Trypanosoma brucei variant surface glycoprotein regulation involves coupled activation/inactivation and chromatin remodeling of expression sites.
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Trypanosoma brucei is an extracellular protozoan parasite that cycles between mammalian hosts and the tsetse vector. In bloodstream-form trypanosomes, only one variant surface glycoprotein gene (VSG) expression site (ES) is active at any time. Transcriptional switching between ESs results in antigenic variation. No VSG is transcribed in the insect procyclic stage. We have used bacteriophage T7 RNA polymerase (T7RNAP) to study the transcriptional accessibility of ES chromatin in vivo. We show that T7RNAP-mediated transcription from chromosomally integrated T7 promoters is repressed along the entire length of the ES in the procyclic form, but not in the bloodstream form, suggesting that the accessible chromatin of inactive bloodstream-form ESs is remodeled upon differentiation to yield a structure that is no longer permissive for T7RNAP-mediated transcription. In the bloodstream form, replacing the active ES promoter with a T7 promoter, which is incapable of sustaining high-level transcription of the entire ES, prompts an ES switch. These data suggest two distinct mechanisms for ES regulation: a chromatin-mediated developmental silencing of the ES in the procyclic form and a rapid coupled mechanism for ES activation and inactivation in the bloodstream form. (+info)
A cell-free assay for glycosylphosphatidylinositol anchoring in African trypanosomes. Demonstration of a transamidation reaction mechanism.
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We established an in vitro assay for the addition of glycosyl-phosphatidylinositol (GPI) anchors to proteins using procyclic trypanosomes engineered to express GPI-anchored variant surface glycoprotein (VSG). The assay is based on the premise that small nucleophiles, such as hydrazine, can substitute for the GPI moiety and effect displacement of the membrane anchor of a GPI-anchored protein or pro-protein causing release of the protein into the aqueous medium. Cell membranes containing pulse-radiolabeled VSG were incubated with hydrazine, and the VSG released from the membranes was measured by carbonate extraction, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis/fluorography. Release of VSG was time- and temperature-dependent, was stimulated by hydrazine, and occurred only for VSG molecules situated in early compartments of the secretory pathway. No nucleophile-induced VSG release was seen in membranes prepared from cells expressing a VSG variant with a conventional transmembrane anchor (i.e. a nonfunctional GPI signal sequence). Pro-VSG was shown to be a substrate in the reaction by assaying membranes prepared from cells treated with mannosamine, a GPI biosynthesis inhibitor. When a biotinylated derivative of hydrazine was used instead of hydrazine, the released VSG could be precipitated with streptavidin-agarose, indicating that the biotin moiety was covalently incorporated into the protein. Hydrazine was shown to block the C terminus of the released VSG hydrazide because the released material, unlike a truncated form of VSG lacking a GPI signal sequence, was not susceptible to proteolysis by carboxypeptidases. These results firmly establish that the released material in our assay is VSG hydrazide and strengthen the proof that GPI anchoring proceeds via a transamidation reaction mechanism. The reaction could be inhibited with sulfhydryl alkylating reagents, suggesting that the transamidase enzyme contains a functionally important sulfhydryl residue. (+info)
The anatomy and transcription of a monocistronic expression site for a metacyclic variant surface glycoprotein gene in Trypanosoma brucei.
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African trypanosomes evade the immune response of their mammalian hosts by switching the expression of their variant surface glycoprotein genes (vsg). The bloodstream trypanosome clone MVAT4 of Trypanosoma brucei rhodesiense expresses a metacyclic vsg as a monocistronic RNA from a promoter located 2 kilobases (kb) upstream of its start codon. Determination of 23 kb of sequence at the metacyclic variant antigen type 4 (MVAT) vsg expression site (ES) revealed an ES-associated gene (esag) 1 preceded by an ingi retroposon and an inverted region containing an unrelated vsg, short stretches of 70-bp repeats and a pseudo esag 3. Nuclear run-on experiments indicate that the 18-kb region upstream of the MVAT4 vsg promoter is transcriptionally silent. However, multiple members of different esag families are expressed from elsewhere in the genome. The MVAT4 vsg promoter is highly repressed in the procyclic stage, in contrast to the known polycistronic vsg ESs which undergo abortive transcription. Activation of the MVAT4 vsg ES occurs in situ without nucleotide sequence changes, although this monocistronic ES undergoes a pattern of base J modifications similar to that reported for the polycistronic ESs. The relative simplicity of the MVAT4 vsg ES and the uncoupled expression of the vsg and esags provide a unique opportunity for investigating the molecular mechanisms responsible for antigenic variation in African trypanosomes. (+info)
Leaky transcription of variant surface glycoprotein gene expression sites in bloodstream african trypanosomes.
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Trypanosoma brucei undergoes antigenic variation by periodically switching the expression of its variant surface glycoprotein (VSG) genes (vsg) among an estimated 20-40 telomere-linked expression sites (ES), only one of which is fully active at a given time. We found that in bloodstream trypanosomes one ES is transcribed at a high level and other ESs are expressed at low levels, resulting in organisms containing one abundant VSG mRNA and several rare VSG RNAs. Some of the rare VSG mRNAs come from monocistronic ESs in which the promoters are situated about 2 kilobases upstream of the vsg, in contrast to the polycistronic ESs in which the promoters are located 45-60 kilobases upstream of the vsg. The monocistronic ES containing the MVAT4 vsg does not include the ES-associated genes (esag) that occur between the promoter and the vsg in polycistronic ESs. However, bloodstream MVAT4 trypanosomes contain the mRNAs for many different ESAGs 6 and 7 (transferrin receptors), suggesting that polycistronic ESs are partially active in this clone. To explain these findings, we propose a model in which both mono- and polycistronic ESs are controlled by a similar mechanism throughout the parasite's life cycle. Certain VSGs are preferentially expressed in metacyclic versus bloodstream stages as a result of differences in ESAG expression and the proximity of the promoters to the vsg and telomere. (+info)
In vitro analysis of alpha-amanitin-resistant transcription from the rRNA, procyclic acidic repetitive protein, and variant surface glycoprotein gene promoters in Trypanosoma brucei.
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In Trypanosoma brucei, transcription resistant to the mushroom toxin alpha-amanitin is not restricted to the rRNA genes (rDNA), as in higher eukaryotes, but extends to genes encoding the major cell surface proteins variant surface glycoprotein (VSG) and procyclin or procyclic acidic repetitive protein (PARP). Here, we report the development of a homologous cell extract from procyclic T. brucei cells in which rDNA and PARP A and VSG gene promoters drive efficient, accurate, and alpha-amanitin-resistant transcription. A comparative analysis revealed that transcription from the three promoters generally required identical reaction conditions for maximal efficiency. Nevertheless, PARP promoter transcription proved to be exceptional by its high efficiency, its lag phase, a high template DNA concentration optimum, and its tolerance to increasing concentrations of Mn(2+). Mutational analysis for both the PARP and rDNA promoters showed that the proximal and distal core elements were essential for efficient transcription in vitro. Deletion of the upstream control regions (UCRs), however, had a different effect. Whereas PARP UCR deletion reduced transcription efficiency almost 10-fold, deletion of the rDNA UCR had only a minor effect on transcription efficiency. (+info)
Predominance of duplicative VSG gene conversion in antigenic variation in African trypanosomes.
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A number of mechanisms have been described by which African trypanosomes undergo the genetic switches that differentially activate their variant surface glycoprotein genes (VSGs) and bring about antigenic variation. These mechanisms have been observed mainly in trypanosome lines adapted, by rapid syringe passaging, to laboratory conditions. Such "monomorphic" lines, which routinely yield only the proliferative bloodstream form and do not develop through their life cycle, have VSG switch rates up to 4 or 5 orders of magnitude lower than those of nonadapted lines. We have proposed that nonadapted, or pleomorphic, trypanosomes normally have an active VSG switch mechanism, involving gene duplication, that is depressed, or from which a component is absent, in monomorphic lines. We have characterized 88 trypanosome clones from the first two relapse peaks of a single rabbit infection with pleomorphic trypanosomes and shown that they represent 11 different variable antigen types (VATs). The pattern of appearance in the first relapse peak was generally reproducible in three more rabbit infections. Nine of these VATs had activated VSGs by gene duplication, the tenth possibly also had done so, and only one had activated a VSG by the transcriptional switch mechanism that predominates in monomorphic lines. At least 10 of the donor genes have telomeric silent copies, and many reside on minichromosomes. It appears that trypanosome antigenic variation is dominated by one, relatively highly active, mechanism rather than by the plethora of pathways described before. (+info)
Control of variant surface glycoprotein gene-expression sites in Trypanosoma brucei.
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Trypanosoma brucei has 20 similar telomeric-expression sites for variant surface glycoprotein genes. Expression sites appear to be controlled at the level of transcription initiation, resulting in only one site being active at any time. Switching between expression sites occurs at a low rate. To analyse the switching mechanism, we used trypanosomes with two expression sites tagged with two different drug-resistance genes and selected these on agarose plates containing both drugs. Double-resistant clones arose at a low frequency of 10(-7) per cell, but these behaved as if they rapidly switched between the two tagged expression sites and lost double resistance in the absence of selection. Using in situ hybridization we found that only 10% of the double-resistant cells had two fluorescent spots corresponding to transcribed expression sites. Our results suggest that: (i) a double expressor is not a stable intermediate in expression site switching; (ii) expression sites are not independently switched on and off; and (iii) expression sites can be in a 'pre-active' silent state from which they can be readily activated. (+info)
N-linked glycans containing linear poly-N-acetyllactosamine as sorting signals in endocytosis in Trypanosoma brucei.
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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. (+info)