Trypanosoma brucei brucei :Aspects moléculaires de l'activation et de l'inactivation de gènes d'antigènes de surface au cours du cycle parasitaire

Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe

Possible DNA modification in GC dinucleotides of Trypanosoma brucei telomeric sequences; relationship with antigen gene transcription.

Polymorphism in restriction site cleavage (PstI, SphI, PvuII, HindIII) has been noticed in several occasions in the telomeric sequences harbouring trypanosome variant-specific antigen genes (1, 2, 3). This polymorphism has been further investigated and seems best interpreted as due to partial DNA modification in GC dinucleotides. The actively transcribed telomeric genes do not exhibit such a polymorphism; furthermore, in at least three independent cases, gene inactivation is linked to the appearance of polymorphism. It could thus be hypothesized that DNA modification prevents antigen gene transcription, or vice-versa. We report however that at least some telomeric antigen-specific sequences of the procyclic trypanosomes (in vitro culture form) are not polymorphic, although they do not synthesize any variant-specific antigen mRNA. There is thus no absolute relationship between the absence of polymorphism and antigen gene transcription.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Telomeric DNA modification linkes to the control of antigen gene expression in trypanosomes

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Transmembrane insertion of the toxoplasma gondii GRA5 protein occurs after soluble secretion into the host cell

The intracelluIar parasite Toxoplasma gondii resides within a specialized compartment, the parasitophorous vacuole (PV), that resists fusion with host cell endocytic and lysosomal compartments. The PV is extensively modified by secretion of parasite proteins, including the dense granule protein GRA5 that is specifically targeted to the delimiting membrane of the PV (PVM). We show here that GRA5 is present both in a soluble form and in hydrophobic aggregates. GRA5 is secreted as a soluble form into the PV after which it becomes stably associated with the PVM. Topological studies demonstrated that GRA5 was inserted into the PVM as a transmembrane protein with its N-terminal domain extending into the cytoplasm and its C terminus in the vacuole lumen. Deletion of 8 of the 18 hydrophobic amino acids of the single predicted transmembrane domain resulted in the failure of GRA5 to associate with the PVM; yet it remained correctly packaged in the dense granules and was secreted as a soluble protein into the PV. Collectively, these studies demonstrate that the secretory pathway in Toxoplasma is unusual in two regards; it allows soluble export of proteins containing typical transmembrane domains and provides a mechanism for their insertion into a host cell membrane after secretion from the parasite.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Characterization of genes coding for two major metacyclic surface antigens in Trypanosoma brucei.

In African trypanosomes, only a very small fraction of the total repertoire of variable antigen types (VATs) is expressed by the metacyclic form. In Trypanosoma brucei stock EATRO 1125, the VATs AnTat 1.30 and 1.45 are reproducibly present in about 15% and 4% of the metacyclic population, respectively. The genes encoding the corresponding antigens or variant surface glycoproteins (VSGs) are in telomeres of large chromosomes, as are some non-metacyclic VSG genes from the same stock. Their activation mechanism has been studied in seven independent clones, 3 of which, referred to as 'first wave' metacyclic VATs (M-VATs), have been cloned from the first wave of parasitemia after cyclic transmission. In all these clones, activation of the antigen gene was linked to the transposition of an expression linked copy (ELC) of the gene to a telomeric expression site. For first wave M-VATs, this site seems variable, although restricted to large chromosomes, and it can be re-used for VSG gene expression in the bloodstream form. In 'late bloodstream' M-VATs, isolated from established chronic infections, the active expression site, at the end of a 200 kb chromosome, is the one preferred for the expression of late antigen types. It can be concluded that no characteristic feature in the genomic location and expression mechanism can distinguish metacyclic antigen genes from those expressed in the bloodstream forms, although the control of their expression must clearly be different.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Modifications of a Trypanosoma b. brucei antigen gene repertoire by different DNA recombinational mechanisms.

In the Trypanosoma b. brucei AnTat 1.1C clone, the gene coding for the variant-specific surface antigen is telomeric and appears as a hybrid sequence, partially modified by gene conversion. This conversion is very similar to that observed in another AnTat 1.1-expressor clone (AnTat 1.1B). This sequence is not activated by duplicative transposition, although it could be activated by duplication in another clone (AnTat 1.10). Instead activation of the AnTat 1.1C gene seems operated by reciprocal recombination between its own telomere and the telomere carrying the previous (AnTat 1.16) ELC. Indeed, from the switch to AnTat 1.1C onward, the AnTat 1.16 ELC becomes a new silent member of its gene family, whereas in the variant directly derived from AnTat 1.1C (AnTat 1.3B), the AnTat 1.1C-containing telomere is lost, probably replaced by a large duplicate, at least 40 kb long, of the AnTat 1.3 gene-containing telomere. Different DNA rearrangement mechanisms used by the trypanosome to change its antigenic type thus contribute, by gain and loss of genes, to the evolution of the repertoire for surface antigens.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Characterization of the membrane tubulation mechanism induced by the Toxoplasma GRA2 protein

International audienceWithin the host cell, the parasitophorous vacuole in which Toxoplasma gondii develops, is characterized by a membranous nanotubular network (MNN). The molecular components, the formation and the function of the MNN remain poorly explored.We had previously demonstrated indirectly that the secreted dense granule GRA2 protein, which contains three amphipathic alpha helices, is a key element of the MNN formation (Mercier et al., 2002; Travier et al., 2008). The aim of this work was to demonstrate directly the role of GRA2 in the MNN formation, using an in vitro system to study the GRA2-membrane interactions. Native and recombinant GRA2 proteins were puri ed from Toxoplasma and from Escherichia coli, respectively. Dynamic light scattering and circular dichroism showed that recombinant GRA2, which folds with an alpha-helical pattern, is puri ed as both a potential dimer and complexes of higher molecular weight. When incubated with Small Unilamellar Vesicles (SUVs) formed with complex lipids, both native and recombinant GRA2 were shown to associate with the SUV membranes and to induce the formation of long membranous tubules observed by transmission electron microscopy (TEM). In the literature, it has been reported that most proteins capable of inducing membrane curvature, interact with phosphatidylinositol (4,5) bi-phosphate (PI(4,5)P2). Fat blots and binding assays of GRA2 to SUVs of de ned lipid composition and containing PI(4,5)P2 showed that GRA2 recognizes phosphoinositides and associates preferentially with PI(4,5)P2-containing SUVs. TEM showed that GRA2 deforms membranes only when PI(4,5)P2 is present. Together, these results allowed us to propose a model of membrane deformation induced by GRA2, the principal effector of the MNN formation

Histone-Modifying Complexes Regulate Gene Expression Pertinent to the Differentiation of the Protozoan Parasite Toxoplasma gondii

Pathogenic apicomplexan parasites like Toxoplasma and Plasmodium (malaria) have complex life cycles consisting of multiple stages. The ability to differentiate from one stage to another requires dramatic transcriptional changes, yet there is a paucity of transcription factors in these protozoa. In contrast, we show here that Toxoplasma possesses extensive chromatin remodeling machinery that modulates gene expression relevant to differentiation. We find that, as in other eukaryotes, histone acetylation and arginine methylation are marks of gene activation in Toxoplasma. We have identified mediators of these histone modifications, as well as a histone deacetylase (HDAC), and correlate their presence at target promoters in a stage-specific manner. We purified the first HDAC complex from apicomplexans, which contains novel components in addition to others previously reported in eukaryotes. A Toxoplasma orthologue of the arginine methyltransferase CARM1 appears to work in concert with the acetylase TgGCN5, which exhibits an unusual bias for H3 [K18] in vitro. Inhibition of TgCARM1 induces differentiation, showing that the parasite life cycle can be manipulated by interfering with epigenetic machinery. This may lead to new approaches for therapy against protozoal diseases and highlights Toxoplasma as an informative model to study the evolution of epigenetics in eukaryotic cells

Unpacking Pandora's Box: Innovative Techniques for Effectively Counseling Asylum Applicants Suffering From Post-Traumatic Stress Disorder

46 p. ; This article was previously published in v.4 no.2 of the Hastings Race and Poverty Law Journal.Many asylum seekers are denied relief in the United States because the asylum officer or immigration judge evaluating the case does not believe the asylum applicant is telling the truth. The applicant may appear not to be credible because his story of persecution lacks sufficient detail and is inconsistent. A victim of severe and often prolonged trauma may develop post-traumatic stress disorder (PTSD), which profoundly affects his ability to tell a consistent and detailed story of past persecution. Thus, an asylum seeker suffering from PTSD as a result of traumatic experiences, desperately in need of a safe haven, may be denied asylum due to the symptoms of his affliction. This article is timely in light of recent changes in immigration law that have considerably raised both evidentiary requirements and the standard for obtaining asylum. These changes create an asylum process that poses significant obstacles for asylum seekers and dramatically reduces their chances of being granted asylum. This article proposes practical methods for use throughout the lawyer-client relationship in order to help an asylum seeker credibly tell his story of past persecution. This article incorporates recent scientific research on PTSD and its affect on memory in recommending the counseling techniques in this article. These techniques may assist an asylum seeker in consistently recalling the details of past persecution that establish a well-founded fear of returning to his home country

Visualization of the Δ<i>gra2-9</i> knockout parasites and PV morphology by transmission electron microscopy.

<p>A) HFFs and parasites were cultured in D10 medium (“Normal D10”) or pre-equilibrated in lipid-free D10 medium (“Lipid-free D10”). Host cells were infected overnight with Δ<i>gra2-9</i> knockout or parental strains, fixed, and then processed for transmission electron microscopy. Scale: 2 μm. B) Magnification of the posterior end of an intracellular Δ<i>gra7</i> knockout parasite in “Normal D10” and in “Lipid-free D10” showing the hyper-formation of the IVN. Scales: 2 μm.</p