Single Variant Surface Glycoprotein Research Articles

Mono-allelic VSG expression by RNA polymerase I in Trypanosoma brucei: Expression site control from both ends?

Trypanosoma brucei is a vector borne, lethal protistan parasite of humans and livestock in sub-Saharan Africa. Antigenic variation of its cell surface coat enables the parasite to evade adaptive immune responses and to live freely in the blood of its mammalian hosts. The coat consists of ten million copies of variant surface glycoprotein (VSG) that is expressed from a single VSG gene, drawn from a large repertoire and located near the telomere at one of fifteen so-called bloodstream expression sites (BESs). Thus, antigenic variation is achieved by switching to the expression of a different VSG gene. A BES is a tandem array of expression site-associated genes and a terminal VSG gene. It is polycistronically transcribed by a multifunctional RNA polymerase I (RNAPI) from a short promoter that is located 45–60kb upstream of the VSG gene. The mechanism(s) restricting VSG expression to a single BES are not well understood. There is convincing evidence that epigenetic silencing and transcription attenuation play important roles. Furthermore, recent data indicated that there is regulation at the level of transcription initiation and that, surprisingly, the VSG mRNA appears to have a role in restricting VSG expression to a single gene. Here, we review BES expression regulation and propose a model in which telomere-directed, epigenetic BES silencing is opposed by BES promoter-directed, activated RNAPI transcription.

Antigenic variation inAfrican trypanosomes: the importance of chromosomal and nuclear context inVSGexpression control

African trypanosomes are lethal human and animal parasites that use antigenic variation for evasion of host adaptive immunity. To facilitate antigenic variation, trypanosomes dedicate approximately one third of their nuclear genome, including many minichromosomes, and possibly all sub-telomeres, to variant surface glycoprotein (VSG) genes and associated sequences. Antigenic variation requires transcription of a single VSG by RNA polymerase I (Pol-I), with silencing of other VSGs, and periodic switching of the expressed gene, typically via DNA recombination with duplicative translocation of a new VSG to the active site. Thus, telomeric location, epigenetic controls and monoallelic transcription by Pol-I at an extranucleolar site are prominent features of VSGs and their expression, with telomeres, chromatin structure and nuclear organization all making vitally important contributions to monoallelic VSG expression control and switching. We discuss VSG transcription, recombination and replication control within this chromosomal and sub-nuclear context.

Antigenic variation in the African trypanosome: molecular mechanisms and phenotypic complexity

Antigenic variation is an immune evasion strategy that has evolved in viral, bacterial and protistan pathogens. In the African trypanosome this involves stochastic switches in the composition of a variant surface glycoprotein (VSG) coat, using a massive archive of silent VSG genes to change the identity of the single VSG expressed at a time. VSG switching is driven primarily by recombination reactions that move silent VSGs into specialized expression sites, though transcription-based switching can also occur. Here we discuss what is being revealed about the machinery that underlies these switching mechanisms, including what parallels can be drawn with other pathogens. In addition, we discuss how such switching reactions act in a hierarchy and contribute to pathogen survival in the face of immune attack, including the establishment and maintenance of chronic infections, leading to host-host transmission.

A pol I transcriptional body associated with VSG mono-allelic expression in Trypanosoma brucei.

In the mammalian host, African trypanosomes generate consecutive waves of parasitaemia by changing their antigenic coat. Because this coat consists of a single type of variant surface glycoprotein (VSG), the question arises of how a trypanosome accomplishes the transcription of only one of a multi-allelic family of VSG expression site loci to display a single VSG type on the surface at any one time. No major differences have been detected between the single active expression site and the cohort of inactive expression sites. Here we identify an extranucleolar body containing RNA polymerase I (pol I) that is transcriptionally active and present only in the bloodstream form of the parasite. Visualization of the active expression site locus by tagging with green fluorescent protein shows that it is specifically located at this unique pol I transcriptional factory. The presence of this transcriptional body in postmitotic nuclei and its stability in the nucleus after DNA digestion provide evidence for a coherent structure. We propose that the recruitment of a single expression site and the concomitant exclusion of inactive loci from a discrete transcriptional body define the mechanism responsible for VSG mono-allelic expression.

Glycosyl-phosphatidylinositol moiety that anchors Trypanosoma brucei variant surface glycoprotein to the membrane.

Two forms of protein-membrane anchor have been described for the externally disposed glycoproteins of eukaryotic plasma membranes; namely, the hydrophobic transmembrane polypeptide and the complex glycosylphosphatidylinositol (G-PI) moiety. The chemical structures of the major species of G-PI anchors found on a single variant surface glycoprotein (VSG) of the parasitic protozoan Trypanosoma brucei were determined by a combination of nuclear magnetic resonance spectroscopy, mass spectrometry, chemical modification, and exoglycosidase digestions. The G-PI anchor was found to be heterogeneous with respect to monosaccharide sequence, and several novel glycosidic linkages were present. The results are pertinent to the mechanism of the biosynthesis of G-PI anchors.

Frequent independent duplicative transpositions activate a single VSG gene.

The expression of several surface antigen genes in Trypanosoma brucei is mediated by the duplicative transposition of a basic-copy variant surface glycoprotein (VSG) gene into an expression site. We determined that the appearance of variant 118, in a parasitemia, resulted from at least four independent duplicative transpositions of the same VSG 118 gene. Variants 117 and 118 both appeared at specific periods but resulted from multiple independent activations. Antigenic variants thus occur in an ordered manner. We show that in the duplicative transpositions of VSG genes, the ends of the transposed segments were homologous between the basic copy and the expression site. Sequences other than the previously reported 70-base-pair (bp) repeats could be involved. In one variant, 118 clone 1, the homology was between a sequence previously transposed into the expression site and a sequence located 6 kilobases upstream of the VSG 118 gene. In variant 118b the homology was presumably in 70-bp repeat arrays, while in a third 118 variant yet another sequence was involved. The possibility that the 70-bp repeats are important in the initial steps of the recombinational events was illustrated by a rearrangement involving a 70-bp repeat array. The data provide strong evidence for the notion that gene conversion mediates the duplicative transposition of VSG genes. We discuss a model that explains how the process of duplicative transposition can occur at random and still produce an ordered appearance of variants.

Two mechanisms of expression of a predominant variant antigen gene of Trypanosoma brucei.

African trypanosomes evade the host immune response by periodically switching their variant surface glycoprotein (VSG) coat. The resulting, serologically distinct variant antigenic types (VATs) appear in a loosely ordered sequence and those arising early in infections are termed predominant VATs. VAT switching reflects the successive transcriptional activation of single VSG genes from a large repertoire. Activation of some VSG genes is accomplished by duplication of a previously silent basic copy (BC) gene and insertion of this expression linked copy (ELC) near a chromosomal telomere where is is expressed. However, other VSG genes, always located near a telomere, use a non-duplication activation ( NDA ) mechanism. We report here that the gene encoding the predominant IsTat 1.A VSG can be activated with or without duplication. Four of six independently derived clones activated the 1.A gene without gene duplication or detectable rearrangement. The other two contained an active 1.A ELC, possibly generated by a gene conversion extending to the end of the telomere. The ability to utilize both NDA and ELC mechanisms of gene activation and perhaps an alternative mechanism for gene duplication may account for the fact that VAT 1.A is the most predominant VAT of the IsTaR 1 serodeme .