Mosaic VSGs and the Scale of Trypanosoma brucei Antigenic Variation

A main determinant of prolonged Trypanosoma brucei infection and transmission and success of the parasite is the interplay between host acquired immunity and antigenic variation of the parasite variant surface glycoprotein (VSG) coat. About 0.1% of trypanosome divisions produce a switch to a different VSG through differential expression of an archive of hundreds of silent VSG genes and pseudogenes, but the patterns and extent of the trypanosome diversity phenotype, particularly in chronic infection, are unclear. We applied longitudinal VSG cDNA sequencing to estimate variant richness and test whether pseudogenes contribute to antigenic variation. We show that individual growth peaks can contain at least 15 distinct variants, are estimated computationally to comprise many more, and that antigenically distinct ‘mosaic’ VSGs arise from segmental gene conversion between donor VSG genes or pseudogenes. The potential for trypanosome antigenic variation is probably much greater than VSG archive size; mosaic VSGs are core to antigenic variation and chronic infection.     

Probabilistic order in antigenic variation of Trypanosoma brucei

Antigenic variation in African trypanosomes displays a degree of order that is usually described as 'semi-predictable' but which has not been analysed in statistical detail. It has been proposed that, during switching, the variable antigen type (VAT) being inactivated can influence which VAT is subsequently activated. Antigenic variation proceeds by the differential activation of members of the large archive of distinct variable surface glycoprotein (VSG) genes. The most popular model for ordered expression of VATs invokes differential activation probabilities for individual VSG genes, dictated in part by which of the four types of genetic locus they occupy. We have shown, in pilot experiments in cattle, correlation between the timing of appearance of VSG-specific mRNA and of lytic antibodies corresponding to seven VSGs encoded by single-copy genes. We have then determined the times of appearance of VAT-specific antibodies, as a measure of appearance of the VATs, in a statistically significant number of mouse infections (n=22). There is a surprisingly high degree of order in temporal appearance of the VATs, indicating that antigenic variation proceeds through order in the probability of activation of each VAT. In addition, for the few examples of each available, the locus type inhabited by the silent 'donor' VSG plays a significant role in determination of order. We have analysed in detail previously published data on VATs appearing in first relapse peaks, and find that the variant being switched off does not influence which one is being switched on. This differs from what has been reported for Plasmodium falciparum var antigenic variation. All these features of trypanosome antigenic variation can be explained by a one-step model in which, following an initial deactivation event, the switch process and the imposition of order early in infection arise from the inherent activation probabilities of the specific VSG being switched on.     

Trypanosoma brucei homologous recombination is dependent on substrate length and homology, though displays a differential dependence on mismatch repair as substrate length decreases

Homologous recombination functions universally in the maintenance of genome stability through the repair of DNA breaks and in ensuring the completion of replication. In some organisms, homologous recombination can perform more specific functions. One example of this is in antigenic variation, a widely conserved mechanism for the evasion of host immunity. Trypanosoma brucei, the causative agent of sleeping sickness in Africa, undergoes antigenic variation by periodic changes in its variant surface glycoprotein (VSG) coat. VSG switches involve the activation of VSG genes, from an enormous silent archive, by recombination into specialized expression sites. These reactions involve homologous recombination, though they are characterized by an unusually high rate of switching and by atypical substrate requirements. Here, we have examined the substrate parameters of T. brucei homologous recombination. We show, first, that the reaction is strictly dependent on substrate length and that it is impeded by base mismatches, features shared by homologous recombination in all organisms characterized. Second, we identify a pathway of homologous recombination that acts preferentially on short substrates and is impeded to a lesser extent by base mismatches and the mismatch repair machinery. Finally, we show that mismatches during T. brucei recombination may be repaired by short-patch mismatch repair.     

Gene conversions mediating antigenic variation in Trypanosoma brucei can occur in variant surface glycoprotein expression sites lacking 70-base-pair repeat sequences.

African trypanosomes undergo antigenic variation of their variant surface glycoprotein (VSG) coat to avoid immune system-mediated killing by their mammalian host. An important mechanism for switching the expressed VSG gene is the duplicative transposition of a silent VSG gene into one of the telomeric VSG expression sites of the trypanosome, resulting in the replacement of the previously expressed VSG gene. This process appears to be a gene conversion reaction, and it has been postulated that sequences within the expression site may act to initiate and direct the reaction. All bloodstream form expression sites contain huge arrays (many kilobase pairs) of 70-bp repeat sequences that act as the 5' boundary of gene conversion reactions involving most silent VSG genes. For this reason, the 70-bp repeats seemed a likely candidate to be involved in the initiation of switching. Here, we show that deletion of the 70-bp repeats from the active expression site does not affect duplicative transposition of VSG genes from silent expression sites. We conclude that the 70-bp repeats do not appear to function as indispensable initiation sites for duplicative transposition and are unlikely to be the recognition sequence for a sequence-specific enzyme which initiates recombination-based VSG switching.     

Trypanosoma brucei BRCA2 acts in antigenic variation and has undergone a recent expansion in BRC repeat number that is important during homologous recombination

Antigenic variation in Trypanosoma brucei has selected for the evolution of a massive archive of silent Variant Surface Glycoprotein (VSG) genes, which are activated by recombination into specialized expression sites. Such VSG switching can occur at rates substantially higher than background mutation and is dependent on homologous recombination, a core DNA repair reaction. A key regulator of homologous recombination is BRCA2, a protein that binds RAD51, the enzyme responsible for DNA strand exchange. Here, we show that T. brucei BRCA2 has undergone a recent, striking expansion in the number of BRC repeats, a sequence element that mediates interaction with RAD51. T. brucei BRCA2 mutants are shown to be significantly impaired in antigenic variation and display genome instability. By generating BRCA2 variants with reduced BRC repeat numbers, we show that the BRC expansion is crucial in determining the efficiency of T. brucei homologous recombination and RAD51 localization. Remarkably, however, this appears not to be a major determinant of the activation of at least some VSG genes.     

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.     

Trypanosoma brucei: frequent loss of a telomeric variant surface glycoprotein gene

We have observed the loss of an inactive telomeric variant surface glycoprotein (VSG) gene that is located on a minichromosome in Trypanosoma brucei. If this is due to gene conversion, it is the third "silent" gene conversion (i.e., one that does not produce an antigenic switch) detected in 19 antigenic switches of the IsTaR 1 serodeme. This is surprisingly frequent since the immune response cannot select against the inactive gene. We estimate that 10(-1) to 10(-3) telomeric VSG gene conversions occur per generation, which is at least 100 times more frequent than antigenic switching. Since all three "silent" gene conversions involved an IsTat 5 VSG gene, the frequency may vary among telomeric VSG genes. However, the high gene conversion frequency for the 5 VSG gene does not ensure a higher antigenic switch frequency than other telomeric VSG genes for which we have probes. These results suggest that gene conversion rapidly alters the repertoire of telomeric VSG genes, possibly including those on minichromosomes, producing a continual variation in the VSG genes that are more likely to be expressed.     

DNA Double-Strand Breaks and Telomeres Play Important Roles in Trypanosoma brucei Antigenic Variation

Human-infecting microbial pathogens all face a serious problem of elimination by the host immune response. Antigenic variation is an effective immune evasion mechanism where the pathogen regularly switches its major surface antigen. In many cases, the major surface antigen is encoded by genes from the same gene family, and its expression is strictly monoallelic. Among pathogens that undergo antigenic variation, Trypanosoma brucei (a kinetoplastid), which causes human African trypanosomiasis, Plasmodium falciparum (an apicomplexan), which causes malaria, Pneumocystis jirovecii (a fungus), which causes pneumonia, and Borrelia burgdorferi (a bacterium), which causes Lyme disease, also express their major surface antigens from loci next to the telomere. Except for Plasmodium, DNA recombination-mediated gene conversion is a major pathway for surface antigen switching in these pathogens. In the last decade, more sophisticated molecular and genetic tools have been developed in T. brucei, and our knowledge of functions of DNA recombination in antigenic variation has been greatly advanced. VSG is the major surface antigen in T. brucei. In subtelomeric VSG expression sites (ESs), VSG genes invariably are flanked by a long stretch of upstream 70-bp repeats. Recent studies have shown that DNA double-strand breaks (DSBs), particularly those in 70-bp repeats in the active ES, are a natural potent trigger for antigenic variation in T. brucei. In addition, telomere proteins can influence VSG switching by reducing the DSB amount at subtelomeric regions. These findings will be summarized and their implications will be discussed in this review.     

Molecular basis of antigenic variation in African trypanosomes

African trypanosomes, which cause sleeping sickness in man and other mammals, are able to evade immune destruction in their hosts by altering the expression of a major cell surface molecule, the variant surface glycoprotein (VSG). The VSGs are encoded by a multigene family, and antigenic variation occurs when the trypanosome switches from expression of one VSG gene to another. This switching process involves changes in the arrangement of the trypanosome genomic DNA.     

The role of duplication in the expression of a variable surface glycoprotein gene of Trypanosoma brucei

The variable surface glycoprotein (VSG) genes of Trypanosoma brucei have been classified into two groups depending upon whether or not duplication of the genes is observed when they are expressed. We report here the observation of duplication apparently linked to expression of the ILTaT 1.3 gene in the ETaR 1 trypanosome stock. In the ILTaR 1 stock, expression of the ILTaT 1.3 VSG did not involve a new duplication, but instead activation of a preexisting gene copy that had been apparently generated earlier by a duplication event analogous to that directly observed in the ETaR 1 trypanosomes. The results suggest that the well-characterised gene duplications found with other VSG genes are common to all VSG genes but are not directly responsible for controlling expression. All currently available data can be accommodated by a model that assumes that gene duplication and replacement occurs independently of antigenic switching.