DNA double strand break position leads to distinct gene expression changes and regulates VSG switching pathway choice.

Antigenic variation is an immune evasion strategy used by Trypanosoma brucei that results in the periodic exchange of the surface protein coat. This process is facilitated by the movement of variant surface glycoprotein genes in or out of a specialized locus known as bloodstream form expression site by homologous recombination, facilitated by blocks of repetitive sequence known as the 70-bp repeats, that provide homology for gene conversion events. DNA double strand breaks are potent drivers of antigenic variation, however where these breaks must fall to elicit a switch is not well understood. To understand how the position of a break influences antigenic variation we established a series of cell lines to study the effect of an I-SceI meganuclease break in the active expression site. We found that a DNA break within repetitive regions is not productive for VSG switching, and show that the break position leads to a distinct gene expression profile and DNA repair response which dictates how antigenic variation proceeds in African trypanosomes.

African trypanosomes expressing multiple VSGs are rapidly eliminated by the host immune system.

Trypanosoma brucei parasites successfully evade the host immune system by periodically switching the dense coat of variant surface glycoprotein (VSG) at the cell surface. Each parasite expresses VSGs in a monoallelic fashion that is tightly regulated. The consequences of exposing multiple VSGs during an infection, in terms of antibody response and disease severity, remain unknown. In this study, we overexpressed a high-mobility group box protein, TDP1, which was sufficient to open the chromatin of silent VSG expression sites, to disrupt VSG monoallelic expression, and to generate viable and healthy parasites with a mixed VSG coat. Mice infected with these parasites mounted a multi-VSG antibody response, which rapidly reduced parasitemia. Consequently, we observed prolonged survival in which nearly 90% of the mice survived a 30-d period of infection with undetectable parasitemia. Immunodeficient RAG2 knock-out mice were unable to control infection with TDP1-overexpressing parasites, showing that the adaptive immune response is critical to reducing disease severity. This study shows that simultaneous exposure of multiple VSGs is highly detrimental to the parasite, even at the very early stages of infection, suggesting that drugs that disrupt VSG monoallelic expression could be used to treat trypanosomiasis.

Variant antigen repertoires in Trypanosoma congolense populations and experimental infections can be profiled from deep sequence data using universal protein motifs.

African trypanosomes are vector-borne hemoparasites of humans and animals. In the mammal, parasites evade the immune response through antigenic variation. Periodic switching of the variant surface glycoprotein (VSG) coat covering their cell surface allows sequential expansion of serologically distinct parasite clones. Trypanosome genomes contain many hundreds of VSG genes, subject to rapid changes in nucleotide sequence, copy number, and chromosomal position. Thus, analyzing, or even quantifying, VSG diversity over space and time presents an enormous challenge to conventional techniques. Indeed, previous population genomic studies have overlooked this vital aspect of pathogen biology for lack of analytical tools. Here we present a method for analyzing population-scale VSG diversity in Trypanosoma congolense from deep sequencing data. Previously, we suggested that T. congolense VSGs segregate into defined "phylotypes" that do not recombine. In our data set comprising 41 T. congolense genome sequences from across Africa, these phylotypes are universal and exhaustive. Screening sequence contigs with diagnostic protein motifs accurately quantifies relative phylotype frequencies, providing a metric of VSG diversity, called the "variant antigen profile." We applied our metric to VSG expression in the tsetse fly, showing that certain, rare VSG phylotypes may be preferentially expressed in infective, metacyclic-stage parasites. Hence, variant antigen profiling accurately and rapidly determines the T. congolense VSG gene and transcript repertoire from sequence data, without need for manual curation or highly contiguous sequences. It offers a tractable approach to measuring VSG diversity across strains and during infections, which is imperative to understanding the host-parasite interaction at population and individual scales.

Ribonuclease H1-targeted R-loops in surface antigen gene expression sites can direct trypanosome immune evasion.

Switching of the Variant Surface Glycoprotein (VSG) in Trypanosoma brucei provides a crucial host immune evasion strategy that is catalysed both by transcription and recombination reactions, each operating within specialised telomeric VSG expression sites (ES). VSG switching is likely triggered by events focused on the single actively transcribed ES, from a repertoire of around 15, but the nature of such events is unclear. Here we show that RNA-DNA hybrids, called R-loops, form preferentially within sequences termed the 70 bp repeats in the actively transcribed ES, but spread throughout the active and inactive ES, in the absence of RNase H1, which degrades R-loops. Loss of RNase H1 also leads to increased levels of VSG coat switching and replication-associated genome damage, some of which accumulates within the active ES. This work indicates VSG ES architecture elicits R-loop formation, and that these RNA-DNA hybrids connect T. brucei immune evasion by transcription and recombination.

Evaluation of mechanisms that may generate DNA lesions triggering antigenic variation in African trypanosomes.

Antigenic variation by variant surface glycoprotein (VSG) coat switching in African trypanosomes is one of the most elaborate immune evasion strategies found among pathogens. Changes in the identity of the transcribed VSG gene, which is always flanked by 70-bp and telomeric repeats, can be achieved either by transcriptional or DNA recombination mechanisms. The major route of VSG switching is DNA recombination, which occurs in the bloodstream VSG expression site (ES), a multigenic site transcribed by RNA polymerase I. Recombinogenic VSG switching is frequently catalyzed by homologous recombination (HR), a reaction normally triggered by DNA breaks. However, a clear understanding of how such breaks arise-including whether there is a dedicated and ES-focused mechanism-is lacking. Here, we synthesize data emerging from recent studies that have proposed a range of mechanisms that could generate these breaks: action of a nuclease or nucleases; repetitive DNA, most notably the 70-bp repeats, providing an intra-ES source of instability; DNA breaks derived from the VSG-adjacent telomere; DNA breaks arising from high transcription levels at the active ES; and DNA lesions arising from replication-transcription conflicts in the ES. We discuss the evidence that underpins these switch-initiation models and consider what features and mechanisms might be shared or might allow the models to be tested further. Evaluation of all these models highlights that we still have much to learn about the earliest acting step in VSG switching, which may have the greatest potential for therapeutic intervention in order to undermine the key reaction used by trypanosomes for their survival and propagation in the mammalian host.

A quorum sensing-independent path to stumpy development in Trypanosoma brucei.

For persistent infections of the mammalian host, African trypanosomes limit their population size by quorum sensing of the parasite-excreted stumpy induction factor (SIF), which induces development to the tsetse-infective stumpy stage. We found that besides this cell density-dependent mechanism, there exists a second path to the stumpy stage that is linked to antigenic variation, the main instrument of parasite virulence. The expression of a second variant surface glycoprotein (VSG) leads to transcriptional attenuation of the VSG expression site (ES) and immediate development to tsetse fly infective stumpy parasites. This path is independent of SIF and solely controlled by the transcriptional status of the ES. In pleomorphic trypanosomes varying degrees of ES-attenuation result in phenotypic plasticity. While full ES-attenuation causes irreversible stumpy development, milder attenuation may open a time window for rescuing an unsuccessful antigenic switch, a scenario that so far has not been considered as important for parasite survival.

A Conserved DNA Repeat Promotes Selection of a Diverse Repertoire of Trypanosoma brucei Surface Antigens from the Genomic Archive.

African trypanosomes are mammalian pathogens that must regularly change their protein coat to survive in the host bloodstream. Chronic trypanosome infections are potentiated by their ability to access a deep genomic repertoire of Variant Surface Glycoprotein (VSG) genes and switch from the expression of one VSG to another. Switching VSG expression is largely based in DNA recombination events that result in chromosome translocations between an acceptor site, which houses the actively transcribed VSG, and a donor gene, drawn from an archive of more than 2,000 silent VSGs. One element implicated in these duplicative gene conversion events is a DNA repeat of approximately 70 bp that is found in long regions within each BES and short iterations proximal to VSGs within the silent archive. Early observations showing that 70-bp repeats can be recombination boundaries during VSG switching led to the prediction that VSG-proximal 70-bp repeats provide recombinatorial homology. Yet, this long held assumption had not been tested and no specific function for the conserved 70-bp repeats had been demonstrated. In the present study, the 70-bp repeats were genetically manipulated under conditions that induce gene conversion. In this manner, we demonstrated that 70-bp repeats promote access to archival VSGs. Synthetic repeat DNA sequences were then employed to identify the length, sequence, and directionality of repeat regions required for this activity. In addition, manipulation of the 70-bp repeats allowed us to observe a link between VSG switching and the cell cycle that had not been appreciated. Together these data provide definitive support for the long-standing hypothesis that 70-bp repeats provide recombinatorial homology during switching. Yet, the fact that silent archival VSGs are selected under these conditions suggests the 70-bp repeats also direct DNA pairing and recombination machinery away from the closest homologs (silent BESs) and toward the rest of the archive.

Blocking Synthesis of the Variant Surface Glycoprotein Coat in Trypanosoma brucei Leads to an Increase in Macrophage Phagocytosis Due to Reduced Clearance of Surface Coat Antibodies.

The extracellular bloodstream form parasite Trypanosoma brucei is supremely adapted to escape the host innate and adaptive immune system. Evasion is mediated through an antigenically variable Variant Surface Glycoprotein (VSG) coat, which is recycled at extraordinarily high rates. Blocking VSG synthesis triggers a precytokinesis arrest where stalled cells persist for days in vitro with superficially intact VSG coats, but are rapidly cleared within hours in mice. We therefore investigated the role of VSG synthesis in trypanosome phagocytosis by activated mouse macrophages. T. brucei normally effectively evades macrophages, and induction of VSG RNAi resulted in little change in phagocytosis of the arrested cells. Halting VSG synthesis resulted in stalled cells which swam directionally rather than tumbling, with a significant increase in swim velocity. This is possibly a consequence of increased rigidity of the cells due to a restricted surface coat in the absence of VSG synthesis. However if VSG RNAi was induced in the presence of anti-VSG221 antibodies, phagocytosis increased significantly. Blocking VSG synthesis resulted in reduced clearance of anti-VSG antibodies from the trypanosome surface, possibly as a consequence of the changed motility. This was particularly marked in cells in the G2/ M cell cycle stage, where the half-life of anti-VSG antibody increased from 39.3 ± 4.2 seconds to 99.2 ± 15.9 seconds after induction of VSG RNAi. The rates of internalisation of bulk surface VSG, or endocytic markers like transferrin, tomato lectin or dextran were not significantly affected by the VSG synthesis block. Efficient elimination of anti-VSG-antibody complexes from the trypanosome cell surface is therefore essential for trypanosome evasion of macrophages. These experiments highlight the essentiality of high rates of VSG recycling for the rapid removal of host opsonins from the parasite surface, and identify this process as a key parasite virulence factor during a chronic infection.

Inositol phosphate pathway controls transcription of telomeric expression sites in trypanosomes.

African trypanosomes evade clearance by host antibodies by periodically changing their variant surface glycoprotein (VSG) coat. They transcribe only one VSG gene at a time from 1 of about 20 telomeric expression sites (ESs). They undergo antigenic variation by switching transcription between telomeric ESs or by recombination of the VSG gene expressed. We show that the inositol phosphate (IP) pathway controls transcription of telomeric ESs and VSG antigenic switching in Trypanosoma brucei. Conditional knockdown of phosphatidylinositol 5-kinase (TbPIP5K) or phosphatidylinositol 5-phosphatase (TbPIP5Pase) or overexpression of phospholipase C (TbPLC) derepresses numerous silent ESs in T. brucei bloodstream forms. The derepression is specific to telomeric ESs, and it coincides with an increase in the number of colocalizing telomeric and RNA polymerase I foci in the nucleus. Monoallelic VSG transcription resumes after reexpression of TbPIP5K; however, most of the resultant cells switched the VSG gene expressed. TbPIP5K, TbPLC, their substrates, and products localize to the plasma membrane, whereas TbPIP5Pase localizes to the nucleus proximal to telomeres. TbPIP5Pase associates with repressor/activator protein 1 (TbRAP1), and their telomeric silencing function is altered by TbPIP5K knockdown. These results show that specific steps in the IP pathway control ES transcription and antigenic switching in T. brucei by epigenetic regulation of telomere silencing.

The utility of an rTeGM6-4r-based immunochromatographic test for the serological diagnosis of non-tsetse-transmitted equine trypanosomosis in rural areas of Mongolia.

Our previous studies report epidemics of non-tsetse-transmitted equine trypanosomosis in Mongolia. However, the current status of non-tsetse-transmitted equine trypanosomosis endemicity remains to be clarified in some parts of Mongolia. We previously reported the potential application of rTeGM6-4r-based diagnostic tools, an rTeGM6-4r-based immunochromatographic test (ICT) and an enzyme-linked immunosorbent assay (ELISA), in the serological surveillance of equine trypanosomosis in Mongolia. In the present study, the utility of the rTeGM6-4r-based ICT was validated. The rTeGM6-4r-based ICT accurately diagnosed positive reference sera that had been prepared from dourine horses in Mongolia, similarly to the rTeGM6-4r-based ELISA. The diagnostic performance of the rTeGM6-4r-based ICT was maintained when the strips were preserved for at least 2 months under dry conditions. The ICT detected 42 positive serum samples from a total of 1701 equine sera that had been collected from all 21 provinces of Mongolia. The κ-value, sensitivity and specificity of rTeGM6-4r-based ICT were 0.58, 50.0% (95% CI, 37.7-62.3%) and 99.3% (95% CI, 98.7-99.6%), respectively, in comparison to the rTeGM6-4r-based ELISA. Our field-friendly rTeGM6-4r-based ICT was found to be useful for the serological diagnosis of non-tsetse-transmitted equine trypanosomosis in rural areas of Mongolia.

The Trypomastigote Small Surface Antigen from Trypanosoma cruzi Improves Treatment Evaluation and Diagnosis in Pediatric Chagas Disease.

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi Assessment of parasitological cure upon treatment with available drugs relies on achieving consistent negative results in conventional parasitological and serological tests, which may take years to assess. Here, we evaluated the use of a recombinant T. cruzi antigen termed trypomastigote small surface antigen (TSSA) as an early serological marker of drug efficacy in T. cruzi-infected children. A cohort of 78 pediatric patients born to T. cruzi-infected mothers was included in this study. Only 39 of the children were infected with T. cruzi, and they were immediately treated with trypanocidal drugs. Serological responses against TSSA were evaluated in infected and noninfected populations during the follow-up period using an in-house enzyme-linked immunosorbent assay (ELISA) and compared to conventional serological methods. Anti-TSSA antibody titers decreased significantly faster than anti-whole parasite antibodies detected by conventional serology both in T. cruzi-infected patients undergoing effective treatment and in those not infected. The differential kinetics allowed a significant reduction in the required follow-up periods to evaluate therapeutic responses or to rule out maternal-fetal transmission. Finally, we present the case of a congenitally infected patient with an atypical course in whom TSSA provided an early marker for T. cruzi infection. In conclusion, we showed that TSSA was efficacious both for rapid assessment of treatment efficiency and for early negative diagnosis in infants at risk of congenital T. cruzi infection. Based upon these findings we propose the inclusion of TSSA for refining the posttherapeutic cure criterion and other diagnostic needs in pediatric Chagas disease.

How Does the VSG Coat of Bloodstream Form African Trypanosomes Interact with External Proteins?

Variations on the statement "the variant surface glycoprotein (VSG) coat that covers the external face of the mammalian bloodstream form of Trypanosoma brucei acts a physical barrier" appear regularly in research articles and reviews. The concept of the impenetrable VSG coat is an attractive one, as it provides a clear model for understanding how a trypanosome population persists; each successive VSG protects the plasma membrane and is immunologically distinct from previous VSGs. What is the evidence that the VSG coat is an impenetrable barrier, and how do antibodies and other extracellular proteins interact with it? In this review, the nature of the extracellular surface of the bloodstream form trypanosome is described, and past experiments that investigated binding of antibodies and lectins to trypanosomes are analysed using knowledge of VSG sequence and structure that was unavailable when the experiments were performed. Epitopes for some VSG monoclonal antibodies are mapped as far as possible from previous experimental data, onto models of VSG structures. The binding of lectins to some, but not to other, VSGs is revisited with more recent knowledge of the location and nature of N-linked oligosaccharides. The conclusions are: (i) Much of the variation observed in earlier experiments can be explained by the identity of the individual VSGs. (ii) Much of an individual VSG is accessible to antibodies, and the barrier that prevents access to the cell surface is probably at the base of the VSG N-terminal domain, approximately 5 nm from the plasma membrane. This second conclusion highlights a gap in our understanding of how the VSG coat works, as several plasma membrane proteins with large extracellular domains are very unlikely to be hidden from host antibodies by VSG.

Suppression of subtelomeric VSG switching by Trypanosoma brucei TRF requires its TTAGGG repeat-binding activity.

Trypanosoma brucei causes human African trypanosomiasis and regularly switches its major surface antigen, VSG, in the bloodstream of its mammalian host to evade the host immune response. VSGs are expressed exclusively from subtelomeric loci, and we have previously shown that telomere proteins TbTIF2 and TbRAP1 play important roles in VSG switching and VSG silencing regulation, respectively. We now discover that the telomere duplex DNA-binding factor, TbTRF, also plays a critical role in VSG switching regulation, as a transient depletion of TbTRF leads to significantly more VSG switching events. We solved the NMR structure of the DNA-binding Myb domain of TbTRF, which folds into a canonical helix-loop-helix structure that is conserved to the Myb domains of mammalian TRF proteins. The TbTRF Myb domain tolerates well the bulky J base in T. brucei telomere DNA, and the DNA-binding affinity of TbTRF is not affected by the presence of J both in vitro and in vivo. In addition, we find that point mutations in TbTRF Myb that significantly reduced its in vivo telomere DNA-binding affinity also led to significantly increased VSG switching frequencies, indicating that the telomere DNA-binding activity is critical for TbTRF's role in VSG switching regulation.

Structure of the trypanosome haptoglobin-hemoglobin receptor and implications for nutrient uptake and innate immunity.

African trypanosomes are protected by a densely packed surface monolayer of variant surface glycoprotein (VSG). A haptoglobin-hemoglobin receptor (HpHbR) within this VSG coat mediates heme acquisition. HpHbR is also exploited by the human host to mediate endocytosis of trypanolytic factor (TLF)1 from serum, contributing to innate immunity. Here, the crystal structure of HpHbR from Trypanosoma congolense has been solved, revealing an elongated three α-helical bundle with a small membrane distal head. To understand the receptor in the context of the VSG layer, the dimensions of Trypanosoma brucei HpHbR and VSG have been determined by small-angle X-ray scattering, revealing the receptor to be more elongated than VSG. It is, therefore, likely that the receptor protrudes above the VSG layer and unlikely that the VSG coat can prevent immunoglobulin binding to the receptor. The HpHb-binding site has been mapped by single-residue mutagenesis and surface plasmon resonance. This site is located where it is readily accessible above the VSG layer. A single HbHpR polymorphism unique to human infective T. brucei gambiense has been shown to be sufficient to reduce binding of both HpHb and TLF1, modulating ligand affinity in a delicate balancing act that allows nutrient acquisition but avoids TLF1 uptake.

A yeast-endonuclease-generated DNA break induces antigenic switching in Trypanosoma brucei.

Trypanosoma brucei is the causative agent of African sleeping sickness in humans and one of the causes of nagana in cattle. This protozoan parasite evades the host immune system by antigenic variation, a periodic switching of its variant surface glycoprotein (VSG) coat. VSG switching is spontaneous and occurs at a rate of about 10(-2)-10(-3) per population doubling in recent isolates from nature, but at a markedly reduced rate (10(-5)-10(-6)) in laboratory-adapted strains. VSG switching is thought to occur predominantly through gene conversion, a form of homologous recombination initiated by a DNA lesion that is used by other pathogens (for example, Candida albicans, Borrelia sp. and Neisseria gonorrhoeae) to generate surface protein diversity, and by B lymphocytes of the vertebrate immune system to generate antibody diversity. Very little is known about the molecular mechanism of VSG switching in T. brucei. Here we demonstrate that the introduction of a DNA double-stranded break (DSB) adjacent to the approximately 70-base-pair (bp) repeats upstream of the transcribed VSG gene increases switching in vitro approximately 250-fold, producing switched clones with a frequency and features similar to those generated early in an infection. We were also able to detect spontaneous DSBs within the 70-bp repeats upstream of the actively transcribed VSG gene, indicating that a DSB is a natural intermediate of VSG gene conversion and that VSG switching is the result of the resolution of this DSB by break-induced replication.

Antigenic diversity is generated by distinct evolutionary mechanisms in African trypanosome species.

Antigenic variation enables pathogens to avoid the host immune response by continual switching of surface proteins. The protozoan blood parasite Trypanosoma brucei causes human African trypanosomiasis ("sleeping sickness") across sub-Saharan Africa and is a model system for antigenic variation, surviving by periodically replacing a monolayer of variant surface glycoproteins (VSG) that covers its cell surface. We compared the genome of Trypanosoma brucei with two closely related parasites Trypanosoma congolense and Trypanosoma vivax, to reveal how the variant antigen repertoire has evolved and how it might affect contemporary antigenic diversity. We reconstruct VSG diversification showing that Trypanosoma congolense uses variant antigens derived from multiple ancestral VSG lineages, whereas in Trypanosoma brucei VSG have recent origins, and ancestral gene lineages have been repeatedly co-opted to novel functions. These historical differences are reflected in fundamental differences between species in the scale and mechanism of recombination. Using phylogenetic incompatibility as a metric for genetic exchange, we show that the frequency of recombination is comparable between Trypanosoma congolense and Trypanosoma brucei but is much lower in Trypanosoma vivax. Furthermore, in showing that the C-terminal domain of Trypanosoma brucei VSG plays a crucial role in facilitating exchange, we reveal substantial species differences in the mechanism of VSG diversification. Our results demonstrate how past VSG evolution indirectly determines the ability of contemporary parasites to generate novel variant antigens through recombination and suggest that the current model for antigenic variation in Trypanosoma brucei is only one means by which these parasites maintain chronic infections.

Histone H1 plays a role in heterochromatin formation and VSG expression site silencing in Trypanosoma brucei.

The African sleeping sickness parasite Trypanosoma brucei evades the host immune system through antigenic variation of its variant surface glycoprotein (VSG) coat. Although the T. brucei genome contains ∼1500 VSGs, only one VSG is expressed at a time from one of about 15 subtelomeric VSG expression sites (ESs). For antigenic variation to work, not only must the vast VSG repertoire be kept silent in a genome that is mainly constitutively transcribed, but the frequency of VSG switching must be strictly controlled. Recently it has become clear that chromatin plays a key role in silencing inactive ESs, thereby ensuring monoallelic expression of VSG. We investigated the role of the linker histone H1 in chromatin organization and ES regulation in T. brucei. T. brucei histone H1 proteins have a different domain structure to H1 proteins in higher eukaryotes. However, we show that they play a key role in the maintenance of higher order chromatin structure in bloodstream form T. brucei as visualised by electron microscopy. In addition, depletion of histone H1 results in chromatin becoming generally more accessible to endonucleases in bloodstream but not in insect form T. brucei. The effect on chromatin following H1 knock-down in bloodstream form T. brucei is particularly evident at transcriptionally silent ES promoters, leading to 6-8 fold derepression of these promoters. T. brucei histone H1 therefore appears to be important for the maintenance of repressed chromatin in bloodstream form T. brucei. In particular H1 plays a role in downregulating silent ESs, arguing that H1-mediated chromatin functions in antigenic variation in T. brucei.

Telomere length affects the frequency and mechanism of antigenic variation in Trypanosoma brucei.

Trypanosoma brucei is a master of antigenic variation and immune response evasion. Utilizing a genomic repertoire of more than 1000 Variant Surface Glycoprotein-encoding genes (VSGs), T. brucei can change its protein coat by "switching" from the expression of one VSG to another. Each active VSG is monoallelically expressed from only one of approximately 15 subtelomeric sites. Switching VSG expression occurs by three predominant mechanisms, arguably the most significant of which is the non-reciprocal exchange of VSG containing DNA by duplicative gene conversion (GC). How T. brucei orchestrates its complex switching mechanisms remains to be elucidated. Recent work has demonstrated that an exogenous DNA break in the active site could initiate a GC based switch, yet the source of the switch-initiating DNA lesion under natural conditions is still unknown. Here we investigated the hypothesis that telomere length directly affects VSG switching. We demonstrate that telomerase deficient strains with short telomeres switch more frequently than genetically identical strains with long telomeres and that, when the telomere is short, switching preferentially occurs by GC. Our data supports the hypothesis that a short telomere at the active VSG expression site results in an increase in subtelomeric DNA breaks, which can initiate GC based switching. In addition to their significance for T. brucei and telomere biology, the findings presented here have implications for the many diverse pathogens that organize their antigenic genes in subtelomeric regions.

Antigenic variation in African trypanosomes: the importance of chromosomal and nuclear context in VSG expression 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.

Limited antigenic variation in the Trypanosoma cruzi candidate vaccine antigen TSA-1.

Chagas disease (American trypanosomiasis caused by Trypanosoma cruzi) is one of the most important neglected tropical diseases in the Western Hemisphere. The toxicities and limited efficacies of current antitrypanosomal drugs have prompted a search for alternative technologies such as a therapeutic vaccine comprised of T. cruzi antigens, including a recombinant antigen encoding the N-terminal 65 kDa portion of Trypomastigote surface antigen-1 (TSA-1). With at least six known genetically distinct T. cruzi lineages, variability between the different lineages poses a unique challenge for the development of broadly effective therapeutic vaccine. The variability across the major lineages in the current vaccine candidate antigen TSA-1 has not previously been addressed. To assess the variation in TSA-1, we cloned and sequenced TSA-1 from several different T. cruzi strains representing three of the most clinically relevant lineages. Analysis of the different alleles showed limited variation in TSA-1 across the different strains and fit with the current theory for the evolution of the different lineages. Additionally, minimal variation in known antigenic epitopes for the HLA-A 02 allele suggests that interlineage variation in TSA-1 would not impair the range and efficacy of a vaccine containing TSA-1.