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Biomedical subjects

E Pays

Publications and source records attributed to E Pays.

At least 19 recordsLinked to original sources
PubMed2006

The function of apolipoproteins L.

The function of the proteins of the apolipoprotein L (apoL) family is largely unknown. These proteins are classically thought to be involved in lipid transport and metabolism, mainly due to the initial discovery that a secreted member of the family, apoL-I, is associated with high-density lipoprotein particles. However, the other members of the family are believed to be intracellular. The recent unravelling of the mechanism by which apoL-I kills African trypanosomes, as well as the increasing evidence for modulation of apoL expression in various pathological processes, provides new insights about the functions of these proteins. ApoLs share structural and functional similarities with proteins of the Bcl-2 family. Based on the activity of apoL-I in trypanosomes and the comparison with Bcl-2 proteins, we propose that apoLs could function as ion channels of intracellular membranes and be involved in mechanisms triggering programmed cell death.

Amino Acid Motifs↗
PubMed2001

A conserved flagellar pocket exposed high mannose moiety is used by African trypanosomes as a host cytokine binding molecule.

Trypanosomes use antigenic variation of their variant-specific surface glycoprotein (VSG) coat as defense against the host immune system. However, in order to sustain their growth, they need to expose conserved epitopes, allowing host macromolecule binding and receptor-mediated endocytosis. Here we show that Trypanosoma brucei uses the conserved chitobiose-oligomannose (GlcNAc(2)-Man(5-9)) moieties of its VSG as a binding ligand for tumor necrosis factor (TNF), a host cytokine with lectin-like properties. As endocytosis in trypanosomes is restricted to the flagellar pocket, we show that soluble flagellar pocket extracts, and in particular soluble VSG, inhibit the binding of (125)I-TNF to trypanosomes. The interaction between TNF and VSG is confirmed by affinity chromatography, biosensor, and dot-blot affinity measurements, and soluble VSG inhibition of TNF-mediated trypanolysis. In all approaches, removal of N-linked carbohydrates abrogates the TNF-VSG interaction. In addition, synthetic high mannose oligosaccharides can block TNF-VSG interactions, and a VSG glycopeptide carrying the GlcNAc(2)-Man(5-9) moiety is shown to inhibit TNF-mediated trypanosome killing in mixed parasite/macrophage cell cultures. Together, these results support the observation that TNF plays a role in growth control of trypanosomes and, moreover, suggest that, by the use of conserved VSG carbohydrates as lectin-binding epitopes, trypanosomes can limit the necessity to express large numbers of invariant surface exposed receptors.

Animals↗
PubMed2001

Control and function of the bloodstream variant surface glycoprotein expression sites in Trypanosoma brucei.

African trypanosomes escape the host immune response through a periodical change of their surface coat made of one major type of protein, the variant surface glycoprotein. From a repertoire of a thousand variant surface glycoprotein genes available, only one is expressed at a time, and this takes place in a specialised expression site itself selected from a collection of an estimated 20-30 sites. As the specialised expression sites are long polycistronic transcription units, the variant surface glycoprotein is co-transcribed with several other genes termed expression site-associated genes. How do the trypanosomes only use a single specialised expression site at a time? Why are there two dozen specialised expression sites? What are the functions of the other genes of these transcription units? We review the currently available answers to these questions.

Animals↗
PubMed2001

Alternative versus classical macrophage activation during experimental African trypanosomosis.

African trypanosomes are extracellular parasites causing sleeping sickness to human or nagana to livestock in sub-Saharan Africa. To gain insight into factors governing resistance/susceptibility to these parasites, the immune responses in mice infected with a Trypanosoma brucei phospholipase C null mutant (PLC(-/-)) or its wild type counterpart (WT) were compared. We found that the T. b. brucei mutant inducing a chronic infection triggers the production of type I cytokines during the early stage of infection, followed by the secretion of type II cytokines in the late/chronic phase of the disease. In contrast, WT-infected mice are killed within 5 weeks and remain locked in a type I cytokine response. The type I/type II cytokine balance may influence the development of different subsets of suppressive macrophages, i.e. classically activated macrophages (type I) versus alternatively activated macrophages (type II) that are antagonistically regulated. Therefore, the phenotype and accessory cell function of macrophages elicited during WT and PLC(-/-) T. b. brucei infections were addressed. Results indicate that classically activated macrophages develop in a type I cytokine environment in the early phase of both WT and PLC(-/-) trypanosome infections. In the late stage of infection, only PLC(-/-)-infected mice resisting the infection develop type II cytokine-associated alternative macrophages. In parallel, we found that mice susceptible to Trypanosoma congolense infection, showing an exponential parasite growth until they die, have a higher level of type II cytokines in the early stage of infection than resistant animals controlling the first peak of parasitaemia. The levels of type I cytokines were comparable in both T. congolense-resistant and -susceptible mice. On the basis of these results, we propose that survival to African trypanosome infection requires a type I cytokine environment and classical macrophage activation in the early stage of infection, enabling mice to control the first peak of parasitaemia. Thereafter, a switch to type II cytokine environment triggering alternative macrophage activation is required to enable progression of the disease into the chronic phase. The possible role of the sequential activation of alternative macrophages in the late/chronic stage of infection in the increased resistance of mice to PLC(-/-) T. b. brucei will be discussed.

Animals↗
PubMed2001

The VSG expression sites of Trypanosoma brucei: multipurpose tools for the adaptation of the parasite to mammalian hosts.

The variant surface glycoprotein (VSG) genes of Trypanosoma brucei are transcribed in telomeric loci termed VSG expression sites (ESs). Despite permanent initiation of transcription in most if not all of these multiple loci, RNA elongation is abortive except in bloodstream forms where full transcription up to the VSG occurs only in a single ES at a time. The ESs active in bloodstream forms are polycistronic and contain several genes in addition to the VSG, named ES-associated genes (ESAGs). So far 12 ESAGs have been identified, some of which are present only in some ESs. Most of these genes encode surface proteins and this list includes different glycosyl phosphatidyl inositol (GPI)-anchored proteins such as the heterodimeric receptor for the host transferrin (ESAG7/6), integral membrane proteins such as the receptor-like transmembrane adenylyl cyclase (ESAG4) and a surface transporter (ESAG10). An interesting exception is ESAG8, which may encode a cell cycle regulator involved in the differentiation of long slender into short stumpy bloodstream forms. Several ESAGs belong to multigene families including pseudogenes and members transcribed out of the ESs, named genes related to ESAGs (GRESAGs). However, some ESAGs (7, 6 and 8) appear to be restricted to the ESs. Most of these genes can be deleted from the active ES without apparently affecting the phenotype of bloodstream form trypanosomes, probably either due to the expression of ESAGs from 'inactive' ESs (ESAG7/6) or due to the expression of GRESAGs (in particular, GRESAGs4 and GRESAGs1). At least three ESAGs (ESAG7, ESAG6 and SRA) share the evolutionary origin of VSGs. The presence of these latter genes in ESs may confer an increased capacity of the parasite for adaptation to various mammalian hosts, as suggested in the case of ESAG7/6 and proven for SRA, which allows T. brucei to infect humans. Similarly, the existence of a collection of slightly different ESAG4s in the multiple ESs might provide the parasite with adenylyl cyclase isoforms that may regulate growth in response to different environmental conditions. The high transcription rate and high recombination level that prevail in VSG ESs may have favored the generation and/or recruitment in these sites of genes whose hyper-evolution allows adaptation to a larger variety of hosts.

Animals↗
PubMed2001

A receptor-like flagellar pocket glycoprotein specific to Trypanosoma brucei gambiense.

Trypanosoma brucei gambiense and T. b. rhodesiense are protozoan parasites causing sleeping sickness in humans due to their resistance to lysis by normal human serum (NHS). Based on the observation that the resistance gene of T. b. rhodesiense encodes a truncated form of the variant specific glycoprotein (VSG), we cloned a similar gene in T. b. gambiense using reverse transcription-linked polymerase chain reaction with VSG-specific primers. This gene, termed TgsGP for T. gambiense-specific glycoprotein, was found to be specific to T. b. gambiense. It is located close to a telomere and is transcribed by a pol II RNA polymerase, only at the bloodstream stage of the parasite development. TgsGP encodes a 47-kDa protein consisting of a N-terminal VSG domain presumably provided with a glycosylphosphatidylinositol (GPI) anchor sequence, similar to the pESAG6 subunit of the trypanosomal transferrin receptor. TgsGP is located in the flagellar pocket, and contains the linear N-linked polyacetyllactosamine characteristic of the endocytotic machinery of T. brucei. These observations strongly suggest that TgsGP is a T. b. gambiense specific receptor. Since stable expression of this protein in T. b. brucei did not confer resistance to NHS, TgsGP may either need another factor to achieve this purpose or fulfils another function linked to adaptation of the parasite to man.

Amino Acid Sequence↗
PubMed2001

An update on antigenic variation in African trypanosomes.

African trypanosomes can spend a long time in the blood of their mammalian host, where they are exposed to the immune system and are thought to take advantage of it to modulate their own numbers. Their major immunogenic protein is the variant surface glycoprotein (VSG), the gene for which must be in one of the 20--40 specialized telomeric expression sites in order to be transcribed. Trypanosomes escape antibody-mediated destruction through periodic changes of the expressed VSG gene from a repertoire of approximately 1000. How do trypanosomes exclusively express only one VSG and how do they switch between them?

Animals↗
PubMed2001

Overlapping sense and antisense transcription units in Trypanosoma brucei.

Procyclins are the major surface glycoproteins of insect-form Trypanosoma brucei. The procyclin expression sites are polycistronic and are transcribed by an alpha-amanitin-resistant polymerase, probably RNA polymerase I (Pol I). The expression sites are flanked by transcription units that are sensitive to alpha-amanitin, which is a hallmark of Pol II-driven transcription. We have analysed a region of 9.5 kb connecting the EP/PAG2 expression site with the downstream transcription unit. The procyclin expression site is longer than was previously realized and contains an additional gene, procyclin-associated gene 4 (PAG4), and a region of unknown function, the T region, that gives rise to trans-spliced, polyadenylated RNAs containing small open reading frames (ORFs). Two new genes, GU1 and GU2, were identified in the downstream transcription unit on the opposite strand. Unexpectedly, the 3' untranslated region of GU2 and the complementary T transcripts overlap by several hundred base pairs. Replacement of GU2 by a unique tag confirmed that sense and antisense transcription occurred from a single chromosomal locus. Overlapping transcription is stage specific and may extend > or = 10 kb in insect-form trypanosomes. The nucleotide composition of the T. brucei genome is such that antisense ORFs occur frequently. If stable mRNAs can be derived from both strands, the coding potential of the genome may be substantially larger than has previously been suspected.

3' Untranslated Regions↗
PubMed2000

A transcript encoding a proteasome beta-subunit and a zinc finger protein in Trypanosoma brucei brucei.

During the screening of a Trypanosoma brucei brucei (T. b. brucei) cDNA library constructed from bloodstream form mRNA, we identified a 2.3kb cDNA encoding a proteasome beta subunit (ORF1) and a putative zinc finger protein (ORF2). Northern blot analysis indicated the presence of a digenic transcript as well as the two individual messengers in both procyclic and bloodstream forms of the parasite. Southern blot analysis showed the relevant locus to be unique. ORF1 encoded a 22.7kDa protein sharing over 50% identity with the eukaryotic PRCE (aka beta5) proteasome beta subunit. This protein contained a beta amino acid signature and residues involved in the catalytic activity. Further phylogenetic analysis indicated that this subunit as well as those from other kinetoplastids could be confidentially assigned to extant eukaryotic subfamilies such as beta1, beta2, and beta5. ORF2 encoded a 14.6kDa putative zinc finger protein containing five repeats of a CCHC motif commonly present in retroviral nucleocapsid proteins as well as proteins involved in vertebrate embryogenesis.

Amino Acid Sequence↗
PubMed2000

A role for the dynamic acylation of a cluster of cysteine residues in regulating the activity of the glycosylphosphatidylinositol-specific phospholipase C of Trypanosoma brucei.

The glycosylphosphatidylinositol-specific phospholipase C or VSG lipase is the enzyme responsible for the cleavage of the glycosylphosphatidylinositol anchor of the variant surface glycoprotein (VSG) and concomitant release of the surface coat in Trypanosoma brucei during osmotic shock or extracellular acidic stress. In Xenopus laevis oocytes the VSG lipase was expressed as a nonacylated and a thioacylated form. This thioacylation occurred within a cluster of three cysteine residues but was not essential for catalytic activity per se. These two forms were also detected in trypanosomes and appeared to be present at roughly equivalent amounts. A reversible shift to the acylated form occurred when cells were triggered to release the VSG by either nonlytic acid stress or osmotic lysis. A wild type VSG lipase or a gene mutated in the three codons for the acylated cysteines were reinserted in the genome of a trypanosome null mutant for this gene. A comparative analysis of these revertant trypanosomes indicated that thioacylation might be involved in regulating enzyme access to the VSG substrate.

Acylation↗
PubMed2000

Characterization of a Trypanosoma brucei SR domain-containing protein bearing homology to cis-spliceosomal U1 70 kDa proteins.

The protozoan parasite Trypanosoma brucei relies on trans-splicing of a common spliced leader (SL) RNA to maturate mRNAs. Using the yeast two-hybrid system a protein (TSR1IP) was identified that interacts with the T. brucei serine-arginine (SR) protein termed TSR1. TSR1IP shows homology to U1 70 kDa proteins, and contains an SR rich domain as well as an acidic/arginine domain homologous to the U1 70 kDa poly(A) polymerase inhibiting domain. This protein is localized in the nucleoplasm and excluded from the nucleolus in trypanosomal bloodstream and procyclic forms. Based on structural modelling predictions and on the identification of a RNA recognition motif (RRM), it was possible to demonstrate by the yeast three-hybrid system that TSR1IP interacts with the 5' splice region of the SL RNA. All the above characteristics suggest that TSR1IP could be involved in trans-splicing.

Amino Acid Sequence↗
PubMed2000

Characterization of a novel alanine-rich protein located in surface microdomains in Trypanosoma brucei.

Heterologous expression in COS cells followed by orientation-specific polymerase chain reaction to select and amplify cDNAs encoding surface proteins in Trypanosoma brucei resulted in the isolation of a cDNA ( approximately 1.4 kilobase) which encodes an acidic, alanine-rich polypeptide that is expressed only in bloodstream forms of the parasite and has been termed bloodstream stage alanine-rich protein (BARP). Analysis of the amino acid sequence predicted the presence of a typical NH(2)-terminal leader sequence as well as a COOH-terminal hydrophobic extension with the potential to be replaced by a glycosylphosphatidylinositol anchor. A search of existing protein sequences revealed partial homology between BARP and the major surface antigen of procyclic forms of Trypanosoma congolense. BARP migrated as a complex, heterogeneous series of bands on Western blots with an apparent molecular mass ( approximately 50-70 kDa) significantly higher than predicted from the amino acid sequence ( approximately 26 kDa). Confocal microscopy demonstrated that BARP was present in small discrete spots that were distributed over the entire cellular surface. Detergent extraction experiments revealed that BARP was recovered in the detergent-insoluble, glycolipid-enriched fraction. These data suggested that BARP may be sequestered in lipid rafts.

Amino Acid Sequence↗
PubMed2000

A single-stranded DNA-binding protein shared by telomeric repeats, the variant surface glycoprotein transcription promoter and the procyclin transcription terminator of Trypanosoma brucei.

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.

Animals↗
PubMed2000

Differential RNA elongation controls the variant surface glycoprotein gene expression sites of Trypanosoma brucei.

The protozoan parasite Trypanosoma brucei develops antigenic variation to escape the immune response of its host. To this end, the trypanosome genome contains multiple telomeric expression sites competent for transcription of variant surface glycoprotein genes, but as a rule only a single antigen is expressed at any time. We used reverse transcription-PCR (RT-PCR) to analyse transcription of different segments of the expression sites in different variant clones of two independent strains of T. brucei. The results indicated that RNA polymerase is installed and active at the beginning of many, if not all, expression sites simultaneously, but that a progressive arrest of RNA elongation occurs in all but one site. This defect is linked to inefficient RNA processing and RNA release from the nucleus. Therefore, functional transcription in the active site appears to depend on the selective recruitment of a RNA elongation/processing machinery.

Animals↗
PubMed2000

Antibodies raised against the flagellar pocket fraction of Trypanosoma brucei preferentially recognize HSP60 in cDNA expression library.

A purified flagellar pocket fraction of the Trypanosoma brucei AnTat 1.1E clone was used for the generation of polyclonal antiserum in rats. Anti-flagellar pocket antibodies present in this serum recognized several proteins distinct from the major variant surface glycoprotein (VSG). In Balb/c mice, flagellar pocket immunization resulted in partial resistance towards the challenge with a low dose of parasites. This was accompanied by the induction of specific IgG2a antibodies. In an attempt to discover protective parasite antigens, antiflagellar pocket serum was used for the screening of a T. brucei bloodstream form cDNA library constructed in the lambdagt11 bacteriophage expression system. Through antibody panning and VSG elimination, 15 specific cDNA inserts were selected. Most intriguing was the observation that in addition to two clones encoding the invariant surface glycoprotein 75 (ISG75), 10 out of 15 independently selected cDNA inserts encoded the trypanosome heat shock protein 60 (tHSP60).

Animals↗
PubMed2000

Slender and stumpy bloodstream forms of Trypanosoma brucei display a differential response to extracellular acidic and proteolytic stress.

Natural infections of mammals with African trypanosomes, such as Trypanosoma brucei, are generally pleomorphic, the population consisting of different forms, termed slender and stumpy forms, that vary in number as the parasitaemia develops. We show that the differentiation of slender into stumpy forms is characterized by the acquisition by the parasite of the ability to regulate its internal pH, even in the face of a large, inwardly directed gradient of H+, as well as a tolerance towards external proteolytic stress. These adaptations effectively abbrogate cellular stress-activated signalling pathways involving adenylate cyclase and glycosylphosphoinositol-specific phospholipase-C mediated release of the surface coat. Although in metabolic terms stumpy forms of the parasite are considered to be preadapted to life in the arthropod vector, these data clearly demonstrate that these forms also possess additional cellular adaptations designed to deal with the immediate and potentially harmful changes in the extracellular environment that occur upon ingestion of a bloodmeal by the tsetse fly vector.

Acid-Base Equilibrium↗