatiana Malherbe, Peter BütikoferAbstract:GPEET procyclin is a major glycosylphosphatidylinositol-anchored protein of Procyclic (insect stage) trypanosomes in culture and is heavily phosphorylated in the GPEET pentapeptide repeat. The phosphorylation reaction is a late event and occurs during maturation and transport of GPEET or on the parasite surface by an ecto-protein kinase. Initial biochemical characterization of the GPEET kinase activity now shows that it depends on bivalent cations for maximal activity, is stimulated by sulfhydryl group reagents, and is specific for ATP as phosphoryl donor. No kinase activity is detected in bloodstream form trypanosomes in culture, whereas strong phosphorylation is observed in early Procyclic Forms. In addition, the GPEET kinase activity is absent from Procyclic trypanosomes that have repressed GPEET synthesis but can be induced in these same stocks by conditions, which also induce GPEET expression. However, the presence of an active kinase does not depend on the presence of (functional) GPEET because it can be detected in parasites expressing a non-phosphorylatable GPEET mutant protein and in procyclin null mutant trypanosomes. Interestingly, the presence of the glycosylphosphatidylinositol lipid moiety seems necessary for GPEET to become phosphorylated. Together, the results demonstrate that GPEET and its kinase are expressed during the same life cycle stages and that factors that induce the expression of GPEET in vitro also induce the expression of the GPEET kinase.
Etienne Pays - One of the best experts on this subject based on the ideXlab platform.
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expression of a variant surface glycoprotein of trypanosoma gambiense in Procyclic Forms of trypanosoma brucei shows that the cell type dictates the nature of the glycosylphosphatidylinositol membrane anchor attached to the glycoprotein
Biochemical Journal, 1997Co-Authors: Francoise Paturiauxhanocq, Michael A. J. Ferguson, Luc Vanhamme, Sylvie Rolin, J Hanocqquertier, Nicole Zitzmann, Maurice Geuskens, Etienne PaysAbstract:Procyclic Forms of Trypanosoma brucei have been genetically modified to express the major metacyclic variant surface glycoprotein (VSG variant AnTat 11.17) of Trypanosoma gambiense. The VSG is expressed in an intact membrane-bound form that can be detected over the entire plasma membrane, together with procyclin, and as a series of lower-molecular-mass fragments that are mostly soluble degradation products. The presence of degraded VSG in the cells and the culture medium suggests that VSG is not efficiently processed and/or efficiently folded when expressed in Procyclic cells. The level of procyclin expressed on the surface of these cells is slightly reduced, although there is no difference in procyclin mRNA levels. The intact membrane-bound form of the VSG is N-glycosylated with oligomannose structures and contains a glycosylphosphatidylinositol (GPI) membrane anchor that can be biosynthetically labelled with [3H]ethanolamine. The anchor is sensitive to mammalian GPI-specific phospholipase D but, like the anchor of procyclin, it is resistant to the action of bacterial phosphatidylinositol-specific phospholipase C. This pattern of phospholipase sensitivity suggests that the GPI anchor acquired by VSG when expressed in Procyclics is acylated on the inositol ring and therefore resembles a Procyclic procyclin-type anchor rather than a trypomastigote VSG-type anchor with respect to the lipid structure. The VSG expressed in Procyclics was sensitive to the action of a mixture of sialidase, beta-galactosidase and beta-hexosaminidase, suggesting that the VSG GPI anchor also contains a sialylated polylactosamine side-chain modification similar to that described for procyclin. These results indicate that the nature of the protein expressed has little influence on the post-translational modifications performed in the secretory pathway of Procyclic trypanosomes.
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the 3 terminal region of the mrnas for vsg and procyclin can confer stage specificity to gene expression in trypanosoma brucei
The EMBO Journal, 1995Co-Authors: Magali Berberof, Annette Pays, Patricia Tebabi, Susan C Welburn, D Jefferies, Luc Vanhamme, Etienne PaysAbstract:The variant surface glycoprotein (VSG) and procyclin are the respective major surface antigens of the bloodstream and the Procyclic Forms of Trypanosoma brucei. These proteins and their mRNAs are both the most abundant and absolutely characteristic of their respective life cycle stages. We show that the 3'-terminal region of these mRNAs regulates expression of a reporter gene in an inverse manner, depending on the developmental form of the parasite. In the case of VSG mRNA, the 97 nt sequence upstream from the polyadenylation site is responsible for these effects. The regulation occurs through a variation of mRNA abundance which is not due to a change in primary transcription. In the bloodstream form this effect is manifested by an increase in RNA stability, whereas in the Procyclic form it seems to be related to a reduction in the efficiency of mRNA maturation. The 3'-end of VSG mRNA can obviate the 5- to 10-fold stimulation of transcription driven by the procyclin promoter during differentiation from the bloodstream to the Procyclic form. The predominance of posttranscriptional over transcriptional controls is probably linked to the organization of the trypanosome genome in polycistronic transcription units.
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transient adenylate cyclase activation accompanies differentiation of trypanosoma brucei from bloodstream to Procyclic Forms
Molecular and Biochemical Parasitology, 1993Co-Authors: Sylvie Rolin, Peter Overath, Pascale Paindavoine, J Hanocqquertier, Francoise Hanocq, Yves Claes, Dominique Le Ray, Etienne PaysAbstract:Pleomorphic bloodstream Forms of Trypanosoma brucei differentiate synchronously into Procyclic Forms when cultivated at 27 degrees C in the presence of citrate/cis-aconitate. The activity of adenylate cyclase was monitored during this process. Two phases of transient stimulation were observed. The first phase occurred 6-10 h after the triggering of differentiation, a period which immediately follows the release of the bulk of the VSG and immediately precedes both the first cell division and the loss of the bloodstream-specific ESAG 4 transmembrane adenylate cyclase. The second phase occurred between 20 and 40 h, when the cells that emerged from the first division began to proliferate. These observations suggest that cAMP may be involved in differentiation/proliferation of the parasite.
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abrupt rna changes precede the first cell division during the differentiation of trypanosoma brucei bloodstream Forms into Procyclic Forms in vitro
Molecular and Biochemical Parasitology, 1993Co-Authors: Etienne Pays, J Hanocqquertier, Francoise Hanocq, Suzanne Van Assel, Derek P Nolan, Sylvie RolinAbstract:We have monitored the timing of DNA and RNA synthesis during the synchronous differentiation of Trypanosoma brucei bloodstream Forms into Procyclic Forms in vitro. Both are triggered after a lag period of 4 h and reach a first peak after 9 h. The division of the kinetoplast precedes that of the nucleus by about 4 h. The first cell divisions are observed after 10 h, and the cell number is doubled after 20 h. The total RNA content per cell increases sharply between 4 and 10 h, then progressively decreases as cell division progresses. The increase in RNA content cannot be due solely to accumulation of rRNA since it is also observed for mRNAs such as actin. The VSG mRNA has almost disappeared within 2 h, while the procyclin mRNA accumulates soon after the triggering of differentiation, with a strong peak between 4 and 6 h. At this moment, the amount of procyclin mRNA per cell is at least 20-fold higher than in established Procyclic culture Forms. The loss of the VSG and the appearance of Procyclic-specific proteins essentially occur before the first cell division. These observations contrast with the progressive transition observed when monomorphic slender Forms are induced to transform under the same conditions.
Diana S Beattie - One of the best experts on this subject based on the ideXlab platform.
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isolation and characterization of complex i rotenone sensitive nadh ubiquinone oxidoreductase from the Procyclic Forms of trypanosoma brucei
FEBS Journal, 2001Co-Authors: Jing Fang, Yudong Wang, Diana S BeattieAbstract:Additional characterization of complex I, rotenone-sensitive NADH:ubiquinone oxidoreductase, in the mitochondria of Trypanosoma brucei brucei has been obtained. Both proline:cytochrome c reductase and NADH:ubiquinone oxidoreductase of Procyclic T. brucei were inhibited by the specific inhibitors of complex I rotenone, piericidin A, and capsaicin. These inhibitors had no effect on succinate: cytochrome c reductase activity. Antimycin A, a specific inhibitor of the cytochrome bc1 complex (ubiquinol:cytochrome c oxidoreductase), blocked almost completely cytochrome c reductase activity with either proline or succinate as electron donor, but had no inhibitory effect on NADH:ubiquinone oxidoreductase activity. The rotenone-sensitive NADH:ubiquinone oxidoreductase of Procyclic T. brucei was partially purified by sucrose density centrifugation of mitochondria solubilized with dodecyl-β-d-maltoside, with an approximately eightfold increase in specific activity compared to that of the mitochondrial membranes. Four polypeptides of the partially purified enzyme were identified as the homologous subunits of complex I (51 kDa, PSST, TYKY, and ND4) by immunoblotting with antibodies raised against subunits of Paracoccus denitrificans and against synthetic peptides predicted from putative complex I subunit genes encoded by mitochondrial and nuclear T. brucei DNA. Blue Native polyacrylamide gel electrophoresis of T. brucei mitochondrial membrane proteins followed by immunoblotting revealed the presence of a putative complex I with a molecular mass of 600 kDa, which contains a minimum of 11 polypeptides determined by second-dimensional Tricine–SDS/PAGE including the 51 kDa, PSST and TYKY subunits.
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the presence of rotenone sensitive nadh dehydrogenase in the long slender bloodstream and the Procyclic Forms of trypanosoma brucei brucei
FEBS Journal, 1996Co-Authors: Diana S Beattie, Marilyn M HowtonAbstract:The mitochondrial electron-transport chain present in the Procyclic and long slender bloodstream Forms of Trypanosoma brucei brucei was investigated by means of several experimental approaches. The oxidation of proline, glycerol and glucose in Procyclic cells was inhibited 80-90% by antimycin A or cyanide, 15-19% by salicylhydroxamic acid, and 30-35% by rotenone. Cytochrom-c-reductase activity, with proline or glycerol 3-phosphate as substrate, in a mitochondrial fraction isolated from these cells was inhibited by antimycin and rotenone, but not by malonate, while cytochrome-c-reductase activity with succinate as substrate was inhibited by antimycin A and malonate, but not by rotenone. In addition, the reduction of dichloroindophenol by NADH was inhibited by rotenone but not by malonate, which suggests that rotenone-sensitive NADH dehydrogenase (complex I) is present in these mitochondria. The presence of three subunits of NADH dehydrogenase was observed in immunoblots of mitochondrial proteins with specific antibodies raised against peptides corresponding to predicted antigenic regions of these proteins, which provides further evidence for the presence of NADH dehydrogenase. In long slender bloodstream Forms, the oxidation of glucose or glycerol was inhibited 100% by salicyhydroxamic acid, unaffected by cyanide or antimycin A, and inhibited 40% or 75%, respectively, by rotenone, which suggests that NADH dehydrogenase is present in these cells. In a mitochondrial fraction isolated from the bloodstream Forms, oxygen uptake with glycerol 3-phosphate as substrate was inhibited 65% by rotenone. Low levels of rotenone-sensitive NADH-dependent reduction of dichloroindophenol and the presence of subunits 7 and 8 of NADH dehydrogenase provided additional evidence for the presence of NADH dehydrogenase in bloodstream Forms of T. brucei.