Exposed epitopes on a Trypanosoma equiperdum variant surface glycoprotein altered by point mutations.

African trypanosomes are covered by a dense protein layer that is immunologically distinct on different trypanosome isolates and is termed the variant surface glycoprotein (VSG). The different VSGs are expressed in a general order, where some VSGs appear preferentially early in infection and others only later. The exposed epitopes on a late antigen, VSG 78, of T.equiperdum were studied by the technique of monoclonal antibody (MAb) escape selection. MAbs that neutralize trypanosomes bearing VSG 78 reacted with the VSG only when it was attached to the trypanosome surface, suggesting that the most immunogenic surface epitopes are conformational. Trypanosome clones resistant to one of the MAbs yet still expressing VSG 78 or 78(20) were isolated in vitro. Two independent variants resistant to MAb H3 changed Ser192 to Arg by a single base change in the VSG gene and a variant resistant to MAb H21 had a single base change that converted Gln172 to Glu. A variant resistant to MAb H7 had several changes in the VSG gene, a gene conversion in the 5' region and an isolated mutation in codon 220 that is proposed to be responsible for the resistance phenotype. The isotypic bias of the MAbs against VSG 78 and an analysis of the natural variants that are resistant to MAb 78H21 suggest that glycosylation plays a role in the immunogenicity of these proteins. The analysis defines some of the exposed amino acid residues and demonstrates that VSG genes are altered by mutations and small gene conversions as well as replaced by large gene conversion-like events. The results provide biological data supporting the model of VSG structure obtained by crystallographic studies.     

The use of incomplete genes for the construction of a Trypanosoma equiperdum variant surface glycoprotein gene.

The expression of Trypanosoma equiperdum variant surface protein (VSG) 78 is accomplished by the duplicative transposition of silent basic copy (BC) genes into a telomer-linked expression site to form an expression-linked copy (ELC). In two independent isolates expressing VSG 78, the ELC is a composite gene. The analysis of VSG 78 cDNA clones from these two Bo Tat 78 isolates and the respective BC genes revealed that both ELCs were constructed from the same three BC genes, a 3' BC which donated the last 255 bp of each ELC and two closely related 5' BCs. Although sequences of both 5' BC genes were found in each ELC, the junction with the 3' BC was provided by the same 5' BC in both cases. This 5' BC is an incomplete gene with insufficient open reading frame to code for a complete VSG and thus can only be used when joined to a competent 3' end. Furthermore, both 5' BC genes lack a conserved 14 nucleotide sequence found on all VSG mRNAs. These results support a model in which composite gene formation plays a role in the determination of the order of VSG expression. They also illustrate similarities between immunoglobulin gene and VSG gene construction.     

DNA rearrangements and antigenic variation in Trypanosoma equiperdum: multiple expression-linked sites in independent isolates of trypanosomes expressing the same antigen.

African trypanosomes resist the immune response of their mammalian hosts by varying the surface glycoprotein which constitutes their antigenic identity. The molecular mechanism of this antigenic variation involves the successive activation of a series of genes which code for different variant surface glycoproteins (VSGs). We have studied the expression of two VSG genes (those of VSG-1 and VSG-28) in Trypanosoma equiperdum, and we report the following findings. (i) The expression of both VSG genes is associated with the duplication and transposition of corresponding basic copy genes. (ii) The duplicated transposed copy appears to be the expressed copy. (iii) Although there are multiple genes which cross-hybridize with the VSG-1 cDNA probe, only one of these appears to be used as a template for the expression-linked copy in four independent BoTat-1 clones. (iv) Analysis of the genomic environments of the expressed VSG-1 genes from each of four independently derived BoTat-1 trypanosome clones revealed that there are at least three different sites into which the expression-linked copy can be inserted.     

VSG 117 gene is conservatively present and early expressed in Trypanosma evansi YNB stock

African trypanosomes, including Trypanosoma brucei and the closely related species Trypanosoma evansi, are flagellated unicellular parasites that proliferate extracellularly in the mammalian bloodstream and tissue spaces. They evade host immune system by periodically switching their variant surface glycoprotein (VSG) coat. Each trypanosome possesses a vast archive of VSGs with distinct sequence identity and different strains contain different archive of VSGs. VSG 117 was reported as a widespread VSG detected in the genomes of all the T. brucei strains. In this study, the presence and expression of VSG 117 gene was observed in T. evansi YNB stock by RT-PCR with VSG-specific primers. We further confirmed that this VSG tends to be expressed in the early stage of T. evansi infections (on day 12-15) by immuno-screening the previously isolated infected blood samples. It is possible that the VSG 117 gene evolved and spread through the African trypanosome population via genetic exchange, before T. evansi lost its ability to infect tsetse fly. Our finding provided an evidence of the close evolutionary relationship between T. evansi and T. brucei, in the terms of VSG genes.     

Size fractionation of Trypanosoma brucei DNA: localization of the 177-bp repeat satellite DNA and a variant surface glycoprotein gene in a mini-chromosomal DNA fraction

We have size-fractionated intact DNA from Trypanosoma brucei into a major large DNA fraction (greater than 350S) and minor middle-sized (60-250S) and small (less than 60S) DNA fractions. Large DNA contains the rRNA genes, the basic copy genes for several variant surface glycoproteins (VSGs), including one which lies near a telomer, and the expression-linked copies of the two VSG genes. The middle-sized DNA contains at least one VSG gene, but the hybridization of this fraction with probes for the conserved repetitive sequences that mark the edges of the transposed segments of VSG genes, suggests that it may contain many VSG genes. The 177-bp repeat satellite DNA is also exclusively found in this fraction.     

Cloning and characterization of a variant surface glycoprotein expression site from Trypanosoma equiperdum.

Variant surface glycoprotein (VSG) genes of African trypanosomes are expressed when they are inserted into one of several telomere-linked expression sites. We cloned and characterized an 11-kilobase (kb) DNA fragment located upstream of an expressed VSG gene. A DNA sequence of 1.8 kb that is located immediately upstream of the inserted VSG gene contains sequences homologous to the 76-base-pair repeats described as being upstream of VSG genes in Trypanosoma brucei (D. A. Campbell, M. P. Van Bree, and J. C. Boothroyd, Nucleic Acids Res. 12:2759-2774). There are no such sequences elsewhere in the 11-kb cloned region. Southern blot analysis using probes from the cloned region revealed multiple unlinked copies of the same or very similar regions. At least three of these are located near telomeres, and two have been shown to be used for the expression of known Trypanosoma equiperdum VSG genes. Like VSG genes, the upstream sequences themselves can be duplicated and deleted. The choice of expression site to be used by a duplicated VSG gene is nonrandom; the site used for expression of the parental VSG gene is strongly favored for use in the daughter variant. Furthermore, even when the parental expression site is not used, the VSG gene occupying it is replaced. Thus, an active expression site is a preferential target for gene conversion in the next variation event.     

Relationship between multiple copies of a T. brucei variable surface glycoprotein gene whose expression is not controlled by duplication

Unlike many other T. brucei variable surface glycoprotein (VSG) genes, the IITat 1.3 gene is not duplicated when it is expressed. Analysis of the multiple copies of this gene present in all IITaR 1 trypanosome clones by restriction enzyme mapping and sequencing shows that the expressed copy may have arisen by duplication and transposition to a telomeric site, as is observed for those VSG genes whose expression is linked to duplication. The existence of a mechanism selecting between a number of complete telomeric VSG gene copies for expression is implied by these results. Comparisons of the nontelomeric copies of the IITat 1.3 gene are consistent with involvement of gene duplication and mutational drift in the evolution of new VSG genes.     

Transcripts coding for variant surface glycoproteins of Trypanosoma brucei have a short,identical exon at their 5′ end

The nucleotide sequence of the 5′ end of the mRNA for variant surface glycoprotein (VSG) 117 has been determined and compared with the sequence of the unexpressed basic copy (BC) of the VSG 117 gene. This shows the existence of an exon 35 nucleotides long at the 5′ end of the mRNA. The evidence suggests that this ‘mini-exon’ is derived from the expression site into which the VSG 117 BC is transposed during activation. The nucleotide sequence of this mini-exon is indistinguishable from that recently found for a different VSG, 118 (Van der Ploeg et al., Nucl. Acids Res. 10 (1982) 3591–3604). Analysis of the 5′ end of the mRNA for another VSG (221) whose gene is thought to be activated by a different mechanism to that of VSGs 117 and 118 indicates that the 5′- most 35 nucleotides of the VSG 221 mRNA are identical to the 117/118 mini-exon sequence. The implications of these results for the mechanism of VSG gene expression are discussed.     

A strand bias occurs in point mutations associated with variant surface glycoprotein gene conversion in Trypanosoma rhodesiense.

We previously described a bloodstream Trypansoma rhodesiense clone, MVAT5-Rx2, whose isolation was based on its cross-reactivity with a monoclonal antibody (MAb) directed against a metacyclic variant surface glycoprotein (VSG). When the duplicated, expressed VSG gene in MVAT5-Rx2 was compared with its donor (basic copy) gene, 11 nucleotide differences were found in the respective 1.5-kb coding regions (Y. Lu, T. Hall, L. S. Gay, and J. E. Donelson, Cell 72:397-406, 1993). Here we describe a characterization of two additional bloodstream trypanosome clones, MVAT5-Rx1 and MVAT5-Rx3, whose VSGs are expressed from duplicated copies of the same donor VSG gene. The three trypanosome clones each react with the MVAT5-specific MAb, but they have different cross-reactivities with a panel of other MAbs, suggesting that their surface epitopes are similar but nonidentical. Each of the three gene duplication events occurs at a different 5' crossover site within a 76-bp repeat and is associated with a different set of point mutations. The 35, 11, and 28 point mutations in the duplicated VSG coding regions of Rx1, Rx2, and Rx3, respectively, exhibit a strand bias. In the sense strand, of the 74 total mutations generated in the three duplications, 54% are A-to-G or G-to-A (A:G) transitions and 7% are C:T transitions, while 26% are C:A transversions and 13% are C:G transversions. No T:G or T:A transversions occurred. Possible models for the generation of these point mutations are discussed.     

An analysis of cosmid clones of nuclear DNA from Trypanosoma brucei shows that the genes for variant surface glycoproteins are clustered in the genome

Trypanosoma brucei contains more than a hundred genes coding for the different variant surface glycoproteins (VSGs). Activation of some of these genes involves the duplication of the gene (the basic copy or BC) and transposition of the duplicate to an expression site (yielding the expression-linked copy or ELC). We have cloned large fragments of genomic DNA in cosmid vectors in Escherichia coli. Cosmids containing the BCs of genes 117, 118 and 121 were readily obtained, but DNA containing the ELCs was strongly selected against in the cosmid and plasmid cloning systems used. We have analysed the distribution of VSG genes in the genome using probes for the sequences at the edges of the transposed segment which are partially homologous among these genes. In genomic cosmid clone banks, about 9% of all colonies hybridize with probes from the 5'- and 3'-edges of the transposed segment, showing that these sequences are linked in the genome. Moreover, the 117 and 118 BC cosmids contain several additional putative VSG genes in tandem, as deduced from hybridization and sequence analyses. We conclude that the VSG genes are highly clustered and share common sequences at the borders of the transposed segment.     

Re-expression of an inactivated variable surface glycoprotein gene in Trypanosoma equiperdum

Variable surface glycoprotein (VSG) genes in African trypanosomes are often activated by the duplicative transposition of a silent basic copy (BC) gene into an unlinked telomerically located expression site, producing an active expression-linked copy (ELC) of that gene. However, some BC genes that are already linked to a telomere are activated without apparent duplication or transposition. We have recently shown that an active VSG ELC can be inactivated in situ, apparently without rearrangement. To explain these observations it has been suggested that VSG genes that are associated with chromosome telomeres are activated by chromosome end exchanges that occur at a considerable distance upstream from the genes themselves and place them cis to a unique VSG expression element. In an attempt to test this model we derived five VSG-1 expressing variants from BoTat-2, a VSG-2 expressing variant of Trypanosoma equiperdum which carries an inactive residual VSG-1 ELC (R-ELC) as well as the active VSG-2 ELC near unlinked chromosome telomeres. We examined the fates of the VSG-2 ELC and the VSG-1 R-ELC in these variants. All five had maintained the VSG-1 R-ELC; three in a reactivated form and two in an inactive state. The latter two variants carried new, active VSG-1 ELCs: one in the site that had previously contained the VSG-2 ELC and one in a previously unidentified site. The VSG-2 ELC was lost in all five of the variants. The results are not consistent with the simple chromosome end exchange model, which predicts that the VSG-2 ELC would be inactivated but not deleted when the VSG-1 R-ELC was reactivated.     

analysis of genomic rearrangements associated with two variable antigen genes of Trypanosoma brucei.

Some variable surface glycoprotein (VSG) genes of Trypanosoma brucei undergo duplication and transposition when they are expressed. We report here the cloning of cDNAs coding for two VSGs from the ILtar 1 repertoire. Analysis of the genomes of trypanosomes expressing these and other antigens shows that there is no additional copy of the sequences coding for eight VSG in expressing clones of trypanosomes, and reveals rearrangements analogous to those previously described for the gene for another VSG from this antigen repertoire. The data indicate that duplication does not accompany the expression of these VSG genes. Transposition to a specific expression site cannot be excluded, but would have to involve either a much larger segment of DNA, or movement to a region of much greater homology with the previous flanking sequences, than is observed for VSG genes that are duplicated when expressed. It is reasoned that the control of expression by coupled duplication and transposition is not sufficient to account for the selection of a single VSG gene for expression.     

Antigenic variation in Trypanosoma brucei analyzed by electrophoretic separation of chromosome-sized DNA molecules

Pulsed field gradient gel electrophoresis fractionates chromosome-sized DNA molecules from T. brucei. About 60% of the DNA remains in or close to the gel slot (large DNA). There are about three chromosomes of approximately 2 Mb, at least six chromosomes of 200-700 kb, and roughly a hundred mini-chromosomes of 50-150 kb. The basic copy genes for VSGs 118 and 221 reside in large DNA. Their activation by duplicative transposition leads to the appearance of an additional copy in the 2 Mb DNA, showing that activation involves an interchromosomal gene transposition. When gene 221 is activated without duplication, it remains in large DNA, proving that at least two sites for expression of VSG genes exist. In support of this, the mini-exons encoding the 5' 35 nucleotides of VSG messenger RNAs are in large and 2 Mb DNA. The mini-chromosomes hybridize strongly to VSG gene probes and are absent in C. fasciculata. We suggest that their main function is to provide a large pool of telomeric VSG genes.     

Characteristics of trypanosome variant antigen genes active in the tsetse fly.

Trypanosoma brucei contains a repertoire of more than 100 different genes for Variant Surface Glycoproteins (VSGs). A small and strain-specific fraction of these genes is expressed in the salivary glands of the tsetse fly (M-genes), giving rise to metacyclic Variable Antigen Types (M-VATs). Antibodies produced in a chronic trypanosome infection initiated by syringe inoculation of bloodstream forms into mammals (i.e. against B-VATs), will react with most of the M-VATs suggesting that these B-VATs express VSG genes that are similar or identical to M-genes. We have cloned DNA complementary to the VSG mRNA of four of such B-VATs and used this to characterize the corresponding VSG genes. In three of the four VATs we find a single VSG gene hybridizing with the cDNA probe and we provide supporting evidence that this gene is expressed as an M-gene. In the bloodstream repertoire these genes appear to be activated by duplicative translocation to another telomere. In all four variants the putative M-genes are telomeric and in the three cases where the location of the genes on chromosome-sized DNA molecules could be determined, the genes were located in large DNA, whereas the majority of the telomeric VSG genes are in chromosomes less than 1000 kb. Our results are best explained by models for M-gene activation involving telomeric expression sites for these genes which are separate from those used by bloodstream forms. The implications of these results for vaccination are discussed.     

Genomic organization and context of a trypanosome variant surface glycoprotein gene family.

We have defined the genomic organization and genomic context of a Trypanosoma brucei brucei gene family encoding variant surface glycoproteins (VSGs). This gene family is neither tandemly repeated nor closely linked in the genome, and is not located on small or intermediate size chromosomes. Two dispersed repeated sequence elements, RIME-ingi and the upstream repeat sequence, are linked to members of this gene family; however, the upstream repeat sequences are closely linked only to the basic copy. In other isolates of T.b. brucei this gene family appears conserved with some variation; a restriction fragment length polymorphism found among these isolates suggests the hypothesis that VSG genes may occasionally be diploid. A model accounting for both the generation of dispersed families of VSG genes, and for the interstrain variability of VSG genes, is proposed.