Variant-specific Surface Antigen Research Articles

The identification of hydroxymethyluracil in DNA of Trypanosoma brucei.

We have previously reported the detection of two unusual nucleotides, pdJ and pdV, in the DNA of Trypanosoma brucei (Gommers-Ampt et al., 1991). pdJ was found to be a novel nucleotide and is possibly involved in the regulation of variant specific surface antigen gene expression in trypanosomes. Recent evidence suggests that V could be a precursor of J, making V a key compound in the study of the biosynthesis and function of J. We have therefore determined the structure of V and here we present proof that V is HOMeU. The identity is based on a detailed comparison of dV(p) with authentic HOMedU(p), showing: I) co-migration in three different liquid chromatography analyses II) identical UV absorbance characteristics III) identical behavior in acetyl-pentafluorobenzyl derivatization and subsequent Gas chromatography/Mass spectrometry (GC/MS). The GC/MS technique has not been used before to analyse HOMedU purified from biological material. Because of its high sensitivity, it may also be useful for the detection of the low amounts of HOMedU resulting from oxidative damage of DNA.

Modifications of a trypanosoma b. brucei antigen gene repertoire by different DNA recombinational mechanisms

In the Trypanosoma b. brucei AnTat 1.1C clone, the gene coding for the variant-specific surface antigen is telomeric and appears as a hybrid sequence, partially modified by gene conversion. This conversion is very similar to that observed in another AnTat 1.1-expressor clone (AnTat 1.1B). This sequence is not activated by duplicative transposition, although it could be activated by duplication in another clone (AnTat 1.10). Instead activation of the AnTat 1.1C gene seems operated by reciprocal recombination between its own telomere and the telomere carrying the previous (AnTat 1.16) ELC. Indeed, from the switch to AnTat 1.1C onward, the AnTat 1.16 ELC becomes a new silent member of its gene family, whereas in the variant directly derived from AnTat 1.1C (AnTat 1.3B), the AnTat 1.1C-containing telomere is lost, probably replaced by a large duplicate, at least 40 kb long, of the AnTat 1.3 gene-containing telomere. Different DNA rearrangement mechanisms used by the trypanosome to change its antigenic type thus contribute, by gain and loss of genes, to the evolution of the repertoire for surface antigens.

DNA rearrangements linked to expression of a predominant surface antigen gene of trypanosomes.

African trypanosomes show an extensive capacity for antigenic variation1–6. The serotype of each antigenic variant is determined by a variant-specific surface antigen (VSA) which forms a continuous coat over the entire surface of the parasite7. All known VSAs are glycoproteins whose antigenic specificity is associated with the protein moiety8,9. The expression of some of the VSA genes is linked to the duplication of a ‘basic’ copy (BC) of the gene and to the transposition of the additional or ‘expression-linked’ copy (ELC) to a new site10–12, where it is transcribed13. Other VSA genes seem to be expressed without being duplicated; they are, however, subject to rearrangements apparently unrelated to their expression14,15. In chronic infections, different variable antigen types (VATs) tend to appear in a loosely ordered sequence: indeed, after either syringe passage or cyclical transmission through the tsetse fly, the same VATs always appear in the early stage of the disease and are therefore known as early or predominant ones16–22. To check whether genes coding for predominant VATs can be characterized by some particular feature of their DNA sequence or gene rearrangement, we have studied here the gene coding for AnTat 1.3, which is the most predominant VAT of our Trypanosoma brucei brucei stock. Three points emerged: (1) when expressed, the AnTat 1.3 gene is duplicated and the ELC transposed in an expression site identical to the one described previously for other VSA genes; (2) its BC is located near the end of a DNA molecule, in a region which is very similar to the expression site and undergoes frequent insertions/deletions unlinked to VSA gene expression; (3) this gene does not belong to a multigene family.

Lymphocyte function in experimental African trypanosomiasis. V. Role of antibody and the mononuclear phagocyte system in variant-specific immunity.

The role of parasite-specific antibody and the mononuclear phagocyte system (MPS) in immunity to the African trypanosomes was examined. For this study C57BL/10SnJ mice were infected with Trypanosoma rhodesiense clone LouTat 1.0. Infected mice were injected with 75Se-labeled LouTat 1.0 trypanosomes, and clearance from the blood upon reexposure was measured throughout the course of infection. Clearance of labeled organisms occurred only on or after day 5, which was the day of natural elimination of LouTat 1.0 from the blood. Clearance was dependent on a functional immune system and correlated with the appearance of antibody to the variant-specific surface antigen (VSSA) of the trypanosomes. The ability to clear trypanosomes was transferred to normal, uninfected mice by immune serum. Both the IgM and IgG fractions of immune serum mediated the clearance, and VSSA-specific IgM fractions were as efficient in clearing LouTat 1.0 as the IgG fractions. Normal levels of complement (C3) were not required for clearance. The liver was the primary organ of clearance, and the ability of the liver to sequester radiolabeled trypanosomes was not impaired in the terminal phase of the disease or by large numbers of circulating trypanosomes present representing different variant antigenic types (VAT). We conclude that in African trypanosomiasis the MPS is not depressed in its ability to clear trypanosomes of the infecting VAT at any time during the course of infection. The observed clearance function requires parasite-specific antibody but normal levels of C3.

Differential size variations between transcriptionally active and inactive telomeres of Trypanosoma brucei.

We have studied the genes coding for the variant-specific surface antigen (VSA) in a series of seven trypanosome clones derived from AnTat 1.1: 1.1 leads to 1.3 leads to 1.6 leads to 1.16 leads to 1.1C leads to 1.3B leads to 1.18 These genes are all telomeric (1-5), and their surrounding, although sometimes similar, differs in each case. The length between these antigen genes and the corresponding DNA end appears to increase at each antigenic switch, with however occasional sharp size reductions, often linked to the involvement of the telomere in gene expression. This increase is due to a constant "growth" of the telomeres, at a rate of about 28 bp per day in at least four cases and probably linked to chromosome duplication. The telomere harbouring the transcribed VSA gene is growing slightly faster (about 36 bp per day), and it is the only one whose size reduction is progressive, leading to a terminal length heterogeneity within a clone. As a result, the active VSA gene is found in a population of telomeres which, as the trypanosomes divide, becomes increasingly heterogeneous, with however a preferred discrete size class about 1.4 kb smaller. The fact that the "active" telomere is the only one in a chromatin conformation highly sensitive to DNAaseI (1-4, 6), suggests that chromatin structure influences the rate and extent of both size increase and shortening of telomeres.

Immunization of cattle with a variant-specific surface antigen of Trypanosoma brucei: influence of different adjuvants.

Nine different adjuvants were examined for their ability to potentiate the humoral and cell-mediated immune responses of cattle to a soluble glycoprotein antigen prepared from Trypanosoma brucei. Serological responses as measured by the Farr assay were best augmented by the oil-based adjuvants and saponin. Cell-mediated immunity, as assessed by specific lymphocyte transformation in vitro, was enhanced by all oil-based adjuvants at different intervals after immunization. Results from a challenge infection of immunized cattle with the homologous clone of T. brucei and from neutralization tests indicated that protection against infection was better correlated with specific antibodies than with cell-mediated responses. From these considerations, and the absence of tissue reactions at the site of inoculation, saponin was considered more practical than the oil-based or bacterial adjuvants for the elicitation in cattle of antibodies to purified soluble antigens.

Conservation of a variant-specific surface antigen gene in different trypanosome species and sub-species

In Trypanosoma brucei brucei, T. b. rhodesiense, T. b. gambiense and T. evansi, the variant-specific antigen (VSA) genes are organized in families of related sequences, one of which is duplicated when expressed. Some of these VSA sequences appear to be conserved in the different species and sub-species: restriction mapping of isotypic genes of AnTat 1.8 VSA (from T. b. brucei) reveals extensive homology in T. b. rhodesiense, T. b. gambiense and T. evansi, although the genetic surrounding differs in each case. By contrast, the AnTat 1.1 sequence (also from T. b. brucei) appears to be absent from T. b. gambiense DNA.

Trypanosoma congolense: Specific transformation in vitro of leukocytes from infected or immunized cattle

An assay to measure the specific proliferation in vitro of peripheral blood leukocytes (PBL) in response to ultrasonicated trypanosomes was adapted for use in cattle. The kinetics of mitosis exhibited by PBL from cattle which had been treated following infection with Trypanosoma congolense paralleled the development of a delayed-type skin reaction elicited with ultrasonicated and Formalin-fixed T. congolense. Responses in both tests were maximal on the fourth day after initiation. Specific proliferation of PBL harvested from cattle which had been immunized with intact, nonviable trypanosomes was also elicited in vitro by trypanosomal antigen. Peripheral blood leukocytes taken from cattle immunized with 50 μg of variant-specific surface antigen (VSSA) from T. brucei and from cattle infected with T. congolense were not stimulated to divide in vitro by ultrasonicated trypanosomes.

Purification and cell-free translation of mRNA coding for a variant specific antigen from Trypanosoma brucei gambiense.

Messenger RNA (mRNA) coding for a variant specific surface antigen (VSA) from Trypanosoma brucei gambiense was isolated from total trypanosomal polyribosomes by indirect immunoprecipitation. An IgG fraction of antisera to purified VSA was obtained by ion exchange chromatography and Protein A-agarose affinity chromatography. These antibodies were then subjected to affinity chromatography on a VSA-agarose column to remove non-specific IgG. Polyribosomes from the same antigenic variant of T. b. gambiense were isolated from a cell lysate and those polysomes bearing nascent VSA were bound to the IgG by gentle mixing and the complexes formed were retrieved by precipitation with fixed Staphylococcus aureus cells. The VSA-specific mRNA was separated from these complexes by dissociation of the polysomes, deproteinization, and affinity chromatography on oligo(dT)-cellulose. The mRNA isolated in this way was shown to be undegraded, active in protein synthesis and homogeneous electrophoretically. The products of the cell-free translation of this mRNA were precipitable by specific IgG but not by antiserum to a heterologous VSA. The translation product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel exclusion chromatography. The molecular weight of the mRNA was measured by electron microscopy, agarose and polyacrylamide gel electrophoresis. Enough highly purified mRNA can be isolated in this manner to be used for hybridization analysis of the VSA gene(s).

Glycosidases in bloodstream forms of Trypanosoma brucei brucei

Abstract 1. 1. Nine glycosidases in bloodstream forms of Trypanosoma brucei brucei S42 were partially characterised. 2. 2. α-Glucosidase had similar physicochemical and enzymic properties to those of α-galactosidase, β-glucosidase, N- acetyl-β-glucosaminidase and N- acetyl-β-galactosaminidase . 3. 3. α-Mannosidase was clearly distinct from the other glycosidases with respect to pH optimum, thermostability, specific activity during the course of parasitaemia, and subcellular location. 4. 4. The glycosidases studied here may play a role in the turnover of trypanosomal glycoproteins, in particular the variant-specific surface antigen.

Immunological purification and partial characterization of variant-specific surface antigen messenger RNA of Trypanosoma brucei brucei.

Polyadenylated RNA isolated from total polyribosomes of two variable antigen types (VATs) of T. brucei brucei were shown to program the synthesis, in mRNA-dependant reticulocyte lysates, of a wide variety of polypeptides. After immunoprecipitation of these cell-free products with an homologous antiserum raised against purified variant-specific surface antigen (VSSA), a major electrophoretic band was apparent on fluorography. It was confirmed that this band corresponds to the variable antigen since only an excess of purified homologous antigen will provoke competition. The apparent molecular weight of the in vitro synthesized antigen is about 63,000 daltons. The VSSA mRNA has been found in membrane-bound polyribosomes and a 15 fold immunological purification of this mRNA has been obtained, using partially purified anti-VSSA IgG in conjunction with inactivated Staphylococcus aureus.

A characterization of mRNA activites and their sequence complexities in Trypanosoma brucei: partial purification and properties of the VSSA mRNA.

Polyadenylated RNA isolated from a clone of Trypanosoma brucei was shown to direct the synthesis of a variety of polypeptides in a cell-free system. A predominant 58,000 dalton polypeptide was immunoprecipitated with antisera to the T. brucei variant specific surface antigen (VSSA). The mRNA that directed the synthesis of the VSSA was 2.0 kilobases (kb) long as measured by polyacrylamide gel electrophoresis in 98% formamide. Complementary DNA was prepared with avian myeloblastosis virus reverse transcriptase and the nucleotide sequence complexities of the total polysomal poly(A)+RNA and a gel purified VSSA mRNA were measured. 20% of the total cellular poly(A)+RNA contained abundant sequences with an apparent complexity of 9.6 kb; 42% of the purified VSSA mRNA contained abundant sequences with a complexity of 7.2 kb. Complementary DNA synthesized from gel purified VSSA mRNA was hybridized to total cellular poly(A)+RNA isolated from an unrelated T. brucei clone expressing a different variant antigen. A portion of the low complexity RNA sequence component was absent in the heterologous mRNA population but the same plateau of hybridization was achieved (93%). The abundance of some of the low complexity mRNAs appears to be T. brucei clone specific.

Activation of complement by variant-specific surface antigen of trypanosoma brucei.

ANTICOMPLEMENT factors have been shown to occur in certain parasitic infections of man and animals. Hammerberg et al.1 demonstrated the presence of a factor from Taenia taeniaeformis capable of activating complement through the alternate pathway. Similar findings have been reported for a substance in the cercarial coat of Schistosoma mansoni2. In trypanosomiasis, no such parasitic activity has been reported although hypocomplementaemia has been recognised as a consistent feature of the disease. Marked reductions in complement C4 and C3 were observed in infections with Trypanosoma lewisi3, Tr. rhodesiense4 and Tr. congolense5. One possible mechanism is that the decrease of these complement components results from activation by antigen–antibody complexes. Deposits of immunoglobulins and complement have been demonstrated in kidneys of mice infected with Trypanosoma brucei6,7 and monkeys infected with Tr. rhodesiense4. We present here evidence for an alternative mechanism operating through direct activation of human complement, via the classical pathway, by an isolated variant-specific surface antigen of Tr. brucei.

Carbohydrate composition of variant-specific surface antigen glycoproteins from Trypanosoma brucei.

The carbohydrate of variant-specific surface antigen glycoproteins from bloodstream forms of 13 cloned variants of Trypanosoma brucei was analyzed by gas-liquid chromatography. The glycoproteins contained from 6 to 17% carbohydrate by weight, and all contained the same 4 sugars: mannose, galactose, glucose, and glucosamine (probably as N-acetylglucosamine). The glycoprotein from variant 048, strain 427 contained (+20%) 11 mannose, 4 galactose, 4 glucose, and 5 glucosamine residues/mole of glycoprotein (molecular weight 65,000). Glucose was an intergral component of the glycoproteins, not dissociable by sodium dodecyl sulphate, 8 M urea, or 1 M acetic acid. Some of the glucose was dissociated by trichloroacetic acid. Most of the glycoproteins formed precipitin with concanavalin A in Ouchterlony double diffusion, but none formed such bands with wheat germ agglutinin or Ricinus communis lectin (molecular weight 120, 000).

The synthesis of a variant-specific antigen by Trypanosoma brucei in vitro.

A variant-specific surface antigen from a cloned population of Trypanosoma brucei S42 has been isolated and partially characterized. [35S]L-methionine was found to be incorporated into this material by cells incubated in vitro in a chemically defined medium. Incorporation of [35S]L-methionine was inhibited by cycloheximide and puromycin at concentrations which are known to specifically inhibit protein synthesis in other systems. The rate of synthesis of the variant-specific antigen in vitro has been estimated to be about 8% of the rate in vivo. Newly synthesized [35S]L-methionine-labelled variant-specific antigen was incorporated into the surface coat.