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E Pays

Publications and source records attributed to E Pays.

At least 37 records · Page 2Linked to original sources
PubMed2000

Attenuation of Trypanosoma brucei is associated with reduced immunosuppression and concomitant production of Th2 lymphokines.

Mechanisms regulating resistance to African trypanosomes were addressed by comparing the immune responses of mice infected with attenuated Trypanosoma brucei brucei lacking the phospholipase C gene (PLC-/-) and those of mice infected with wild-type (WT) parasites. Inhibition of concanavalin A (ConA)-induced T cell proliferation occurred in spleen and lymph nodes of PLC-/-- and WT-infected mice. Although suppressive cells were elicited in spleen and lymph nodes of WT-infected animals, such cells were not detected in lymph nodes of PLC-/--infected mice. PLC-/--infected mice had more interleukin-4 and -10 in their blood than did WT-infected mice. Correspondingly, PLC-/--infected mice had higher IgG1 antibody levels against variant surface glycoprotein than did WT-infected mice. These data indicate that attenuation of T. b. brucei correlates with the absence of cells suppressing ConA-induced T cell proliferation in the lymph nodes, with increased production of Th2 cytokines and a stronger IgG1 antibody response to trypanosome antigens.

Animals↗
PubMed2000

Comparative analysis of antibody responses against HSP60, invariant surface glycoprotein 70, and variant surface glycoprotein reveals a complex antigen-specific pattern of immunoglobulin isotype switching during infection by Trypanosoma brucei.

During Trypanosoma brucei infections, the response against the variant surface glycoprotein (VSG) of the parasite represents a major interaction between the mammalian host immune system and the parasite surface. Since immune recognition of other parasite derived factors also occurs, we examined the humoral host response against trypanosome heat shock protein 60 (HSP60), a conserved antigen with an autoimmune character. During experimental T. brucei infection in BALB/c mice, the anti-HSP60 response was induced when parasites differentiated into stumpy forms. This response was characterized by a stage-specific immunoglobulin isotype switching as well as by the induction of an autoimmune response. Specific recognition of trypanosome HSP60 was found to occur during the entire course of infection. Immunoglobulin G2a (IgG2a) and IgG2b antibodies, induced mainly in a T-cell-independent manner, were observed during the first peak of parasitemia, whereas IgG1 and IgG3 antibodies were found at the end of the infection, due to a specific T-cell-mediated response. Comparative analysis of the kinetics of anti-HSP60, anti-invariant surface glycoprotein 70 (ISG70), and anti-VSG antibody responses indicated that the three trypanosome antigens give rise to specific and independent patterns of immunoglobulin isotype switching.

Animals↗
PubMed1999

N-linked glycans containing linear poly-N-acetyllactosamine as sorting signals in endocytosis in Trypanosoma brucei.

African trypanosomes, such as Trypanosoma brucei, are protozoan parasites that are transmitted by the tsetse fly and cause sleeping sickness in humans and Nagana in cattle. Trypanosomes evade the immune responses of their hosts by varying their surface coat protein (VSG) and restricting exocytosis and endocytosis to an invagination of the plasma membrane called the flagellar pocket (FP). The FP represents only 0.5% of the cellular surface but membrane turnover here occurs at high rates [1] [2] [3]. No model has yet been proposed to account for the sequestration of membrane proteins and the rate of membrane turnover that occur in the FP. Recent data have suggested that glycans are involved in the sorting of membrane proteins in polarized cells [4] [5] [6] [7]. Here, we show that N-linked glycans containing linear poly-N-acetyllactosamine (pNAL) are only associated with proteins of the FP/endocytic pathway in T. brucei and are present only in bloodstream forms of the parasite. These glycoproteins bind to tomato lectin (TL), a property that allowed their single-step isolation. Chito-oligosaccharides that compete specifically for pNAL binding to TL also inhibited receptor-mediated uptake of several ligands. These results suggest a model in which N-linked linear pNAL acts as a sorting signal for endocytosis in trypanosomes.

Animals↗
PubMed1999

Differential regulation of ESAG transcripts in Trypanosoma brucei.

In Trypanosoma brucei, several genes termed ESAGs for expression site-associated genes are contained within the polycistronic transcription units of the VSG gene, and their transcription is coordinately regulated during the parasite life-cycle. Since the VSG mRNA is characterized by a drastic destabilization under conditions where translation is inhibited, we examined if this post-transcriptional control also applies to the ESAG mRNAs. While the ESAG 7/6 mRNA behaved like the VSG mRNA, the ESAG 8 and ESAG 3 mRNAs did not. We ascribe this differential behaviour to the residual transcription that still occurs only in the ESAG 7/6 region of the VSG unit under conditions where this unit is down-regulated.

Animals↗
PubMed1999

Characterization of a SR protein from Trypanosoma brucei with homology to RNA-binding cis-splicing proteins.

The protozoan parasite Trypanosoma brucei relies on trans-splicing to process its mRNAs. A novel nuclear serine/arginine (SR)-rich trypanosomal protein (TSR1) was characterized which contains two RNA recognition motifs. The TSR1 protein appears to be homologous to RNA-binding SR proteins of the cis-splicing machinery from higher eukaryotes. Moreover, in the yeast two-hybrid system, TSR1 is able to interact with the human splicing factors involved in the recognition of the 3' splicing site (U2AF35/U2AF65). In both procyclic and bloodstream forms of T. brucei, TSR1 was found to localize in the nucleus. In the bloodstream stage TSR1 showed the speckles pattern characteristic of SR proteins involved in cis-splicing. Moreover, TSR1 was able to specifically bind the spliced leader (SL) RNA involved in trans-splicing in trypanosomes by the yeast three-hybrid system. These and other observations suggest that TSR1 may be involved in trans-splicing in T. brucei.

Amino Acid Sequence↗
PubMed1999

A protein linked to mitochondrion development in Trypanosoma brucei.

The use of the two-hybrid system in yeast allowed us to isolate a new mitochondrial protein of Trypanosoma brucei, termed PIE8, for putative protein interacting with ESAG8. This protein was found to localize progressively in the single mitochondrion of the parasite during the mitochondrial reactivation needed to adapt the parasite from the glycolysis-based metabolism in the mammalian host, to the cytochrome-mediated respiration in the fly vector. Once this reactivation is established, PIE8 is lost from the mitochondrion. Thus, the temporary presence of PIE8 in the mitochondrion is linked to mitochondrial reactivation.

Amino Acid Sequence↗
PubMed1999

Regulated transcription of the histone H2B genes of Trypanosoma brucei.

In Trypanosoma brucei, the genes encoding histone H2B are organized in a cluster of about 10-15 tandemly linked copies per haploid genome. The H2B transcripts are processed by trans-splicing and polyadenylation, and encode a polypeptide of 111 residues with a molecular mass of 12.5 kDa. H2B mRNAs are differentially expressed during the parasite life-cycle and are present at higher levels in dividing procyclic and bloodstream slender forms than in the nondividing bloodstream stumpy forms. Analysis of H2B mRNA levels during the synchronous differentiation from stumpy to procyclics forms revealed that the abundance of these transcripts is regulated through the cell-cycle, reaching maximum levels during S-phase. Addition of hydroxyurea to procyclic forms in culture specifically decreased H2B mRNA levels by about twofold, an effect not linked to its 3' untranslated region. Inhibition of protein synthesis prevented this decrease.

Amino Acid Sequence↗
PubMed1999

Cell lysis induces redistribution of the GPI-anchored variant surface glycoprotein on both faces of the plasma membrane of Trypanosoma brucei.

African trypanosomes are coated by 10 million copies of a single variant specific glycoprotein (VSG) which are anchored in the plasma membrane by glycosylphosphatidylinositol (GPI). A GPI-specific phospholipase C (GPI-PLC) triggers fast VSG release upon cell lysis but in vivo it is safely controlled and topologically concealed from its substrate by being intracellular. One enigmatic aspect of GPI-PLC action therefore consists of how it could gain access to the VSG in the exoplasmic leaflet of the membrane. The data presented herewith disclose an unexpected possible solution for this puzzle: upon cell rupture the VSG invades the cytoplasmic face of the plasma membrane which thus becomes double coated. This unusual VSG rearrangement was stable in ruptured plasma membrane from GPI-PLC null mutant trypanosomes but transiently preceded VSG release in wild-type parasites. The formation of double coat membrane (DCM) was independent of the presence or activation of GPI-PLC, occurred both at 4 degrees C and 30 degrees C and was unaffected by the classical inhibitor of VSG release, p-choromercuryphenylsulfonic acid (PCM). DCMs conserved the same coat thickness and association with subpellicular microtubules as in intact cells and were prone to form vesicles following gradual detachment of the latter. Our data also demonstrate that: (i) GPI-PLC expressed by one trypanosome only targets its own plasma membrane, being unable to release VSG of another parasite; (ii) DCMs concomitantly formed from trypanosomes expressing different VSGs do not intermix, an indication that DCM might be refractory to membrane fusion.

Animals↗
PubMed1999

Characterization of a coiled coil protein present in the basal body of Trypanosoma brucei.

TBBC (for Trypanosoma brucei basal body component) is a unique gene transcribed in a 4.8 kb mRNA encoding a 1,410 amino acid protein that consists almost entirely of a coiled coil structure. This protein appeared to localize in the basal body, with an accessory presence at the posterior end of the cell, the nucleus and over the flagellum. Since the two other known components of the trypanosome basal body are (gamma)-tubulin and an uncharacterized component termed BBA4 we performed double immunofluorescence experiments with anti-TBBC and either anti-BBA4 or anti-(gamma)-tubulin antibodies. These three components did not colocalize but were very closely associated, BBA4 being the most proximal to the kinetoplast DNA. Anti-TBBC antibodies detected a 170 kDa protein in western blots of total HeLa cell extracts. Moreover, these antibodies stained the centriole of HeLa and COS cells as well as the centriole of mouse spermatozoa, indicating that a TBBC-like centriolar component has been conserved during the evolution of eukaryotes.

Amino Acid Sequence↗
PubMed1998

A VSG expression site-associated gene confers resistance to human serum in Trypanosoma rhodesiense.

Infectivity of Trypanosoma brucei rhodesiense to humans is due to its resistance to a lytic factor present in human serum. In the ETat 1 strain this character was associated with antigenic variation, since expression of the ETat 1.10 variant surface glycoprotein was required to generate resistant (R) clones. In addition, in this strain transcription of a gene termed SRA was detected in R clones only. We show that the ETat 1.10 expression site is the one selectively transcribed in R variants. This expression site contains SRA as an expression site-associated gene (ESAG) and is characterized by the deletion of several ESAGs. Transfection of SRA into T.b. brucei was sufficient to confer resistance to human serum, identifying this gene as one of those responsible for T.b. rhodesiense adaptation to humans.

Animals↗
PubMed1998

Trypanosoma brucei TBRGG1, a mitochondrial oligo(U)-binding protein that co-localizes with an in vitro RNA editing activity.

We report the characterization of a Trypanosoma brucei 75-kDa protein of the RGG (Arg-Gly-Gly) type, termed TBRGG1. Dicistronic and monocistronic transcripts of the TBRGG1 gene were produced by both alternative splicing and polyadenylation. TBRGG1 was found in two or three forms that differ in their electrophoretic mobility on SDS-polyacrylamide gel electrophoresis gels, one of which was more abundant in the procyclic form of the parasite. TBRGG1 was localized to the mitochondrion and appeared to be more abundant in bloodstream intermediate and stumpy forms in which the mitochondrion reactivates and during the procyclic stage, which possesses a fully functional mitochondrion. This protein was characterized to display oligo(U) binding characteristics and was found to co-localize with an in vitro RNA editing activity in a sedimentation analysis. TBRGG1 most likely corresponds to the 83-kDa oligo(U)-binding protein previously identified by UV cross-linking of guide RNA to mitochondrial lysates (Leegwater, P., Speijer, D., and Benne, R. (1995) Eur. J. Biochem. 227, 780-786).

Alternative Splicing↗
PubMed1998

Conserved organization of genes in trypanosomatids.

Trypanosomatids are unicellular protozoan parasites which constitute some of the most primitive eukaryotes. Leishmania spp, Trypanosoma cruzi and members of the Trypanosoma brucei group, which cause human diseases, are the most studied representatives of this large family. Here we report a comparative analysis of a large genomic region containing glucose transporter genes in three Salivarian trypanosomes (T. brucei, T. congolense and T. vivax), T. cruzi and Leishmania donovani. In T. brucei, the 8 kb (upstream) and 14 kb (downstream) regions flanking the glucose transporter genes cluster contain two and six new genes, respectively, six of them encoding proteins homologous to known eukaryotic proteins (phosphatidylinositol 3 kinase, ribosomal protein S12, DNAJ and three small G-proteins--Rab1, YPT6 and ARL3). This gene organization is identical in T. brucei, T. congolense and T. vivax suggesting that Salivarian trypanosomes have a high level of conservation in gene organization. In T. cruzi and Leishmania, the overall organization of this cluster is conserved, with insertion of additional genes when compared with T. brucei. Phylogenetic reconstitution based on glucose transporters is in accord with the monophyly of the genus Trypanosoma and the early separation of T. vivax within Salivarian trypanosomes. On the basis of gene organization, biochemical characteristics of isoforms and phylogeny, we discuss the genesis of the glucose transporter multigene family in Salivarian trypanosomes.

Amino Acid Sequence↗
PubMed1998

Mild acid stress as a differentiation trigger in Trypanosoma brucei.

In vitro differentiation of Trypanosoma brucei from the bloodstream to the procyclic form is efficiently induced by the combination of cold shock from 37 to 27 degrees C and the addition of citrate/cis-aconitate (CCA) to the incubation medium. Here it is reported that exposure of pleomorphic bloodstream trypanosomes to mild acidic conditions (pH 5.5 for 2 h at 37 degrees C) not only accelerated the process of morphological transformation from long slender and intermediate to short stumpy bloodstream forms but also allowed their subsequent differentiation into procyclic forms even in the absence of CCA. This process appeared to involve the glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC), since null GPI-PLC mutants (PLC-) appeared to be largely refractory to acid stress-induced differentiation. However, an effective response was restored upon reintegration of the GPI-PLC gene in the genome (PLC+).

Aconitic Acid↗
PubMed1998

Controls of the expression of the Vsg in Trypanosoma brucei.

We present an overview of the regulation of vsg expression, focusing on initiation and elongation of transcription as well as processing and stabilization of the transcripts. We propose a model where common factors are involved in the reverse controls of the genes for the two main stage-specific antigens, the Vsg and procyclin: a cross-talk between the two transcription units would allow a fast rerouting of limiting factors at differentiation, thereby allowing the expression of only one type of antigen at a time. A similar mechanism would ensure that only one vsg ES is fully expressed at a time in bloodstream forms.

Animals↗
PubMed1998

The glycosyl-inositol-phosphate and dimyristoylglycerol moieties of the glycosylphosphatidylinositol anchor of the trypanosome variant-specific surface glycoprotein are distinct macrophage-activating factors.

The TNF-alpha-inducing capacity of different trypanosome components was analyzed in vitro, using as indicator cells a macrophage cell line (2C11/12) or peritoneal exudate cells from LPS-resistant C3H/HeJ mice and LPS-sensitive C3H/HeN mice. The variant-specific surface glycoprotein (VSG) was identified as the major TNF-alpha-inducing component present in trypanosome-soluble extracts. Both soluble (sVSG) and membrane-bound VSG (mfVSG) were shown to manifest similar TNF-alpha-inducing capacities, indicating that the dimyristoylglycerol (DMG) compound of the mfVSG anchor was not required for TNF-alpha triggering. Detailed analysis indicated that the glycosyl-inositol-phosphate (GIP) moiety was responsible for the TNF-alpha-inducing activity of VSG and that the presence of the GIP-associated galactose side chain was essential for optimal TNF-alpha production. Furthermore, the results showed that the responsiveness of macrophages toward the TNF-alpha-inducing activity of VSG was strictly dependent on the activation state of the macrophages, since resident macrophages required IFN-gamma preactivation to become responsive. Comparative analysis of the ability of both forms of VSG to activate macrophages revealed that mfVSG but not sVSG stimulates macrophages toward IL-1alpha secretion and acquisition of LPS responsiveness. The priming activity of mfVSG toward LPS responsiveness was also demonstrated in vivo and may be relevant during trypanosome infections, since Trypanosoma brucei-infected mice became gradually LPS-hypersensitive during the course of infection. Collectively, the VSG of trypanosomes encompasses two distinct macrophage-activating components: while the GIP moiety of sVSG mediates TNF-alpha induction, the DMG compound of the mfVSG anchor contributes to IL-1 alpha induction and LPS sensitization.

Animals↗
PubMed1997

Characterization of the ligand-binding site of the transferrin receptor in Trypanosoma brucei demonstrates a structural relationship with the N-terminal domain of the variant surface glycoprotein.

The Trypanosoma brucei transferrin (Tf) receptor is a heterodimer encoded by ESAG7 and ESAG6, two genes contained in the different polycistronic transcription units of the variant surface glycoprotein (VSG) gene. The sequence of ESAG7/6 differs slightly between different units, so that receptors with different affinities for Tf are expressed alternatively following transcriptional switching of VSG expression sites during antigenic variation of the parasite. Based on the sequence homology between pESAG7/6 and the N-terminal domain of VSGs, it can be predicted that the four blocks containing the major sequence differences between pESAG7 and pESAG6 form surface-exposed loops and generate the ligand-binding site. The exchange of a few amino acids in this region between pESAG6s encoded by different VSG units greatly increased the affinity for bovine Tf. Similar changes in other regions were ineffective, while mutations predicted to alter the VSG-like structure abolished the binding. Chimeric proteins containing the N-terminal dimerization domain of VSG and the C-terminal half of either pESAG7 or pESAG6, which contains the ligand-binding domain, can form heterodimers that bind Tf. Taken together, these data provided evidence that the T.brucei Tf receptor is structurally related to the N-terminal domain of the VSG and that the ligand-binding site corresponds to the exposed surface loops of the protein.

Amino Acid Sequence↗
PubMed1997

Characterization of a novel, stage-specific, invariant surface protein in Trypanosoma brucei containing an internal, serine-rich, repetitive motif.

A new surface membrane protein, invariant surface glycoprotein termed ISG100, was identified in Trypanosoma brucei, using catalyzed surface, radioiodination of intact cells. This integral membrane glycoprotein was purified by a combination of detergent extraction, lectin-affinity, and ion-exchange chromatography followed by preparative SDS-polyacrylamide gel electrophoresis. The protein was expressed only in bloodstream forms of the parasite, was heavily N-glycosylated, and was present in different clonal variants of the same serodeme as well as in different serodemes. The gene for this protein was isolated by screening a cDNA expression library with antibodies against the purified protein followed by screening of a genomic library. The nucleotide sequence of the gene (4050 base pairs) predicted a highly reiterative polypeptide containing three distinct domains, a unique N-terminal domain of about 10 kDa containing three potential N-glycosylation sites, which was followed by a large internal domain consisting entirely of 72 consecutive copies of a serine-rich, 17-amino acid motif (approximately 113 kDa) and terminated with an apparent transmembrane spanning region of about 3.3 kDa. The internal repeat region of this gene (3672 base pairs) represents the largest reiterative coding sequence to be fully characterized in any species of trypanosome. There was no significant homology with other known proteins, and overall the predicted protein was extremely hydrophobic. Unlike the genes for other surface proteins, the gene encoding ISG100 was present as a single copy. Although present in the flagellar pocket, ISG100 was predominantly associated with components of the pathways for endo/exocytosis, such as intracellular vesicles located in the proximity of the pocket as well a large, electron-lucent perinuclear digestive vacuole.

Amino Acid Sequence↗