Targeting the variable surface of African trypanosomes with variant surface glycoprotein-specific,serum-stable RNA aptamers

African trypanosomes cause sleeping sickness in humans and Nagana in cattle. The parasites multiply in the blood and escape the immune response of the infected host by antigenic variation. Antigenic variation is characterized by a periodic change of the parasite protein surface, which consists of a variant glycoprotein known as variant surface glycoprotein (VSG). Using a SELEX (systematic evolution of ligands by exponential enrichment) approach, we report the selection of small, serum-stable RNAs, so-called aptamers, that bind to VSGs with subnanomolar affinity. The RNAs are able to recognize different VSG variants and bind to the surface of live trypanosomes. Aptamers tethered to an antigenic side group are capable of directing antibodies to the surface of the parasite in vitro. In this manner, the RNAs might provide a new strategy for a therapeutic intervention to fight sleeping sickness.     

Glycoproteins of trypanosomes: their biosynthesis and biological significance

1. Trypanosomes are unicellular parasites that cause human sleeping sickness in Africa and Chagas' disease in South America. Glycoproteins are important components of their plasma membrane. 2. The bloodstream form of the extracellular salivarian African trypanosome (e.g. Trypanosoma brucei) has the ability to express on its cell surface a repertoire of variant surface glycoproteins (VSGs) and in so doing, evades the immune response of the host (antigenic variation). 3. The VSG is probably synthesized initially in a manner like that of the membrane-bound glycoproteins of mammalian systems, but it also undergoes some novel post-translational modifications. 4. The stercorarian South American trypanosome (Trypanosoma cruzi) is an intracellular parasite which expresses different glycoproteins on its plasma membrane at various stages of its life-cycle, but does not exhibit antigenic variation. 5. The biosynthesis and functions of trypanosomal glycoproteins are compared with those of mammalian glycoproteins, and are discussed with particular reference to potential targets for chemotherapy and immunotherapy of trypanosomiasis.     

Identification of a tryptophan-like epitope borne by the variable surface glycoprotein (VSG) of African trypanosomes

Antibodies (Ab) directed against a tryptophan-like epitope (WE) were previously detected in patients with human African trypanosomiasis (HAT). We investigated whether or not these Ab resulted from immunization against trypanosome antigen(s) expressing a WE. By Western blotting, we identified an antigen having an apparent molecular weight ranging from 60 to 65 kDa, recognized by purified rabbit anti-WE Ab. This antigen, present in trypomastigote forms, was absent in procyclic forms and Trypanosoma cruzi trypomastigotes. Using purified variable surface glycoproteins (VSG) from various trypanosomes, we showed that VSG was the parasite antigen recognized by these rabbit Ab. Anti-WE and anti-VSG Ab were purified from HAT sera by affinity chromatography. Immunoreactivity of purified antibodies eluted from affinity columns and of depleted fractions showed that WE was one of the epitopes borne by VSG. These data underline the existence of an invariant WE in the structure of VSG from several species of African trypanosomes.     

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.     

Genetics of resistance to the African trypanosomes. VII. Trypanosome virulence is not linked to variable surface glycoprotein expression

The question of linkage of virulence traits to variable surface glycoprotein (VSG) expression in African trypanosomiasis was addressed. Previously we demonstrated that daughter cells arising in mice infected with a genetically homogeneous trypanosome population of Trypanosoma brucei rhodesiense were more virulent than the infecting population (J. A. Inverso and J. M. Mansfield, J. Immunol. 130:412, 1983). These virulent trypanosomes expressed differences in surface phenotype compared with the infecting variant types, and we proposed that virulence may be "linked" to VSG expression. In the present study, however, we have shown that expression of virulence is independent of the VSG phenotype displayed by trypanosome populations. A VSG-identical but highly virulent subpopulation of T. b. rhodesiense LouTat 1 was derived by rapid subpassage and subcloning in immunosuppressed mice. The virulent LouTat 1A subclone derived in this manner killed B10.BR/SgSnJ mice in 3 to 4 days postinfection compared with approximately 60 days for the parent clone, LouTat 1. The virulent subclone LouTat 1A appears to express the same VSG as the less virulent LouTat 1 population, as determined by polyspecific and monoclonal antibody-binding assays, cross-protection tests, and amino acid sequence analyses of the N-terminal portion of the VSG molecules. When LouTat 1 and subclone LouTat 1A were injected into a heterologous host species, multiple variant antigenic types (VATs) arising from each inoculum were isolated and characterized. VATs derived from the virulent subclone were as uniformly virulent for B10.BR mice as LouTat 1A. In summary, these results demonstrate that trypanosome virulence, once expressed, is a stable phenotype that does not seem to be associated with a particular VSG phenotype, nor does virulence change with the expression of different VSG genes.     

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.     

The PARP and rRNA promoters of Trypanosoma brucei are composed of dissimilar sequence elements that are functionally interchangeable.

The African trypanosomes express two major surface proteins, the variant surface glycoprotein (VSG) and the procyclic acidic repetitive protein (PARP). The RNA polymerase that transcribes the VSG and PARP genes shares many characteristics with RNA polymerase I. We show that although there is very little similarity in nucleotide sequence, the functional structure of a trypanosome rRNA promoter is almost identical to that of the PARP promoter. Further, domains from the PARP promoter can functionally substitute for the corresponding parts of the rRNA promoter, and vice versa.     

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.     

The dynamics of antigenic variation and growth of African trypanosomes

Antigenic variation in African trypanosomes, which is a simple strategy for survival in the immune host, is rendered complex by its magnitude. For protection from nonspecific immunity and escape from specific immunity, each trypanosome is covered by a replaceable surface coat composed of the variant surface glycoprotein (VSG), which specifies the variable antigen type (VAT) of the trypanosome. Antigenic variation is the process by which the trypanosome switches from one coat to another. Here, David Barry and Michael Turner consider this phenomenon within the context of the course of trypanosome infection.     

Exposed epitopes on a Trypanosoma equiperdum variant surface glycoprotein altered by point mutations.

African trypanosomes are covered by a dense protein layer that is immunologically distinct on different trypanosome isolates and is termed the variant surface glycoprotein (VSG). The different VSGs are expressed in a general order, where some VSGs appear preferentially early in infection and others only later. The exposed epitopes on a late antigen, VSG 78, of T.equiperdum were studied by the technique of monoclonal antibody (MAb) escape selection. MAbs that neutralize trypanosomes bearing VSG 78 reacted with the VSG only when it was attached to the trypanosome surface, suggesting that the most immunogenic surface epitopes are conformational. Trypanosome clones resistant to one of the MAbs yet still expressing VSG 78 or 78(20) were isolated in vitro. Two independent variants resistant to MAb H3 changed Ser192 to Arg by a single base change in the VSG gene and a variant resistant to MAb H21 had a single base change that converted Gln172 to Glu. A variant resistant to MAb H7 had several changes in the VSG gene, a gene conversion in the 5' region and an isolated mutation in codon 220 that is proposed to be responsible for the resistance phenotype. The isotypic bias of the MAbs against VSG 78 and an analysis of the natural variants that are resistant to MAb 78H21 suggest that glycosylation plays a role in the immunogenicity of these proteins. The analysis defines some of the exposed amino acid residues and demonstrates that VSG genes are altered by mutations and small gene conversions as well as replaced by large gene conversion-like events. The results provide biological data supporting the model of VSG structure obtained by crystallographic studies.     

Expression and function of the Trypanosoma brucei major surface protease (GP63) genes

The genome of the African trypanosome Trypanosoma brucei (Tb) contains at least three gene families (TbMSP-A, -B, and -C) encoding homologues of the abundant major surface protease (MSP, previously called GP63), which is found in all Leishmania species. TbMSP-B mRNA occurs in both procyclic and bloodstream trypanosomes, whereas TbMSP-A and -C mRNAs are detected only in bloodstream organisms. RNA interference (RNAi)-mediated gene silencing was used to investigate the function of TbMSP-B protein. RNAi directed against TbMSP-B but not TbMSP-A ablated the steady state TbMSP-B mRNA levels in both procyclic and bloodstream cells but had no effect on the kinetics of cultured trypanosome growth in either stage. Procyclic trypanosomes have been shown previously to have an uncharacterized cell surface metalloprotease activity that can release ectopically expressed surface proteins. To determine whether TbMSP-B is responsible for this release, transgenic variant surface glycoprotein 117 (VSG117) was expressed constitutively in T. brucei procyclic TbMSP-RNAi cell lines, and the amount of surface VSG117 was determined using a surface biotinylation assay. Ablation of TbMSP-B but not TbMSP-A mRNA resulted in a marked decrease in VSG release with a concomitant increase in steady state cell-associated VSG117, indicating that TbMSP-B mediates the surface protease activity of procyclic trypanosomes. This finding is consistent with previous pharmacological studies showing that peptidomimetic collagenase inhibitors block release of transgenic VSG from procyclic trypanosomes and are toxic for bloodstream but not procyclic organisms.     

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.     

Independent regulation of B cell responses to surface and subsurface epitopes of African trypanosome variable surface glycoproteins

Regulation of B cell responses to the trypanosome surface Ag was examined in H-2k compatible "responder" B10.BR and "nonresponder" C3H mice after infection with two variant clones of Trypanosoma brucei rhodesiense. Development of a selective RIA for independent detection of antibody binding to surface (exposed) and subsurface (buried) epitopes of the trypanosome variable surface glycoprotein (VSG) molecule permitted sensitive quantitation and kinetic characterization of immune responses to these epitopes. The infected B10.BR mice responded to both exposed and buried VSG epitopes of clone LouTat 1 trypanosomes, whereas a B cell response by C3H mice to exposed VSG epitopes was not detected by RIA analyses at any time. However, VSG-specific IgM and IgG responses were produced to buried VSG epitopes, demonstrating that LouTat 1 induced immunoregulation was specific only for the B cell responses to exposed VSG epitopes. Subsequently, comparisons of B10.BR and C3H B cell responses to a heterologous variant, LouTat 1.5, were made. The results revealed that both infected mouse strains produced VSG 1.5-specific antibody to exposed and buried epitopes with different kinetics and maximal sera concentrations, showing, therefore, that these responses are not coordinately regulated. In addition, it was clear that the observed immunosuppression to exposed LouTat 1 VSG epitopes in C3H mice could be regulated by the parasite since functional C3H B cell responses were mounted against exposed VSG epitopes of a closely related variant (LouTat 1.5) after infection.