What has DNA sequencing revealedabout the VSG expression sites ofAfrican trypanosomes?Richard McCulloch1 and David Horn2

1 University of Glasgow, Wellcome Centre for Molecular Parasitology and Division of Infection and Immunity,

Glasgow Biomedical Research Centre, 120 University Place, Glasgow, G12 8TA, UK2 London School of Hygiene and Tropical Medicine, Keppel Street, London, WC1E 7HT, UK

Opinion

Antigenic variation is crucial for the survival of Africantrypanosomes in mammals and involves switches inexpression of variant surface glycoprotein genes, whichare co-transcribed with a number of expression-site-associated genes (ESAGs) from loci termed ‘blood-stream expression sites’ (BESs). Trypanosomes possessmultiple BESs, although the reason for this (and whyESAGs are resident in these loci) has remained a subjectof debate. The genome sequence of Trypanosoma bru-cei, released in 2005, did not include the BESs because oftheir telomeric disposition. This gap in our knowledgehas now been bridged by two new studies, which wediscuss here, asking what has been revealed about thebiological significance of BES multiplicity and ESAGfunction and evolution.

Trypanosomes and tsetse fliesAfrican trypanosomes infect a range of mammals, in-cluding humans and their domestic cattle, causing try-panosomiasis disease that continues to afflict the peopleof the sub-Saharan region of Africa and impose aneconomic burden [1]. Trypanosoma brucei exists as threesubspecies: Trypanosoma brucei rhodesiense, Trypano-soma brucei gambiense and Trypanosoma brucei brucei,each of which is transmitted between successive hostsby tsetse flies. The distribution of mammals the sub-species infect is variable, and T. b. gambiense has arelatively restricted host range [2] compared with T. b.rhodesiense, which parasitizes many African gameanimals [3]. T. b. brucei, the subspecies whose genomesequence was reported in 2005 [4], is not human-infec-tive but is otherwise genetically closely related to T. b.rhodesiense [5]. Many other non-human-infective Africantrypanosomes have been described, some of which do notcycle through tsetse flies. One example is Trypanosomaequiperdum, which is sexually transmitted betweenequines and normally restricted to the urogenital tract,severely limiting its range of host mammals [6].The genetic features underlying the major differencesbetween these species and subspecies remain onlypartially characterized.

Corresponding author: McCulloch, R. (rmc9z@udcf.gla.ac.uk).

1471-4922/$ – see front matter � 2009 Published by Elsevier Ltd. doi:10.1016/j.pt.2009.05.007

T. brucei antigenic variation and the role of VSG

expression sitesAfrican trypanosomes live extracellularly in the blood-stream and tissue fluids of their mammalian hosts where,in common with many pathogens, survival in the face ofattack by the host immune system is achieved by antigenicvariation, the pre-emptive switching of surface antigens. Intrypanosomes, the variant antigen is called variant surfaceglycoprotein (VSG), which forms a dense coat on the cellsurface that shields invariant surface antigens fromimmune recognition. Host antibodies directed againstthe expressed VSG kill the trypanosome, but continualswitching to antigenically distinct VSG coats enables theparasite to avoid elimination of the whole population,leading to long-term infections characterized by fluctuat-ing parasitaemia.

Antigenic variation, in any non-viral pathogen, is de-pendent on three key features [7]: a family of variantsurface antigen genes, a mechanism to express only onesuch gene at a time and a mechanism to switch expression.The genome sequence of T. b. brucei revealed that Africantrypanosomes have evolved an enormous family of VSGgenes (estimated at �2000) [4], dwarfing the size of anti-genically variant gene families described elsewhere. Acti-vation of the majority of these genes occurs byrecombination, normally by gene conversion reactions thatcopy a silentVSG intoVSG expression sites (ESs), deletingthe resident VSG [8]. Trypanosome ESs are the sites thatprovide for singular VSG expression, and they have anumber of surprising features. First, trypanosomes pos-sess two distinct classes of ES. Those that are used toexpress VSG in the mammalian bloodstream are termed‘bloodstream ES’ (BESs). However, the VSG coat is alsosynthesized in metacyclic form cells in the tsetse salivarygland; here, the VSG is expressed from metacyclic ESs(MESs). Second, trypanosomes possess multiple copies ofeach class of ES (see below). This is surprising because inmost other pathogens that rely on recombination for anti-genic variation, such as Borrelia hermsii, Anaplasma mar-ginale [9] or Neisseria species [10], a single equivalentantigen gene ES suffices. Only one T. brucei BES is nor-mally active in the cell at a time, but BES multiplicitymeans that, by silencing transcription from the active BESand co-ordinatedly activating transcription from one of the

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Figure 1. A generic bloodstream variant surface glycoprotein expression site in the African trypanosome. A chromosome containing a telomeric bloodstream variant

surface glycoprotein (VSG) expression site (BES) is represented by a black line; the typical size of a BES is indicated. The RNA polymerase I promoter that directs

transcription of all downstream BES genes is shown by a flag. Arrays of DNA repeats associated with the BES are represented by hatched grey boxes: 70 bp repeats (varying

in size from �0.1 kbp to 7.0 kbp) are found upstream of most VSG genes in T. brucei and are thought to provide upstream homology during gene conversion recombination

(a typical ‘cassette’ of sequence that is copied during such a switching reaction is indicated); telomere repeats mark the chromosome end and are found �0.2-1.6 kbp

downstream of the VSG; 50 bp repeats are found upstream of the BES promoter. The VSG (red box) is always found adjacent to the telomere, and VSG pseudogenes and

VSG-related genes are occasionally found upstream (not shown). Expression-site-associated genes (ESAGs) are found between the promoter and 70 bp repeats; dark blue

boxes represent functional genes, and light blue boxes represent pseudogenes (c). Depicted is the canonical order of widely conserved ESAGs; variations in this order

include inversions, duplications and truncations. Further ESAGs that have been described in only some ESs (see Table 1) are not shown: ESAG9 is a small gene family, and

one BES in T. brucei has been shown to harbour a copy upstream of the 70 bp repeats; promoter duplication is frequent in BESs, and in these cases, ESAG10 is found

between the duplicated promoters; ESAG12 has been described downstream of ESAG4 in five BESs.

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silent BESs [11], a change in the expressed VSG can occur[12,13]. Finally, the BESs do not simply express the VSG(Figure 1). Instead, multiple expression-site-associatedgenes (ESAGs) are co-transcribed with the VSG (whichis always found proximal to the telomere repeats) from asingle developmentally regulated RNA polymerase (Pol) Ipromoter [14]. MESs are also transcribed by RNA Pol I butdiffer from the BES in not containing ESAGs and havingdistinct RNA Pol I promoter sequences, perhaps reflectingdiffering regulatorymechanisms [15]. Nevertheless,ESAGgenes and pseudogenes are found upstream of the MES,suggesting this ES class arose from the BES [8].

Figure 1 presents a graphical depiction of a BES, show-ing a generalization of the gene organisation. Although thisorganisation, as well as the multiplicity of the ESs andtheir split into two developmental classes, has been knownfor some time, many questions remain. For instance, in theabsence of comprehensive BES sequences (see below), howrepresentative is Figure 1 of the cohort of trypanosomeBESs within a single cell and in the population? Why dotrypanosomes possess multiple ESs and why are ESAGsassociated with the BES? We still know surprisingly littleabout the functions of the ESAGs (Table 1), but surfacereceptor, signalling and transporter functions have beenestablished or suggested. It has been proposed that VSGsare at subtelomeres because this genomic location facili-tates recombination [16], but little work has askedwhetherthe ESAGs are also prone to recombination or what se-lective pressures might act upon them. Through directedsequencing of the BES, Hertz-Fowler et al. [17] and Younget al. [18] have begun to consider such questions, askingwhether the BESs provide the capacity to alter BES-associated gene expression in a coordinated way, drivenby host adaptive immunity and/or by a requirement toaccess polymorphic host nutrients.

Sequencing VSG expression sitesVSG ESs present an obstacle to conventional genomesequencing. Because they are telomeric, ESs are under-represented in bacterial artificial chromosome librariesand, therefore, the T. b. brucei genome sequence did not

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reveal the repertoire of available ESs [4]. To circumventthis, Becker et al. [19] devised a transformation-associatedrecombination (TAR) strategy to isolate ES-containingtelomeres in the yeast Saccharomyces cerevisiae. As usedto date, this relies upon recombination using the BESpromoter sequence to generate artificial chromosomes,meaning that the MES repertoire was not isolated. UsingTAR, 14 distinct BESs were characterized in T. b. bruceistrain Lister 427 [17] (slightly fewer than the 20 estimatedpreviously [20]), and 23 distinct BES were described in T.b. brucei strain EATRO 2340 [18], 13 in T. b. gambienseDAL927 and 16 in T. equiperdum STIB818. This variationin numbers is consistent with the dynamic nature of sub-telomeres in potentially all organisms [16,21]. We do notknow, however, whether all T. brucei telomeres in themegabase-sized chromosomes are occupied by ESs. Forinstance, in those strains and/or subspecies in whichBES number is reduced, is this balanced by greater num-bers of MESs? If correct, this would imply that telomericrecombination occurs both within and between the BESand MES repertoires [22]. It is not clear that this is thecase, however. Antibody labelling and neutralization stu-dies suggest the MES repertoire is rather invariant [23],although it does display a turnover in content [24]. T.brucei has also evolved multicopy intermediate-sizedand mini chromosomes for antigenic variation [25].Although we know that the intermediate chromosometelomeres harbour BESs [26], it has not been determinedwhether MESs also populate these chromosomes.

The study of Hertz-Fowler et al. [17] provides a systema-tic comparison of a complete set of BESs within a givenstrain; a ‘snapshot’ ofBES composition at the time of cloningand sequencing. This indicates the BESs have remarkablyconservedgeneorder [27] (Figure 1).Nevertheless, diversityis found within this order. Numerous examples of BESswith ESAG duplications were apparent, as were sites thathave undergone truncations (in one case, leaving only theVSG and promoter-proximal ESAG7). In addition, manyexamples of VSG and ESAG pseudogenes were found:ESAG3 and ESAG11 pseudogenes were particularly preva-lent (indeed, a functional copy ofESAG11wasnot observed).

Table 1. Summary of the number and predicted function of ESAGs in the bloodstream VSG expression sites of T. b. brucei Lister 427

ESAG Number of T. b. brucei Lister 427 BESs that

contain specified ESAGaFunction or properties Refs

1 11 Membrane glycoprotein [46]

2 11 GPI-anchored glycoprotein? [36]

3 13a Membrane glycoprotein? [36]

4 10c Receptor-like adenylate cyclase [47]

5 13d Lipid transfer/lipopolysaccharide binding? [43]

6 13 Transferrin receptor subunit [38]

7 13 Transferrin receptor subunit [38]

8 10e Nucleolar RNA stability regulator? [48]

9 1 Unknown [49]

10 7 Biopterin transporter? [50]

11 11f GPI-anchored glycoprotein? [51]

12 5 Unknown [17]

SRA 0g Resistance to human serum [28]bSix BESs contain only ESAG3 pseudogenes.aFrom a total repertoire predicted to comprise 14 BESs.cTwo BESs contain only ESAG4 pseudogenes.dTwo BESs contain only ESAG5 pseudogenes.eFound downstream of a duplicated BES promoter.fOnly ESAG11 pseudogenes detected.gOnly described in T. b. rhodesiense.

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BES structural conservation also appears to be true in T. b.rhodesiense, at least for the small number of BESs charac-terized [27,28], although we cannot yet say whether moredistantly related trypanosomes differ. What do thesesequence data tell us?

Sequence variability suggests that recombinationwithin the BESs occurs frequently. On one level, this isnot surprising because the BESs are the recipients of novelVSGs during recombination-driven antigenic variation.Although some of these reactions localize to the telomericVSG sequence and involve the 70 bp repeats that flankmost VSGs [29], other reactions use silent BESs assequence donors and might affect ESAG sequence organ-isation [30–32]. Indeed, instances of long-range gene con-versions, complex recombination reactions using multipleBESs as substrates and examples of BES sequence losshave been seen [17,33]. Beyond this VSG-focused recom-bination and/or rearrangement, evidence for recombina-tion of ESAGs independently of the VSGs is inferred bymapping of recombination breakpoints through sequencecomparisons [17,18] and has been observed experimentally[34]. This could be consistent with the ESAGs being underselective pressure (see below). The abundance of VSG andESAG pseudogenes in the BESs might also be instructive.Most VSGs outside of the BESs are pseudogenes [4], andthese are crucial in antigenic variation, enabling the gener-ation of novel ‘mosaic’ VSGs through segmental gene con-versions, thereby enhancing parasite transmission [29,35].One hypothesis that has been suggested to explain whytrypanosomes possess multiple BESs is that the silentBESs provide locations in which the novel mosaic VSGsare assembled [8], meaning theVSG pseudogenesmight beassembly intermediates. However, it is striking that eachsequenced BES had a functional VSG adjacent to thetelomere and the VSG pseudogenes were always moredistal to the chromosome end and flanked by 70 bp repeats.This suggests a selective pressure to maintain intact VSGsat the telomeres of the BESs, presumably through frequentactivation, and that VSG pseudogenes are frequently car-ried into the BESs in gene conversion reactions from the

silent VSG arrays that target the activation of down-stream, intact VSGs. Of course, if this interpretation iscorrect, the question remains: where does VSG mosaicassembly occur? The presence ofESAG pseudogenesmightbe due to genetic redundancy, which might be exacerbatedrelative to the VSGs because many ESAGs are part ofconstitutively transcribed gene families with copies in thehousekeeping core of the genome [36]. However, this doesnot exclude the untested, interesting suggestion thatESAG pseudogenes, like VSG pseudogenes, provide agenetic resource for recombination-driven sequencechange [17]. The near-invariant positional conservationof ESAG3 and ESAG11 pseudogenes in most BESs isparticularly striking [17,27] and might be related to thishypothesis. Testing this suggestion will be complicated,however, given the current sketchy understanding ofESAG functions (Table 1).

Do these data reveal why ESAGs are located withinBESs? Perhaps surprisingly, several BESs that have lostnumerous ESAGs through truncation seem to support T.brucei growth in vitro [17] and in vivo [28] when activelytranscribed. This might indicate that most ESAGs aredispensable and could provide an indication of theminimalESAG complement of a BES, but such a conclusionmust betempered by observations that transcription can occurfrom ‘silent’ BESs in some settings [37]. One hypothesis,proposed by Bitter et al. [38], has sought to explain the co-expression of ESAGs with VSG and provides anotherexplanation for the existence of multiple BESs. This workexamined ESAG6 and ESAG7, promoter-proximal genesthat together encode the trypanosome receptor for hosttransferrin, providing a source of iron for the parasite. Itwas proposed that sequence differences in ESAG6 andESAG7 genes in the BESs provide receptor variants thatenable T. brucei to bind the different transferrin moleculesfound in the range of host mammals the parasite infects.BES transcriptional switching, therefore, would allow T.brucei to express the highest affinity receptor for hosttransferrin, enabling efficient uptake in the presence ofhost antibodies against the transferrin receptor [38,39].

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This hypothesis has been extended by other workers tosuggest that host-adaptive functions might be provided byother ESAGs [36]. However, although a host-specific rolehas been demonstrated for one ESAG, the human serum-resistance-associated gene (SRA), the ‘host-range’ hypoth-esis has remained controversial. Notably, SRA is unlikemost other ESAGs in that it is limited to only some BESsand only in T. b. rhodesiense [28]. Moreover, for ESAG6and ESAG7, it has been suggested that transferrin con-centrations in vivo are sufficiently high to allow sufficientiron uptake, even when the trypanosome expresses a BESwith a low-affinity transferrin receptor [40], and that anti-transferrin receptor antibodies are generated to only alimited and transient extent in vivo [41]. Indeed, somework has argued that T. brucei growth differences indifferent host serum, including the selection for parasitesthat have switched BES transcription, are not simply dueto transferrin uptake efficiency but might involve otherESAGs [42].

Young et al. [18] now provide a further test of the hostrange hypotheses by asking whether there is a correlationbetween ESAG sequence variation and host range for T. b.brucei, T. b. gambiense and T. equiperdum. The authorslooked not only at ESAG6 (transferrin receptor) but also atESAG5 (possible role in lipid transfer and/or lipopolysac-charide binding) [43] and ESAG2 (function unknown). Nocorrelation between BES number and host range wasfound: even in T. equiperdum, which has a very limitedhost range, multiple BESs were present. Similarly, ESAGdiversity and host range did not seem to correlate:sequence variation of the three ESAGs was actuallygreater in T. equiperdum than in T. b. gambiense. More-over, evidence could be found for adaptive evolutionarypressures having shaped the sequences of each ESAG in T.equiperdum, whereas only ESAG2 in T. b. gambiense dis-played similar signs. At face value, these data seem tosuggest the BES repertoire has not evolved to provide arange of ESAGs, including ESAG6 and ESAG7, whichallow T. brucei to match these gene products to differingfacets of the host mammals the parasites infect. However,any predicted correlation might be confounded by a num-ber of factors, such as differing rates of recombination(including genetic exchange during mating) and the evol-utionary timing of trypanosome species and subspeciesemergence. It is also complicated by our lack of functionalcharacterization of ESAG5 and ESAG2, most notablywhether they act at the host–parasite interface (forexample by being cell surface proteins, like ESAG6).

Concluding remarksOur understanding of the biology underlying the ESAG–

VSG association at telomeres remains incomplete, but thenew sequence data represent an important step in the questto understand the roles of theESAGs and their interactionswith mammalian hosts. Not least, these data will facilitatedirected experimentalmanipulation of theBESsand amoredetailed analysis of ES rearrangements. Several studies areunder way to explore recombination, repair and switchingamong BESs. Conditional meganuclease expression nowallows DNA double-strand breaks to be introduced at adesired location [44,45] and might inform us of the mech-

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anisms that underlie antigenic variation, as well as facil-itating functional and genetic dissection of the machineryinvolved. Ultimately, such experiments might provideanswers to the questions raised above.

AcknowledgementsThe number of references cited in this article is limited, and we apologizeto those people whose important contributions have not been identified.R.M.’s laboratory is supported by grants from the Wellcome Trust andMedical Research Council; D.H.’s laboratory is supported by grants fromthe Wellcome Trust.

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