Mismatch repair in Trypanosoma brucei: heterologous expression of MSH2 from Trypanosoma cruzi provides new insights into the response to oxidative damage

Trypanosomes are unicellular eukaryotes that cause disease in humans and other mammals. Trypanosoma cruzi and Trypanosoma brucei are the causative agents, respectively, of Chagas disease in the Americas and sleeping sickness in sub-Saharan Africa. To better comprehend the interaction of these parasites with their hosts, understanding the mechanisms involved in the generation of genetic variability is critical. One such mechanism is mismatch repair (MMR), which has a crucial, evolutionarily conserved role in maintaining the fidelity of DNA replication, as well as acting in other cellular processes, such as DNA recombination. Here we have attempted to complement T. brucei MMR through the expression of MSH2 from T. cruzi. Our results show that T. brucei MSH2-null mutants are more sensitive to hydrogen peroxide (H2O2) than wild type cells, suggesting the involvement of MSH2 in the response to oxidative stress in this parasite. This phenotype is reverted by the expression of either the T. cruzi or the T. brucei MSH2 protein in the MSH2-null mutants. In contrast, MMR complementation, as assessed by resistance to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and microsatellite instability, was not achieved by the heterologous expression of T. cruzi MSH2. This finding, associated to the demonstration that mutation of MLH1, another component of the MMR system, did not affect sensitivity of T. brucei cells to H2O2, suggests an additional role of MSH2 in dealing with oxidative damage in these parasites, which may occur independently of MMR.     

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.     

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.     

Trypanosoma brucei BRCA2 acts in antigenic variation and has undergone a recent expansion in BRC repeat number that is important during homologous recombination

Antigenic variation in Trypanosoma brucei has selected for the evolution of a massive archive of silent Variant Surface Glycoprotein (VSG) genes, which are activated by recombination into specialized expression sites. Such VSG switching can occur at rates substantially higher than background mutation and is dependent on homologous recombination, a core DNA repair reaction. A key regulator of homologous recombination is BRCA2, a protein that binds RAD51, the enzyme responsible for DNA strand exchange. Here, we show that T. brucei BRCA2 has undergone a recent, striking expansion in the number of BRC repeats, a sequence element that mediates interaction with RAD51. T. brucei BRCA2 mutants are shown to be significantly impaired in antigenic variation and display genome instability. By generating BRCA2 variants with reduced BRC repeat numbers, we show that the BRC expansion is crucial in determining the efficiency of T. brucei homologous recombination and RAD51 localization. Remarkably, however, this appears not to be a major determinant of the activation of at least some VSG genes.     

Trypanosoma congolense: structure and molecular organization of the surface glycoproteins of two early bloodstream variants

The complete primary structures of two variant specific glycoproteins (VSGs) of the nannomonad Trypanosoma (N.) congolense are presented. These coat proteins subserve the function of antigenic variation. The secondary structure potentials of both VSGs have been calculated. The amino acid sequences and secondary structure potentials of these VSGs have been compared with the primary structures and secondary structure potentials of several Trypanosoma brucei complex VSGs. In homologous regions, the T. brucei complex VSGs show a pattern of sharply contrasting secondary structure potentials. It has been suggested previously that this pattern gives rise to different folding structures in different members of this polygene protein family. Thus, different short regions of the polypeptide sequence are exposed as antigenic "caps" on the solvent-exposed surface of intact trypanosomes. A sharply contrasting secondary structure potential pattern is also found in regions of the two T. congolense VSGs. However, there is little homology of primary structure between each of the two T. congolense VSGs and any member of the T. brucei complex VSG polygene family whose primary structure has been determined.     

Interactions among Trypanosoma brucei RAD51 paralogues in DNA repair and antigenic variation

Homologous recombination in Trypanosoma brucei is used for moving variant surface glycoprotein (VSG) genes into expression sites during immune evasion by antigenic variation. A major route for such VSG switching is gene conversion reactions in which RAD51, a universally conserved recombinase, catalyses homology-directed strand exchange. In any eukaryote, RAD51-directed strand exchange in vivo is mediated by further factors, including RAD51-related proteins termed Rad51 paralogues. These appear to be ubiquitously conserved, although their detailed roles in recombination remain unclear. In T. brucei, four putative RAD51 paralogue genes have been identified by sequence homology. Here we show that all four RAD51 paralogues act in DNA repair, recombination and RAD51 subnuclear dynamics, though not equivalently, while mutation of only one RAD51 paralogue gene significantly impedes VSG switching. We also show that the T. brucei RAD51 paralogues interact, and that the complexes they form may explain the distinct phenotypes of the mutants as well as observed expression interdependency. Finally, we document the Rad51 paralogues that are encoded by a wide range of protists, demonstrating that the Rad51 paralogue repertoire in T. brucei is unusually large among microbial eukaryotes and that one member of the protein family corresponds with a key, conserved eukaryotic Rad51 paralogue.     

The identification of circular extrachromosomal DNA in the nuclear genome of Trypanosoma brucei

Nuclear extrachromosomal DNA elements have been identified in several kinetoplastids such as Leishmania and Trypanosoma cruzi, but never in Trypanosoma brucei. They can occur naturally or arise spontaneously as the result of sublethal drug exposure of parasites. In most cases, they are represented as circular elements and are mitotically unstable. In this study we describe the presence of circular DNA in the nucleus of Trypanosoma brucei. This novel type of DNA was termed NR-element (NlaIII repeat element). In contrast to drug-induced episomes in other kinetoplastids, the T. brucei extrachromosomal NR-element is not generated by drug selection. Furthermore, the element is stable during mitosis over many generations. Restriction analysis of tagged NR-element DNA, unusual migration patterns during pulsed field gel electrophoresis (PFGE) and CsCl/ethidium bromide equilibrium centrifugation demonstrates that the NR-element represents circular DNA. Whereas it has been found in all field isolates of the parasites we analysed, it is not detectable in some laboratory strains notably the genome reference strain 927. The DNA sequence of this element is related to a 29 bp repeat present in the subtelomeric region of VSG-bearing chromosomes of T. brucei. It has been suggested that this subtelomeric region is part of a transition zone on chromosomes separating the relatively stable telomeric repeats from the recombinationaly active region downstream of VSG genes. Therefore, we discuss a functional connection between the occurrence of this circular DNA and subtelomeric recombination events in T. brucei.     

Crystallization of amino-terminal domains and domain fragments of variant surface glycoproteins from Trypanosoma brucei brucei

Crystals were produced from variant surface glycoproteins (VSG) of Trypanosoma brucei brucei antigenic variants MITat 1.2, 1.6, and ILTat 1.22, 1.23, 1.24, 1.25, and 1.26. Purified VSGs had molecular weights from 60,000 to 68,000 on sodium dodecyl sulfate-polyacrylamide gels, whereas the crystals obtained were composed of polypeptides of approximate Mr 40,000-50,000. Amino-terminal amino acid sequences determined from the crystallized VSGs were identical to sequences obtained from the respective intact proteins, indicating that the crystals contained VSG amino-terminal fragments. Crystallization conditions and lattice dimensions of the crystals are given.     

Trypanosoma evansi: Identification and characterization of a variant surface glycoprotein lacking cysteine residues in its C-terminal domain

African trypanosomes are flagellated unicellular parasites which proliferate extracellularly in the mammalian host blood-stream and tissue spaces. They evade the hosts’ antibody-mediated lyses by sequentially changing their variant surface glycoprotein (VSG). VSG tightly coats the entire parasite body, serving as a physical barrier. In Trypanosoma brucei and the closely related species Trypanosoma evansi, Trypanosoma equiperdum, each VSG polypeptide can be divided into N- and C-terminal domains, based on cysteine distribution and sequence homology. N-terminal domain, the basis of antigenic variation, is hypervariable and contains all the exposed epitopes; C-terminal domain is relatively conserved and a full set of four or eight cysteines were generally observed. We cloned two genes from two distinct variants of T. evansi, utilizing RT-PCR with VSG-specific primers. One contained a VSG type A N-terminal domain followed a C-terminal domain lacking cysteine residues. To confirm that this gene is expressed as a functional VSG, the expression and localization of the corresponding gene product were characterized using Western blotting and immunofluorescent staining of living trypanosomes. Expression analysis showed that this protein was highly expressed, variant-specific, and had a ubiquitous cellular surface localization. All these results indicated that it was expressed as a functional VSG. Our finding showed that cysteine residues in VSG C-terminal domain were not essential; the conserved C-terminal domain generally in T. brucei like VSGs would possibly evolve for regulating the VSG expression.     

Characterization of Trypanosoma brucei gambiense variant surface glycoprotein LiTat 1.5

At present, all available diagnostic antibody detection tests for Trypanosoma brucei gambiense human African trypanosomiasis are based on predominant variant surface glycoproteins (VSGs), such as VSG LiTat 1.5. During investigations aiming at replacement of the native VSGs by recombinant proteins or synthetic peptides, the sequence of VSG LiTat 1.5 was derived from cDNA and direct N-terminal amino acid sequencing. Characterization of the VSG based on cysteine distribution in the amino acid sequence revealed an unusual cysteine pattern identical to that of VSG Kinu 1 of T. b. brucei. Even though both VSGs lack the third of four conserved cysteines typical for type A N-terminal domains, they can be classified as type A.     

Distinct roles for two RAD51-related genes in Trypanosoma brucei antigenic variation

In Trypanosoma brucei, DNA recombination is crucial in antigenic variation, a strategy for evading the mammalian host immune system found in a wide variety of pathogens. T.brucei has the capacity to encode >1000 antigenically distinct variant surface glycoproteins (VSGs). By ensuring that only one VSG is expressed on the cell surface at one time, and by periodically switching the VSG gene that is expressed, T.brucei can evade immune killing for prolonged periods. Much of VSG switching appears to rely on a widely conserved DNA repair pathway called homologous recombination, driven by RAD51. Here, we demonstrate that T.brucei encodes a further five RAD51-related proteins, more than has been identified in other single-celled eukaryotes to date. We have investigated the roles of two of the RAD51-related proteins in T.brucei, and show that they contribute to DNA repair, homologous recombination and RAD51 function in the cell. Surprisingly, however, only one of the two proteins contributes to VSG switching, suggesting that the family of diverged RAD51 proteins present in T.brucei have assumed specialized functions in homologous recombination, analogous to related proteins in metazoan eukaryotes.     

Antigenic variation during the developmental cycle of Trypanosoma brucei

During the complex life cycle of Trypanosoma brucei, changes in the exposed surface antigens occur in both the mammalian host and the insect vector (Glossina spp.). These antigenic changes are associated with alterations of the variant surface glycoprotein (VSG) composition or with the loss of the VSG. In the bloodstream of the mammalian host, trypanosomes successfully evade destruction by the host's immune response by continuously expressing alternative VSGs, at low frequency, which are not destroyed by host antibodies. When ingested by the tsetse fly, the bloodstream trypanosomes rapidly lose their surface coat and surface membrane antigens are exposed which are normally covered in the bloodstream. In the salivary glands of the tsetse fly, the trypanosomes differentiate to the metacyclic stage, which reacquires a surface coat. The antigenic composition of the metacyclics is heterogeneous. The same metacyclic types are expressed regardless of the bloodstream antigenic type ingested by the tsetse fly. In the mammal the metacyclics differentiate to long-slender bloodstream forms but continue to express the metacyclic VSG for at least three days. The next VSGs expressed in the mammalian host appear to be influenced by the antigenic type ingested by the tsetse. The ingested antigenic type is often expressed in the first parasitemia following expression of the metacyclic antigenic types.