Antigenic variation in trypansosomes: Secrets surface slowly

Among pathogenic micro-organisms that evade the mammalian immune responses, Trypanosoma brucei has developed the most elaborate capacity for antigenic variation. Trypanosomes branched early during eukaryotic evolution. They are characterized by many aberrations, ranging from the unusual compartmentation of metabolic pathways to the heresy of RNA editing. The ubiquitous phenomenon of glycosylphosphatidylinositol-anchoring of eukaryotic plasma membrane proteins and RNA trans-splicing (trypanosome genes contain no introns), which adds an identical leader sequence to all trypanosome mRNAs, were first defined during studies of antigenic variation. Genetic transformation of trypanosomes and the high efficiency of gene targeting provide new opportunities to investigate the regulation of antigenic variation. There is every reason to expect trypanosomes to provide further surprises and insights into the evolution of genetic regulatory mechanisms.     

The GPI-Phospholipase C of Trypanosoma brucei Is Nonessential But Influences Parasitemia in Mice

In the mammalian host, the cell surface of Trypanosoma brucei is protected by a variant surface glycoprotein that is anchored in the plasma membrane through covalent attachment of the COOH terminus to a glycosylphosphatidylinositol. The trypanosome also contains a phospholipase C (GPI-PLC) that cleaves this anchor and could thus potentially enable the trypanosome to shed the surface coat of VSG. Indeed, release of the surface VSG can be observed within a few minutes on lysis of trypanosomes in vitro. To investigate whether the ability to cleave the membrane anchor of the VSG is an essential function of the enzyme in vivo, a GPI-PLC null mutant trypanosome has been generated by targeted gene deletion. The mutant trypanosomes are fully viable; they can go through an entire life cycle and maintain a persistent infection in mice. Thus the GPI-PLC is not an essential activity and is not necessary for antigenic variation. However, mice infected with the mutant trypanosomes have a reduced parasitemia and survive longer than those infected with control trypanosomes. This phenotype is partially alleviated when the null mutant is modified to express low levels of GPI-PLC.     

Trypanosoma brucei: peptide mapping of partially homologous variable surface glycoproteins.

Antigenic variation in Trypanosoma brucei is caused by amino acid sequence changes in a major surface glycoprotein. Each trypanosome may contain between 100 and 1000 genes coding for this glycoprotein. Some of these genes are members of partially homologous gene families. In addition, segments of different genes may combine to form 'hybrid' or 'mosaic' genes. Thus, surface glycoproteins exist containing varying amounts of amino acid sequence homology. For investigations of molecular mechanisms of antigenic diversity it is important to identify sub-sets of trypanosomes expressing related surface glycoproteins. We describe here a simple method based on trypanosome surface labeling followed by peptide mapping to indicate homologous peptides present in one sub-set of T. brucei surface glycoproteins.     

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.     

Mapping of segmental antigenic determinants on structurally related variant surface glycoproteins of Trypanosoma brucei

To study common and variant specific antigenic determinants on variant surface glycoproteins from Trypanosoma brucei, we have selected four serologically cross-reacting variant populations. Monoclonal antibodies were raised against the purified variant surface glycoproteins from each variant trypanosome population. Six monoclonal antibodies bind to segmental epitopes and one binds to a topographically assembled epitope. Amino acid compositions of these variant surface glycoproteins reveal striking conservation of certain residues including cysteine and charged amino acids. We also find that all seven monoclonal antibodies used in this study bind to protein determinants not exposed on the surface of the living trypanosome. Only one monoclonal antibody exhibits homologous specificity, while the remainder display cross-reactivity for three or all four variant surface glycoproteins. In addition, polyacrylamide gel electrophoresis peptide mapping and Western blots probed with each monoclonal antibody reveal significant peptide homologies. Furthermore, two pairs of monoclonal antibodies recognize two epitopes that are possibly immunodominant. The significance of these findings is discussed in terms of the structural similarities and differences among variant surface glycoproteins.     

The GPI biosynthetic pathway as a therapeutic target for African sleeping sickness

African sleeping sickness is a debilitating and often fatal disease caused by tsetse fly transmitted African trypanosomes. These extracellular protozoan parasites survive in the human bloodstream by virtue of a dense cell surface coat made of variant surface glycoprotein. The parasites have a repertoire of several hundred immunologically distinct variant surface glycoproteins and they evade the host immune response by antigenic variation. All variant surface glycoproteins are anchored to the plasma membrane via glycosylphosphatidylinositol membrane anchors and compounds that inhibit the assembly or transfer of these anchors could have trypanocidal potential. This article compares glycosylphosphatidylinositol biosynthesis in African trypanosomes and mammalian cells and identifies several steps that could be targets for the development of parasite-specific therapeutic agents.     

Purification and characterization of a novel glycan-phosphatidylinositol-specific phospholipase C from Trypanosoma brucei

A novel membrane-bound glycan-phosphatidylinositol-specific phospholipase C, which catalyzes the conversion of membrane form variant surface glycoproteins to soluble variant surface glycoproteins, with the release of sn-1,2-dimyristylglycerol, has been isolated from Trypanosoma brucei. The activity was solubilized from trypanosome membrane fractions in non-ionic detergent and purified by anion exchange chromatography on DEAE-cellulose followed by chromatography on phosphatidylinositol-Sepharose. The enzyme constitutes about 0.1% of the total cellular protein and has an apparent molecular weight of 39,800. The enzyme shows a head group specificity for molecules containing carbohydrate covalently linked to glycan-phosphatidylinositol, but can also act on the monoacyl derivative of membrane form variant surface glycoprotein. It shows no specific ion requirements but is stimulated by thiol-reducing agents and inhibited by ions that thiols chelate.     

The surface structure of trypanosomes in relation to their molecular phylogeny

Molecular phylogenetic analysis using genes coding for ribosomal RNA and proteins suggests that trypanosomes are monophyletic. Salivarian trypanosomes showing antigenic variation of the variant surface glycoprotein (VSG) diverged from non-Salivarian trypanosomes some 200-300 million years ago. Representatives of the non-Salivarian group, the mammalian parasite, Trypanosoma cruzi, and the fresh-water fish trypanosome, T. carassii, are characterised by surfaces dominated by carbohydrate-rich mucin-like glycoproteins, which are not subject to antigenetic variation. It is suggested that this latter surface structure is typical for non-Salivarian trypanosomes as well as members of the other Kinetoplastid suborder, the Bodonina. This would imply that at some point in time in the evolution of the Salivaria the highly abundant and comparatively poorly immunogenetic mucin-like molecules must have been replaced for equally abundant but highly immunogenic VSG-like molecules. While the selective advantage for such a unique transition is difficult to imagine, the subsequent diversification of VSG genes/molecules may have been comparatively straightforward because even the most limited form of antigenic variation would have extended the duration of infection in the vertebrate and thus would have increased the chance for transfer to the vector.     

Trypanosome Glycosylphosphatidylinositol Biosynthesis

Trypanosoma brucei, a protozoan parasite, causes sleeping sickness in humans and Nagana disease in domestic animals in central Africa. The trypanosome surface is extensively covered by glycosylphosphatidylinositol (GPI)-anchored proteins known as variant surface glycoproteins and procyclins. GPI anchoring is suggested to be important for trypanosome survival and establishment of infection. Trypanosomes are not only pathogenically important, but also constitute a useful model for elucidating the GPI biosynthesis pathway. This review focuses on the trypanosome GPI biosynthesis pathway. Studies on GPI that will be described indicate the potential for the design of drugs that specifically inhibit trypanosome GPI biosynthesis.     

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.     

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.     

Rapid lateral diffusion of the variant surface glycoprotein in the coat of Trypanosoma brucei

The membrane form of the variant surface glycoprotein (mfVSG) is anchored in the plasma membrane of Trypanosoma brucei by a dimyristoylphosphatidylinositol residue connected via a glycan to the COOH-terminal amino acid. The glycoprotein molecules are tightly packed, forming a coat that is impenetrable to lytic serum components. Lateral diffusion of mfVSG was measured by the fluorescence recovery after photobleaching technique. mfVSG labeled on the cell surface with rhodamine-conjugated anti-VSG Fab fragments showed a diffusion coefficient of 1 X 10(-10) cm2/s at 37 degrees C and of 0.7 X 10(-10) cm2/s at 27 degrees C. About 80% of the molecules were mobile. Affinity-purified mfVSG molecules implanted into the plasma membrane of baby hamster kidney cells exhibited a similar mobility to that found in the trypanosome coat [D = (0.4-0.7) X 10(-10) cm2/s at 4 degrees C]. Phospholipid mobility in the plasma membrane of trypanosomes was characterized by a diffusion coefficient of 2.2 X 10(-9) cm2/s at 37 degrees C. It is concluded that mfVSG mobility in the surface coat of the parasite is rapid and comparable to that of other membrane-bound glycoproteins but slower than that of phospholipids.     

The glycoforms of a Trypanosoma brucei variant surface glycoprotein and molecular modeling of a glycosylated surface coat

The plasma membrane of the African sleeping sickness parasite Trypanosoma brucei is covered with a dense, protective surface coat. This surface coat is a monolayer of five million variant surface glycoprotein (VSG) dimers that form a macromolecular diffusion barrier. The surface coat protects the parasite from the innate immune system and, through antigenic variation, the specific host immune response. There are several hundred VSG genes per parasite, and they encode glycoproteins that vary in primary amino acid sequence, the number of N-glycosylation sites, and the types of N-linked oligosaccharides and glycosylphosphatidylinositol membrane anchors they contain. In this study, we show that VSG MITat.1.5 is glycosylated at all three potential N-glycosylation sites, and we assign the oligosaccharides present at each site. Using the most abundant oligosaccharides at each site, we construct a molecular model of the glycoprotein to assess the role of N-linked oligosaccharides in the architecture of the surface coat.     

GPI-anchored proteins and glycoconjugates segregate into lipid rafts in Kinetoplastida

The plasma membranes of the divergent eukaryotic parasites, Leishmania and Trypanosoma, are highly specialised, with a thick coat of glycoconjugates and glycoproteins playing a central role in virulence. Unusually, the majority of these surface macro-molecules are attached to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. In mammalian cells and yeast, many GPI-anchored molecules associate with sphingolipid and cholesterol-rich detergent-resistant membranes, known as lipid rafts. Here we show that GPI-anchored parasite macro-molecules (but not the dual acylated Leishmania surface protein (hydrophilic acylated surface protein) or a subset of the GPI-anchored glycoinositol phospholipid glycolipids) are enriched in a sphingolipid/sterol-rich fraction resistant to cold detergent extraction. This observation is consistent with the presence of functional lipid rafts in these ancient, highly polarised organisms.     

Limitation of Trypanosoma brucei parasitaemia results from density-dependent parasite differentiation and parasite killing by the host immune response.

In the bloodstream of its mammalian host, the "slender" form of Trypanosoma brucei replicates extracellularly, producing a parasitaemia. At high density, the level of parasitaemia is limited at a sublethal level by differentiation to the non-replicative "stumpy" form and by the host immune response. Here, we derive continuous time equations to model the time-course, cell types and level of trypanosome parasitaemia, and compare the best fits with experimental data. The best fits that were obtained favour a model in which both density-dependent trypanosome differentiation and host immune response have a role in limiting the increase of parasites, much poorer fits being obtained when differentiation and immune response are considered independently of one another. Best fits also favour a model in which the slender-to-stumpy differentiation progresses in a manner that is essentially independent of the cell cycle. Finally, these models also make the prediction that the density-dependent trypanosome differentiation mechanism can give rise to oscillations in parasitaemia level. These oscillations are independent of the immune system and are not due to antigenic variation.     

The taxonomic position and evolutionary relationships of Trypanosoma rangeli.

This paper presents a re-evaluation of the taxonomic position and evolutionary relationships of Trypanosoma (Herpetosoma) rangeli based on the phylogenetic analysis of ssrRNA sequences of 64 Trypanosoma species and comparison of mini-exon sequences. All five isolates of T. rangeli grouped together in a clade containing Trypanosoma (Schizotrypanum) cruzi and a range of closely related trypanosome species from bats [Trypanosoma (Schizotrypanum) dionisii, Trypanosoma (Schizotrypanum) vespertilionis] and other South American mammals [Trypanosoma (Herpetosoma) leeuwenhoeki, Trypanosoma (Megatrypanum) minasense, Trypanosoma (Megatrypanum) conorhini] and an as yet unidentified species of trypanosome from an Australian kangaroo. Significantly T. rangeli failed to group with (a) species of subgenus Herpetosoma, other than those which are probably synonyms of T. rangeli, or (b) species transmitted via the salivarian route, although either of these outcomes would have been more consistent with the current taxonomic and biological status of T. rangeli. We propose that use of the names Herpetosoma and Megatrypanum should be discontinued, since these subgenera are clearly polyphyletic and lack evolutionary and taxonomic relevance. We hypothesise that T. rangeli and T. cruzi represent a group of mammalian trypanosomes which completed their early evolution and diversification in South America.