Jeremy Sternberg
8.3 Variant surface glycoprotein – the key to trypanosome-host interactions
One parasite molecule, the variant surface glycoprotein (VSG), dominates the interactions of trypanosomes with their mammalian hosts and defines the im- munology of infection (Figure 8.2). About 5 × 106 glycosylphosphatidyl in- ositol (GPI)-anchored VSG molecules coat the surface of the cell, comprising more than 95 per cent of all surface membrane proteins and some 15 per cent
GIP-VSG
DMG
C-terminal domain N-terminal domain
GPI Anchor
CH2-CH-CH2
[GPI-PLC Cleavage]
Asp4B7
VSG
NH-CH2-CH2-0 C=0
0=P=0-
6MANα1-2MANα1-6MANα1-4GlcNH2α1-6Myo-Inositol1 αGal2-4
0 0=P=0-
0 0=C
CH3 (CH2)13 (CH2)13
0 C=0
CH3
Figure 8.2 Variant surface glycoprotein and the GPI anchor.The upper panel represents VSG polypeptide dimers (shown as space filling models) and the GPI anchor, the acyl groups of which are inserted in the lipid bilayer membrane. The lower panel shows the chemical structure of the anchor and the GPI-PLC cleavage point Adapted from Paulnock, DM & Coller, SP (2001). Analysis of macrophage activation in African trypanosomiasis.Journal of Leukocyte Biology69, 685–690; and Field, MC and Carrington, M (2004). Intracellular membrane transport systems inTrypanosoma brucei.Traffic5, 905–913.
of total cellular protein – a clear indication of the biological importance of this molecule to the trypanosome. In order to enable antigenic variation, VSG genes represent 20 per cent of the protein-coding capacity of the trypanosome genome, and they are highly divergent in primary sequence, while retaining similar tertiary structure resulting from conserved cysteine residues. It is this sequence, and therefore epitopic diversity, that underpins the role of VSG in antigenic variation.
VSG molecules and their GPI anchor components also mediate immunopathol- ogy in trypanosomiasis, and a truncated VSG gene product (termed Serum Re- sistance Associated (SRA)) gene plays a critical role in defence against innate immunity. The VSG is a 400–500 amino acid protein linked via its carboxyl ter- minus to a GPI anchor. The N terminal domain, most of which is exposed exter- nally, forms the majority of the protein, and this presents a uniform molecular coat surrounding all exposed areas of plasma membrane of the parasite. The VSG coat is a dynamic structure in which all VSG molecules are cycled through the active endocytic system of the trypanosomes every 12 minutes.
VSG is a highly immunogenic protein, eliciting a strong T cell independent IgM response that yields cross-linking and opsonising antibodies that are more than sufficient to eliminate the infection. However, the membrane cycling of VSG actively removes bound IgG and IgM and slows the accumulation of potentially trypanocidal levels of immunoglobulin on the cell surface.
As indicated in Figure 8.2, the GPI anchor of the VSG is cleaved by glycosyl- phosphatidylinositol-specific phospholipase-C (GPI-PLC) in African trypano- somes, releasing a soluble form of VSG carrying the glycosylinositolphosphate residue of the GPI anchor (GIP-VSG), with the dimyristoylglycerol (DMG) com- ponent remaining associated with the membrane. Current research suggests that this enzyme is located on the exterior of the flagellar membrane, but con- tact with the GPI anchors of VSG molecules must be tightly regulated, as GIP- VSG release from trypanosomes only takes place in stressed cells.
8.3.1 VSG and antigenic variation
In 1910, Ross and Thomson conducted the first study on the development of sleeping sickness in a human host, during the course of the unsuccessful treat- ment of a patient who had been infected with trypanosomes in modern-day Zambia. It was noted that the parasite numbers in the blood fluctuated, with waves of parasitaemia followed by periods when parasites were undetectable (Figure 8.3). Remarkably, given that the biological basis of antigenicity and the action of antibodies were unknown at that time, these early pioneers had the prescience to propose that the undulating course of parasitaemia resulted from antigenic variation. Later, once the biology of the immune system was under- stood, the role of antigenic variation in defining the course of trypanosome parasitaemia in the infected mammalian host was confirmed using serological and, subsequently, molecular biological methods.
VSG molecules are immunogenic and elicit high-titre lytic IgM responses which would be expected to effectively opsonise trypanosomes. However,
1600 1400 1200 1000 800 600 400 200 0
Day of Infection Parasitaemia (/mm3)
130 140 150 160 170
100 110 120
Figure 8.3 Typical parasitaemia profile in human African trypanosomiasis.Each wave of
parasitaemia is the result of antigenic variation, with one or more novel VATs being expressed.
trypanosome infections persist because a small number of cells in the popu- lation undergo antigenic variation to express a new VSG coat that is not anti- genically cross-reactive with its predecessors.
It is important to note that antigenic variation is a stochastic process, occur- ring independently of any immunological challenge to the parasite, at a rate of between 10−2 and 10−6 per cell division for any individual member of the trypanosome population in an infected host. Each immunologically distinct VSG (and its set of epitopes) is known as a Variant Antigen Type (VAT). The full complement of VSGs that may be expressed in a trypanosome clone, typically in excess of 1,600, is known as the VAT Repertoire. VAT repertoires are subject to rapid evolution, and trypanosome clones isolated from geographically close areas may have quite different repertoires.
8.3.1.1 The mechanism of antigenic variation
The mechanism of antigenic variation in African trypanosomes has been the subject of intense and detailed research, and our understanding of the pro- cess at the molecular level is summarised in Figure 8.4. Essentially, one gene is selected for expression from a repertoire of non-expressed ‘basic copy’ genes (that act as a ‘library’ of VATs). A critical feature of this system is that VSG expres- sion is mono-allelic, so only a single VAT may be expressed at any one time; it is thought that mixed VAT expression would disrupt the surface coat structure.
The basic copy genes are distributed with up to 1,500 copies in large tan- dem arrays of VSG genes on large chromosomes and a further 200 copies at sub-telomeric sites on minichromosomes (Figure 8.4a). Of the basic copy genes in arrays in large chromosomes, a substantial proportion do not code for
VSGe VSGx (B)
(C)
(D)
(E)
Gene conversion
Segmental gene conversion
Transcriponal Switching
Telomere Exchange Sub-telomeric Arrays of VSG genes and pseudogenes (ψ), n =1500
VSGa VSGb
VSGc VSGd
VSGz VSGx
Expression site associated genes
Bloodstream VSG Expression Site n =14-23
= acve promoter
= repeve DNA
= polycistronic RNA transcript (A)
VSGe
Minichromosomal telomeres VSG n =200
VSGf
VSGmosaic VSGc
VSGx
VSGz
ψ ψ ψ
ψ ψ ψ
Figure 8.4 Antigenic variation mechanisms inT. brucei.a: Genetic organisation of variant surface glycoprotein (VSG) genes in trypanosome expressing hypothetical VSGx in active expression site. Basic copy genes are in arrays (both coding sequences, pseudogene and gene fragments) on large chromosomes, at inactive expression sites and at the telomeres of minichromosomes. b and c: Gene conversion leads to expression of intact or segmental basic copy genes.
d: Transcriptional switching of expression sites. e: Reciprocal recombination, leading to exchange of telomeric VSG.
functional VSGs but, rather, exist as gene fragments or pseudogenes that re- quire segmental gene conversion to enable expression.
Although one might expect that the requirement for mono-allelic VSG expres- sion could be satisfied with a single expression site, there are in fact are least 14 bloodstream-stage VSG expression sites and up to a further 25 metacyclic-stage VSG expression sites per diploid genome. These expression sites are under tight control, with only one actively expressed at any one time and, unusually, VSG gene transcription is carried out by RNA polymerase I. The control of which VSG gene expression site is active is at the level of transcription elongation, and it also appears to be connected to a specialised region of chromatin in the try- panosome nucleus, known as the expression site body.
Antigenic variation thus involves either the activation of alternative VSG ex- pression sites (Figure 8.4d), or the switching of a new basic copy gene into an expression site. The latter process primarily involves a duplicative transposi- tion (or gene conversion), although reciprocal telomere recombination may also take place (Figure 8.4e). Duplicative transposition may involve intact basic copy VSG genes (Figure 8.4b) or may involve the reassembly of VSG gene and VSG pseudogene fragments to produce novel chimaeric VSGs (Figure 8.4c); this is thought to be important late in chronic infections as the basic copy repertoire becomes exhausted.
8.3.1.2 Expression site associated genes
The VSG expression sites of bloodstream form trypanosomes are not simply vehicles for VSG gene expression but, rather, are part of a much larger tran- scription unit with the promoter far (45-50kB) upstream from the VSG coding sequence. Within this large transcription unit are a series of genes and pseu- dogenes presumed to be derived from ancestral VSG gene duplication events.
These genes are known as Expression Site Associated genes (ESAGS), and they contribute to a polycistronic transcription unit that includes the cognate VSG gene. ESAGs include the heterodimeric transferrin receptor and the Serum Re- sistance Associated (SRA) gene specifically found inT. b. rhodesienseand dis- cussed further below.
The transferrin receptor, as with other eukaryotic and prokaryotic pathogens, scavenges iron-transferrin complexes from the host plasma and may provide a clue to the reason why trypanosomes utilise multiple expression sites. It has been suggested that ESAG gene polymorphisms between different expression sites allow trypanosomes to ‘select’ transferrin receptors optimal for capture of the transferrin variants in each of their natural hosts, and this may be an adaptive explanation for the existence of multiple VSG expression sites in try- panosomes.