Rapid change of the repertoire of variant surface glycoprotein genes in trypanosomes by gene duplication and deletion

To study the evolution of the variant surface glycoprotein (VSG) repertoire of trypanosomes we have analysed the DNA region surrounding the VSG 118 gene in different trypanosome strains. We find a remarkable degree of variation in this area. Downstream from the 118 gene a 5.7 X 10(3) base-pair DNA segment containing a potential VSG gene has been quadruplicated in strain 427 of Trypanosoma brucei, but not in most other strains analysed. The VSG 1.1000 gene, located immediately upstream from the 118 gene in one trypanosome strain, has been cleanly deleted in another. Our results are most easily explained by multiple unequal cross-overs between sister chromatids and are the first indication that sister chromatid exchange occurs in trypanosomes.     

Analysis of a donor gene region for a variant surface glycoprotein and its expression site in African trypanosomes

African trypanosomes evade the immune response of their mammalian hosts by sequentially expressing genes for different variant surface glycoproteins (VSGs) from telomere-linked VSG expression sites. In the Trypanosoma brucei clone whose genome is being sequenced (GUTat 10.1), we show that the expressed VSG (VSG 10.1) is duplicated from a silent donor VSG located at another telomere-linked site. We have determined two 130 kb sequences representing the VSG 10.1 donor and expression sites. The telomere-linked donor VSG 10.1 resembles metacyclic VSG expression sites, and is preceded by a cluster of 35 or more tandem housekeeping genes, all of which are transcribed away from the telomere. The 45 kb telomere-linked VSG 10.1 expression site contains a promoter followed by seven expression site-associated genes (ESAGs), three pseudo ESAGs, two pseudo VSGs and VSG 10.1. The 80 kb preceding the expression site has few, if any, functional ORFs, but contains 50 bp repeats, INGI retrotransposon-like elements, and novel 4–12 kb repeats found near other telomeres. This analysis provides the first look over a 130 kb range of a telomere-linked donor VSG and its corresponding telomere-linked VSG expression site and forms the basis for studies on antigenic variation in the context of a completely sequenced genome.     

Subcellular localization of a variable surface glycoprotein phosphatidylinositol-specific phospholipase-C in African trypanosomes

African trypanosomes contain a membrane-bound enzyme capable of removing dimyristylglycerol from the membrane-attached form of the variable surface glycoprotein (mfVSG; Ferguson, M. A. J., K. Halder, and G. A. M. Cross, 1985, J. Biol Chem., 260:4963-4968). Although mfVSG phospholipase-C has been implicated in the removal of the VSG from the trypanosome surface (Cardoso de Almeida, M. L., and M. J. Turner, 1983, Nature (Lond.)., 302:349-352; Ferguson, M. A. J., K. Halder, and G. A. M. Cross, 1985, J. Biol Chem., 260:4963-4968), its precise function and subcellular location have not been determined. We have developed a procedure for the separation of the cell fractions and organelles of Trypanosoma brucei brucei (and other trypanosome species) by differential sucrose and isopycnic PercollR centrifugation. These fractions were tested for mfVSG phospholipase activity using Trypanosoma brucei mfVSG labeled with 3H-myristic acid as substrate. The highest enzyme-specific activity was associated with the flagella and evidence is presented to suggest that it is localized in the flagellar pocket. Some activity was also associated with the Golgi complex. These results suggest that the mfVSG phospholipase is localized primarily in the membrane of the flagella pocket and possibly other membrane organelles derived from and associated with this structure, and may be part of the VSG-membrane recycling system in African trypanosomes. The activity of mfVSG phospholipase amongst various trypanosome species was determined. We show that, in contrast to the bloodstream forms of Trypanosoma brucei, cultured procyclic Trypanosoma brucei and bloodstream Trypanosoma vivax had little or no mfVSG phospholipase activity. The activity found in bloodstream forms of Trypanosoma congolense was intermediate between Trypanosoma vivax and Trypanosoma brucei.     

Targeting the variable surface of African trypanosomes with variant surface glycoprotein-specific,serum-stable RNA aptamers

African trypanosomes cause sleeping sickness in humans and Nagana in cattle. The parasites multiply in the blood and escape the immune response of the infected host by antigenic variation. Antigenic variation is characterized by a periodic change of the parasite protein surface, which consists of a variant glycoprotein known as variant surface glycoprotein (VSG). Using a SELEX (systematic evolution of ligands by exponential enrichment) approach, we report the selection of small, serum-stable RNAs, so-called aptamers, that bind to VSGs with subnanomolar affinity. The RNAs are able to recognize different VSG variants and bind to the surface of live trypanosomes. Aptamers tethered to an antigenic side group are capable of directing antibodies to the surface of the parasite in vitro. In this manner, the RNAs might provide a new strategy for a therapeutic intervention to fight sleeping sickness.     

The surface of the African trypanosomes

The African trypanosomes bear on the outside of their cell membrane a single 10-15 nm thick coat of a glycoprotein. This glycoprotein may differ in structure in the predominant populations of parasitemic waves found in relapsing infections. Variant Specific Glycoprotein (VSG) range in MW between 53,000-63,000 d and may have variable amounts of carbohydrate attached at one, two, or several loci. Such differences in carbohydrate content may account in part for their range in molecular size. Approximately 30 C-terminal residues demonstrate isotypy ; i.e. these regions fall into classes having similar amino acid sequence. Modest homology has been demonstrated in two VSGs of T. congolense arising in relapsing infections although comparison of many VSG show little or no obvious homology. More recently, lipid-associated forms of VSG have been described and it is believed that these forms may be transmembrane proteins. Different VSGs appear to have different amounts of the primary sequence which have alpha-helix-forming potential. In some VSG, in excess of 80% of the structure is helical as judged by both Chou-Fasman calculations and by circular dichroism. This raises the possibility that different VSG may have different folding patterns. The arrangement of VSG on the trypanosome surface probably places the basic amino acid-rich carbohydrate-bearing C-terminus of the polypeptide chain close to the membrane. There is some protein-protein association between VSGs for which (in T. evansi) the C-terminal tail is not required. The importance of VSG structure lies not only in the fact that the molecule mediates the phenomenon of antigenic variation but also in the recent observation that VSG may act on the cellular immune system to suppress the humoral immune responses of the host.     

Trypanosome variant surface glycoprotein genes expressed early in infection

We have studied further the genes for trypanosomal variant surface glycoproteins expressed during a chronic infection of rabbits with Trypanosoma brucei, strain 427. We show that there are three closely related chromosomal-internal isogenes for VSG 121; expression of one of these genes is accompanied by the duplicate transposition of the gene to a telomeric expression site, also used by other chromosome-internal VSG genes. The 3' end of the 121 gene is replaced during transposition with another sequence, also found in the VSG mRNAs of two other variants. We infer that an incoming VSG gene duplicate recombines with the resident gene in the expression site and may exchange ends in this process. The extra expression-linked copy of the 121 gene is lost when another gene enters the expression site. However, when the telomeric VSG gene 221 is activated without duplication the extra 121 gene copy is inactivated without detectable alterations in or around the gene. We have also analysed the VSG genes expressed very early when trypanosomes are introduced into rats or tissue culture. The five genes identified in 24 independent switching events were all found to be telomeric genes and we calculate that the telomeric 1.8 gene has a 50% chance of being activated in this trypanosome strain when the trypanosome switches the VSG that is synthesized. We argue that the preferential expression of telomeric VSG genes is due to two factors: first, some telomeric genes reside in an inactive expression site, that can be reactivated; second, telomeric genes can enter an active expression site by a duplicative telomere conversion and this process occurs more frequently than the duplicative transposition of chromosome-internal genes to an expression site.     

Leaky transcription of variant surface glycoprotein gene expression sites in bloodstream african trypanosomes.

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.     

Sequence divergence in a family of variant surface glycoprotein genes from trypanosomes: Coding region hypervariability and downstream recombinogenic repeats

The surface of the parasitic protozoanTrypanosoma brucei spp. is covered with a dense coat consisting of a single type of glycoprotein molecule, the variant surface glycoprotein (VSG). There may be as many as 1,000 genes for VSG within the genome ofT. brucei, and the switch of expression from one to another is the phenomenon of antigenic variation. As an approach to understanding the evolution of VSG genes we have determined the genomic DNA sequences of the eight genes encoding the variant surface glycoprotein 117 (VSG) family. From these data we have observed a number of features concerning the relationships between these genes: (1) there is a region of high variability confined to the N-terminus of the coding sequence, and comparison of the sequences with the available X-ray diffraction crystal structures suggests that two of the most variable stretches within the N-terminal domain are present on surface-exposed loops, indicating a role for epitope selection in evolution of these genes; (2) the 29 nucleotides surrounding the splice acceptor site are absolutely conserved in all eight 117 VSG genes; (3) numerous insertion/deletion mutations are located within or immediately downstream of the C-terminal protein-coding sequences: (4) within 500 by downstream of the insertion/deletion mutations are one or two copies of a repeat motif highly homologous to the recombinogenic 76-bp repeat sequences present upstream of many VSG basic copy genes and the expression-linked copy. Correspondence to: J.C. Boothroyd     

Genetics of resistance to the African trypanosomes. VII. Trypanosome virulence is not linked to variable surface glycoprotein expression

The question of linkage of virulence traits to variable surface glycoprotein (VSG) expression in African trypanosomiasis was addressed. Previously we demonstrated that daughter cells arising in mice infected with a genetically homogeneous trypanosome population of Trypanosoma brucei rhodesiense were more virulent than the infecting population (J. A. Inverso and J. M. Mansfield, J. Immunol. 130:412, 1983). These virulent trypanosomes expressed differences in surface phenotype compared with the infecting variant types, and we proposed that virulence may be "linked" to VSG expression. In the present study, however, we have shown that expression of virulence is independent of the VSG phenotype displayed by trypanosome populations. A VSG-identical but highly virulent subpopulation of T. b. rhodesiense LouTat 1 was derived by rapid subpassage and subcloning in immunosuppressed mice. The virulent LouTat 1A subclone derived in this manner killed B10.BR/SgSnJ mice in 3 to 4 days postinfection compared with approximately 60 days for the parent clone, LouTat 1. The virulent subclone LouTat 1A appears to express the same VSG as the less virulent LouTat 1 population, as determined by polyspecific and monoclonal antibody-binding assays, cross-protection tests, and amino acid sequence analyses of the N-terminal portion of the VSG molecules. When LouTat 1 and subclone LouTat 1A were injected into a heterologous host species, multiple variant antigenic types (VATs) arising from each inoculum were isolated and characterized. VATs derived from the virulent subclone were as uniformly virulent for B10.BR mice as LouTat 1A. In summary, these results demonstrate that trypanosome virulence, once expressed, is a stable phenotype that does not seem to be associated with a particular VSG phenotype, nor does virulence change with the expression of different VSG genes.     

Secondary structure of the variant surface glycoproteins of trypanosomes

The secondary structure of seven variant surface glycoproteins (VSGs) of trypanosomes has been determined by Raman spectroscopy. They are all predominantly alpha-helical, the alpha-helix content varying between 50 and 60%. The beta-strand content varies between 20 and 25%, and the content of beta-turn and nonregular structures is about 25%. For three VSGs the N-terminal domain obtained by proteolytic cleavage was found to have essentially the same secondary structure as the complete VSGs. For three VSGs a secondary structure prediction has been performed applying the rules of Chou and Fasman. In all cases, two long alpha-helices extending over about 50 residues or 80 A are predicted in agreement with the X-ray diffraction data of Freymann et al. [(1984) Nature 311, 167-169] and Metcalf et al. [(1987) Nature 325, 84-86]. The region between the two alpha-helical segments exhibits a high potential of beta-turns, suggesting that this segment may be exposed on the cell surface and carry major antigenic determinants.     

Identification of a tryptophan-like epitope borne by the variable surface glycoprotein (VSG) of African trypanosomes

Antibodies (Ab) directed against a tryptophan-like epitope (WE) were previously detected in patients with human African trypanosomiasis (HAT). We investigated whether or not these Ab resulted from immunization against trypanosome antigen(s) expressing a WE. By Western blotting, we identified an antigen having an apparent molecular weight ranging from 60 to 65 kDa, recognized by purified rabbit anti-WE Ab. This antigen, present in trypomastigote forms, was absent in procyclic forms and Trypanosoma cruzi trypomastigotes. Using purified variable surface glycoproteins (VSG) from various trypanosomes, we showed that VSG was the parasite antigen recognized by these rabbit Ab. Anti-WE and anti-VSG Ab were purified from HAT sera by affinity chromatography. Immunoreactivity of purified antibodies eluted from affinity columns and of depleted fractions showed that WE was one of the epitopes borne by VSG. These data underline the existence of an invariant WE in the structure of VSG from several species of African trypanosomes.     

African trypanosomes contain 5-methylcytosine in nuclear DNA

It is currently unclear if there are modified DNA bases in Trypanosoma brucei other than J-base. We identify herein a cytosine-5 DNA methyltransferase gene and report the presence and location of 5-methylcytosine in genomic DNA. Our data demonstrate that African trypanosomes contain a functional cytosine DNA methylation pathway.