The Experts below are selected from a list of 321 Experts worldwide ranked by ideXlab platform

Isabel Roditi - One of the best experts on this subject based on the ideXlab platform.

  • RNA-Seq analysis validates the use of culture-derived Trypanosoma brucei and provides new markers for mammalian and insect life-cycle stages
    BMC Genomics, 2018
    Co-Authors: Arunasalam Naguleswaran, Nicholas Doiron, Isabel Roditi
    Abstract:

    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.

  • Analysis and growth of T. brucei bloodstream and procyclic form TbMCP14 (conditional) null mutants.
    2015
    Co-Authors: Juan P. De Macêdo, Isabel Roditi, Gabriela Schumann Burkard, Moritz Niemann, Michael P. Barrett, Henri Vial, Pascal Mäser, André Schneider, Peter Bütikofer
    Abstract:

    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.

  • social motility of african trypanosomes is a property of a distinct life cycle stage that occurs early in tsetse fly transmission
    PLOS Pathogens, 2014
    Co-Authors: Simon Imhof, Xuan Lan Vu, Kapila Gunasekera, Sebastian Knusel, Isabel Roditi
    Abstract:

    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.

  • insights into the regulation of gpeet Procyclin during differentiation from early to late procyclic forms of trypanosoma brucei
    Molecular and Biochemical Parasitology, 2013
    Co-Authors: Sebastian Knusel, Isabel Roditi
    Abstract:

    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.

  • a family of stage specific alanine rich proteins on the surface of epimastigote forms of trypanosoma brucei
    Molecular Microbiology, 2007
    Co-Authors: Simon Urwyler, Erwin Studer, Christina Kunz Renggli, Isabel Roditi
    Abstract:

    A 'two coat' model of the life cycle of Trypanosoma brucei has prevailed for more than 15 years. Metacyclic forms transmitted by infected tsetse flies and mammalian bloodstream forms are covered by variant surface glycoproteins. All other life cycle stages were believed to have a Procyclin coat, until it was shown recently that epimastigote forms in tsetse salivary glands express Procyclin mRNAs without translating them. As epimastigote forms cannot be cultured, a procedure was devised to compare the transcriptomes of parasites in different fly tissues. Transcripts encoding a family of glycosylphosphatidyl inositol-anchored proteins, BARPs (previously called bloodstream alanine-rich proteins), were 20-fold more abundant in salivary gland than midgut (procyclic) trypanosomes. Anti-BARP antisera reacted strongly and exclusively with salivary gland parasites and a BARP 3' flanking region directed epimastigote-specific expression of reporter genes in the fly, but inhibited expression in bloodstream and procyclic forms. In contrast to an earlier report, we could not detect BARPs in bloodstream forms. We propose that BARPs form a stage-specific coat for epimastigote forms and suggest renaming them brucei alanine-rich proteins.

Christine Clayton - One of the best experts on this subject based on the ideXlab platform.

  • the zinc finger proteins zc3h20 and zc3h21 stabilise mrnas encoding membrane proteins and mitochondrial proteins in insect form trypanosoma brucei
    Molecular Microbiology, 2020
    Co-Authors: Bin Liu, Kevin Kamanyi Marucha, Christine Clayton
    Abstract:

    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.

  • the zinc finger proteins zc3h20 and zc3h21 stabilise mrnas encoding membrane proteins and mitochondrial proteins in insect form trypanosoma brucei
    bioRxiv, 2019
    Co-Authors: Bin Liu, Kevin Kamanyi Marucha, Christine Clayton
    Abstract:

    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.

  • the zinc finger proteins zc3h20 and zc3h21 stabilise mrnas encoding membrane proteins and mitochondrial proteins in insect form trypanosoma brucei
    bioRxiv, 2019
    Co-Authors: Bin Liu, Kevin Kamanyi Marucha, Christine Clayton
    Abstract:

    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.

  • expression of the rna binding protein rbp10 promotes the bloodstream form differentiation state in trypanosoma brucei
    PLOS Pathogens, 2017
    Co-Authors: Elisha Mugo, Christine Clayton
    Abstract:

    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.

  • the rna binding protein rbp10 controls a regulatory cascade that defines bloodstream form trypanosome identity
    bioRxiv, 2016
    Co-Authors: Elisha Mugo, Christine Clayton
    Abstract:

    Gene expression control in the pathogen Trypanosoma brucei 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, defines 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: consequently, alterations in RBP10 trigger a regulatory cascade.

Michael A. J. Ferguson - One of the best experts on this subject based on the ideXlab platform.

  • molecular control of irreversible bistability during trypanosome developmental commitment
    Journal of Cell Biology, 2015
    Co-Authors: Maria Rosa Domingosananes, Michael D Urbaniak, Michael A. J. Ferguson, Balazs Szoőr, Keith R Matthews
    Abstract:

    The life cycle of Trypanosoma brucei involves developmental transitions that allow survival, proliferation, and transmission of these parasites. One of these, the differentiation of growth-arrested stumpy forms in the mammalian blood into insect-stage procyclic forms, can be induced synchronously in vitro with cis-aconitate. Here, we show that this transition is an irreversible bistable switch, and we map the point of commitment to differentiation after exposure to cis-aconitate. This irreversibility implies that positive feedback mechanisms operate to allow commitment (i.e., the establishment of “memory” of exposure to the differentiation signal). Using the reversible translational inhibitor cycloheximide, we show that this signal memory requires new protein synthesis. We further performed stable isotope labeling by amino acids in cell culture to analyze synchronized parasite populations, establishing the protein and phosphorylation profile of parasites pre- and postcommitment, thereby defining the “commitment proteome.” Functional interrogation of this data set identified Nek-related kinase as the first-discovered protein kinase controlling the initiation of differentiation to procyclic forms.

  • global quantitative silac phosphoproteomics reveals differential phosphorylation is widespread between the procyclic and bloodstream form lifecycle stages of trypanosoma brucei
    Journal of Proteome Research, 2013
    Co-Authors: Michael D Urbaniak, David M A Martin, Michael A. J. Ferguson
    Abstract:

    We report a global quantitative phosphoproteomic study of bloodstream and procyclic form Trypanosoma brucei using SILAC labeling of each lifecycle stage. Phosphopeptide enrichment by SCX and TiO2 led to the identification of a total of 10096 phosphorylation sites on 2551 protein groups and quantified the ratios of 8275 phosphorylation sites between the two lifecycle stages. More than 9300 of these sites (92%) have not previously been reported. Model-based gene enrichment analysis identified over representation of Gene Ontology terms relating to the flagella, protein kinase activity, and the regulation of gene expression. The quantitative data reveal that differential protein phosphorylation is widespread between bloodstream and procyclic form trypanosomes, with significant intraprotein differential phosphorylation. Despite a lack of dedicated tyrosine kinases, 234 phosphotyrosine residues were identified, and these were 3–4 fold over-represented among site changing >10-fold between the two lifecycle stage...

  • comparative silac proteomic analysis of trypanosoma brucei bloodstream and procyclic lifecycle stages
    PLOS ONE, 2012
    Co-Authors: Michael D Urbaniak, Lucia M S Guther, Michael A. J. Ferguson
    Abstract:

    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.

  • fate of glycosylphosphatidylinositol gpi less Procyclin and characterization of sialylated non gpi anchored surface coat molecules of procyclic form trypanosoma brucei
    Eukaryotic Cell, 2009
    Co-Authors: Maria Lucia S Guther, Alan R Prescott, Kenneth A Beattie, Douglas J Lamont, John James, Michael A. J. Ferguson
    Abstract:

    A Trypanosoma brucei TbGPI12 null mutant that is unable to express cell surface Procyclins and free glycosylphosphatidylinositols (GPI) revealed that these are not the only surface coat molecules of the procyclic life cycle stage. Here, we show that non-GPI-anchored Procyclins are N-glycosylated, accumulate in the lysosome, and appear as proteolytic fragments in the medium. We also show, using lectin agglutination and galactose oxidase-NaB(3)H(4) labeling, that the cell surface of the TbGPI12 null parasites contains glycoconjugates that terminate in sialic acid linked to galactose. Following desialylation, a high-apparent-molecular-weight glycoconjugate fraction was purified by ricin affinity chromatography and gel filtration and shown to contain mannose, galactose, N-acetylglucosamine, and fucose. The latter has not been previously reported in T. brucei glycoproteins. A proteomic analysis of this fraction revealed a mixture of polytopic transmembrane proteins, including P-type ATPase and vacuolar proton-translocating pyrophosphatase. Immunolocalization studies showed that both could be labeled on the surfaces of wild-type and TbGPI12 null cells. Neither galactose oxidase-NaB(3)H(4) labeling of the non-GPI-anchored surface glycoconjugates nor immunogold labeling of the P-type ATPase was affected by the presence of Procyclins in the wild-type cells, suggesting that the Procyclins do not, by themselves, form a macromolecular barrier.

  • gpi anchored proteins and free gpi glycolipids of procyclic form trypanosoma brucei are nonessential for growth are required for colonization of the tsetse fly and are not the only components of the surface coat
    Molecular Biology of the Cell, 2006
    Co-Authors: Maria Lucia S Guther, Alvaro Acostaserrano, Sylvia Lee, Laurence Tetley, Michael A. J. Ferguson
    Abstract:

    The procyclic form of Trypanosoma brucei exists in the midgut of the tsetse fly. The current model of its surface glycocalyx is an array of rod-like Procyclin glycoproteins with glycosylphosphatidylinositol (GPI) anchors carrying sialylated poly-N-acetyllactosamine side chains interspersed with smaller sialylated poly-N-acetyllactosamine–containing free GPI glycolipids. Mutants for TbGPI12, deficient in the second step of GPI biosynthesis, were devoid of cell surface Procyclins and poly-N-acetyllactosamine–containing free GPI glycolipids. This major disruption to their surface architecture severely impaired their ability to colonize tsetse fly midguts but, surprisingly, had no effect on their morphology and growth characteristics in vitro. Transmission electron microscopy showed that the mutants retained a cell surface glycocalyx. This structure, and the viability of the mutants in vitro, prompted us to look for non-GPI–anchored parasite molecules and/or the adsorption of serum components. Neither were apparent from cell surface biotinylation experiments but [3H]glucosamine biosynthetic labeling revealed a group of previously unidentified high apparent molecular weight glycoconjugates that might contribute to the surface coat. While characterizing GlcNAc-PI that accumulates in the TbGPI12 mutant, we observed inositolphosphoceramides for the first time in this organism.

Etienne Pays - One of the best experts on this subject based on the ideXlab platform.

  • a single stranded dna binding protein shared by telomeric repeats the variant surface glycoprotein transcription promoter and the Procyclin transcription terminator of trypanosoma brucei
    Nucleic Acids Research, 2000
    Co-Authors: Magali Berberof, Patricia Tebabi, Stéphane Lips, Luc Vanhamme, Sylvie Alexandre, Etienne Pays
    Abstract:

    In Trypanosoma brucei the genes are organised into long polycistronic transcription units and only three promoters for protein-encoding genes and a single terminator have been characterised. These promoters recruit a polI-like RNA polymerase for the transcription units encoding the two major stage-specific antigens of the parasite, the variant surface glycoprotein (VSG) of the bloodstream form and Procyclin of the insect-specific procyclic form, while the terminator is that of a Procyclin transcription unit. By deletional and mutational analysis we defined the two DNA sequences essential for the activity of the VSG promoter from a bloodstream form transcription unit and one of the functional elements of the Procyclin terminator. These three short sequences are similar, and their C-rich strand binds the same protein of 40 kDa. In addition, this factor also binds to the C-rich strand of the telomeric repeats, the consensus target sequence being 5′-CCCTNN-3′. The factor-binding sequences are functionally interchangeable in chimeric promoter or terminator constructs, although additional elements are required for full activity.

  • Characterization of a transcription terminator of the Procyclin PARP A unit of Trypanosoma brucei.
    Molecular and Cellular Biology, 1996
    Co-Authors: Magali Berberof, Patricia Tebabi, Annette Pays, Stéphane Lips, Etienne Pays
    Abstract:

    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.

  • specific binding of proteins to the noncoding strand of a crucial element of the variant surface glycoprotein Procyclin and ribosomal promoters of trypanosoma brucei
    Molecular and Cellular Biology, 1995
    Co-Authors: Luc Vanhamme, Patricia Tebabi, Annette Pays, Sylvie Alexandre, Etienne Pays
    Abstract:

    The variant surface glycoprotein (VSG) and Procyclin promoters of Trypanosoma brucei recruit an RNA polymerase sharing characteristic with polymerase I, but there is no sequence homology between them nor between these promoters and ribosomal promoters. We report the detailed characterization of the VSG promoter. The 70-bp region upstream of the transcription start site was sufficient for full promoter activity. Mutational analysis revealed three short critical stretches at positions -61 to -59 (box 1), -38 to -35 (box 2), and -1 to +1 (start site), the spacing of which was essential. These elements were conserved in the promoter for a metacyclic VSG gene. Hybrid sequences containing box 1 of the VSG promoter and box 2 of the ribosomal promoter were active. A specific binding of proteins to the noncoding strand of box 2, but not to double-stranded DNA, occurred. Competition experiments indicated that these proteins also bind to the corresponding region of the metacyclic VSG, Procyclin, and ribosomal promoters. Binding of such a protein, of 40 kDa, appeared to be shared by these promoters.

  • the 3 terminal region of the mrnas for vsg and Procyclin can confer stage specificity to gene expression in trypanosoma brucei
    The EMBO Journal, 1995
    Co-Authors: Magali Berberof, Annette Pays, Patricia Tebabi, Susan C Welburn, D Jefferies, Luc Vanhamme, Etienne Pays
    Abstract:

    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.

  • a similar gene is shared by both the variant surface glycoprotein and Procyclin gene transcription units of trypanosoma brucei
    Molecular and Cellular Biology, 1991
    Co-Authors: Magali Berberof, Annette Pays, Etienne Pays
    Abstract:

    The genes for the variant surface glycoprotein (VSG) and Procyclin are expressed in a mutually exclusive manner during the life cycle of Trypanosoma brucei and synthesize the most abundant mRNAs specific to the bloodstream and procyclic stages of the parasite, respectively. Genes belonging to the polycistronic transcription unit of the VSG gene (expression site-associated genes [ESAGs]) are uniquely expressed in the bloodstream form, but some members of ESAG families (genes related to ESAGs [GRESAGs]) are independently transcribed outside the VSG gene expression site. We report here that a gene related to ESAG 2, GRESAG 2.1, is present and expressed in a Procyclin gene transcription unit (PARP A locus), which is polycistronic. Members of the ESAG 2 family are thus present in the two major differentially stage-regulated transcription units of this parasite.

Peter Bütikofer - One of the best experts on this subject based on the ideXlab platform.

  • phosphatidylserine synthase 2 and phosphatidylserine decarboxylase are essential for aminophospholipid synthesis in trypanosoma brucei
    Molecular Microbiology, 2017
    Co-Authors: Luce Farine, Jennifer Jelk, Terry K. Smith, Jaeyeon Choi, Dennis R Voelker, Jon Nunes, Peter Bütikofer
    Abstract:

    Phosphatidylethanolamine (PE) and phosphatidylserine (PS) are ubiquitously expressed and metabolically interconnected glycerophospholipids in eukaryotes and prokaryotes. In Trypanosoma brucei, PE synthesis has been shown to occur mainly via the Kennedy pathway, one of the three routes leading to PE synthesis in eukaryotes, while PS synthesis has not been studied experimentally. We now reveal the importance of T. brucei PS synthase 2 (TbPSS2) and T. brucei PS decarboxylase (TbPSD), two key enzymes involved in aminophospholipid synthesis, for trypanosome viability. By using tetracycline-inducible down-regulation of gene expression and in vivo and in vitro metabolic labeling, we found that TbPSS2 (i) is necessary for normal growth of procyclic trypanosomes, (ii) localizes to the endoplasmic reticulum and (iii) represents the unique route for PS formation in T. brucei. In addition, we identified TbPSD as type I PS decarboxylase in the mitochondrion and found that it is processed proteolytically at a WGSS cleavage site into a heterodimer. Down-regulation of TbPSD expression affected mitochondrial integrity in both procyclic and bloodstream form trypanosomes, decreased ATP production via oxidative phosphorylation in procyclic form and affected parasite growth.

  • Analysis and growth of T. brucei bloodstream and procyclic form TbMCP14 (conditional) null mutants.
    2015
    Co-Authors: Juan P. De Macêdo, Isabel Roditi, Gabriela Schumann Burkard, Moritz Niemann, Michael P. Barrett, Henri Vial, Pascal Mäser, André Schneider, Peter Bütikofer
    Abstract:

    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.

  • characterization of choline uptake in trypanosoma brucei procyclic and bloodstream forms
    Molecular and Biochemical Parasitology, 2013
    Co-Authors: Juan Pereira De Macedo, Henri Vial, Pascal Mäser, Remo S Schmidt, Doris Rentsch, Erwin Sigel, Peter Bütikofer
    Abstract:

    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.

  • Trypanosoma congolense Procyclins: Unmasking Cryptic Major Surface Glycoproteins in Procyclic Forms
    Eukaryotic cell, 2006
    Co-Authors: Silvia Utz, Christina Kunz Renggli, Isabel Roditi, Igor C. Almeida, Alvaro Acosta-serrano, Peter Bütikofer
    Abstract:

    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.

  • coordinate expression of gpeet Procyclin and its membrane associated kinase in trypanosoma brucei procyclic forms
    Journal of Biological Chemistry, 2003
    Co-Authors: Annecatherine Schlaeppi, Tatiana Malherbe, Peter Bütikofer
    Abstract:

    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.