J. David Barry

Telomeric Expression Sites Are Highly Conserved in Trypanosoma brucei

Subtelomeric regions are often under-represented in genome sequences of eukaryotes. One of the best known examples of the use of telomere proximity for adaptive purposes are the bloodstream expression sites (BESs) of the African trypanosome Trypanosoma brucei. To enhance our understanding of BES structure and function in host adaptation and immune evasion, the BES repertoire from the Lister 427 strain of T. brucei were independently tagged and sequenced. BESs are polymorphic in size and structure but reveal a surprisingly conserved architecture in the context of extensive recombination. Very small BESs do exist and many functioning BESs do not contain the full complement of expression site associated genes (ESAGs). The consequences of duplicated or missing ESAGs, including ESAG9, a newly named ESAG12, and additional variant surface glycoprotein genes (VSGs) were evaluated by functional assays after BESs were tagged with a drug-resistance gene. Phylogenetic analysis of constituent ESAG families suggests that BESs are sequence mosaics and that extensive recombination has shaped the evolution of the BES repertoire. This work opens important perspectives in understanding the molecular mechanisms of antigenic variation, a widely used strategy for immune evasion in pathogens, and telomere biology.

A cysteine proteinase cDNA from Trypanosoma brucei predicts an enzyme with an unusual C-terminal extension.

A cDNA for a Trypanosoma brucei cysteine proteinase has been cloned and sequenced. The deduced protein can be divided into four domains, based on homologies with other cysteine proteinases: the pre‐, pro‐ and central regions show considerable homology to the cathepsin L class of mammalian enzymes, whilst the long C‐terminal extension distinguishes the trypanosome enzyme from all mammalian cysteine proteinases reported. This 108 amino acid extension, which includes 9 contiguous prolines near the junction with the central domain, appears likely to be processed in part to produce the mature enzyme, and may be involved in targetting the protein within the cell. The trypanosome genome contains more than 20 copies of the cysteine proteinase gene arranged in a long tandem array.

The Genome Sequence of Trypanosoma brucei gambiense , Causative Agent of Chronic Human African Trypanosomiasis

Background Trypanosoma brucei gambiense is the causative agent of chronic Human African Trypanosomiasis or sleeping sickness, a disease endemic across often poor and rural areas of Western and Central Africa. We have previously published the genome sequence of a T. b. brucei isolate, and have now employed a comparative genomics approach to understand the scale of genomic variation between T. b. gambiense and the reference genome. We sought to identify features that were uniquely associated with T. b. gambiense and its ability to infect humans. Methods and Findings An improved high-quality draft genome sequence for the group 1 T. b. gambiense DAL 972 isolate was produced using a whole-genome shotgun strategy. Comparison with T. b. brucei showed that sequence identity averages 99.2% in coding regions, and gene order is largely collinear. However, variation associated with segmental duplications and tandem gene arrays suggests some reduction of functional repertoire in T. b. gambiense DAL 972. A comparison of the variant surface glycoproteins (VSG) in T. b. brucei with all T. b. gambiense sequence reads showed that the essential structural repertoire of VSG domains is conserved across T. brucei. Conclusions This study provides the first estimate of intraspecific genomic variation within T. brucei, and so has important consequences for future population genomics studies. We have shown that the T. b. gambiense genome corresponds closely with the reference, which should therefore be an effective scaffold for any T. brucei genome sequence data. As VSG repertoire is also well conserved, it may be feasible to describe the total diversity of variant antigens. While we describe several as yet uncharacterized gene families with predicted cell surface roles that were expanded in number in T. b. brucei, no T. b. gambiense-specific gene was identified outside of the subtelomeres that could explain the ability to infect humans.

An update on antigenic variation in African trypanosomes

African trypanosomes can spend a long time in the blood of their mammalian host, where they are exposed to the immune system and are thought to take advantage of it to modulate their own numbers. Their major immunogenic protein is the variant surface glycoprotein (VSG), the gene for which must be in one of the 20--40 specialized telomeric expression sites in order to be transcribed. Trypanosomes escape antibody-mediated destruction through periodic changes of the expressed VSG gene from a repertoire of approximately 1000. How do trypanosomes exclusively express only one VSG and how do they switch between them?

Antigenic diversity is generated by distinct evolutionary mechanisms in African trypanosome species

Antigenic variation enables pathogens to avoid the host immune response by continual switching of surface proteins. The protozoan blood parasite Trypanosoma brucei causes human African trypanosomiasis (“sleeping sickness”) across sub-Saharan Africa and is a model system for antigenic variation, surviving by periodically replacing a monolayer of variant surface glycoproteins (VSG) that covers its cell surface. We compared the genome of Trypanosoma brucei with two closely related parasites Trypanosoma congolense and Trypanosoma vivax, to reveal how the variant antigen repertoire has evolved and how it might affect contemporary antigenic diversity. We reconstruct VSG diversification showing that Trypanosoma congolense uses variant antigens derived from multiple ancestral VSG lineages, whereas in Trypanosoma brucei VSG have recent origins, and ancestral gene lineages have been repeatedly co-opted to novel functions. These historical differences are reflected in fundamental differences between species in the scale and mechanism of recombination. Using phylogenetic incompatibility as a metric for genetic exchange, we show that the frequency of recombination is comparable between Trypanosoma congolense and Trypanosoma brucei but is much lower in Trypanosoma vivax. Furthermore, in showing that the C-terminal domain of Trypanosoma brucei VSG plays a crucial role in facilitating exchange, we reveal substantial species differences in the mechanism of VSG diversification. Our results demonstrate how past VSG evolution indirectly determines the ability of contemporary parasites to generate novel variant antigens through recombination and suggest that the current model for antigenic variation in Trypanosoma brucei is only one means by which these parasites maintain chronic infections.

The identification of Trypanosoma brucei subspecies using repetitive DNA sequences.

We describe the use of repetitive DNA probes to characterise the relationships between different stocks of African trypanosomes representing the subspecies of Trypanosoma brucei. Probes derived from the ribosomal RNA genes (coding region and nontranscribed spacer) and another repetitive DNA sequence were used to characterise trypanosome stocks by Southern blotting. Numerical taxonomy methods applied to the resulting restriction enzyme patterns were used to derive a dendrogram depicting the relationships between the stocks examined. We show that three groups of West African human infective stocks can be distinguished: firstly, a group containing exclusively T. b. gambiense; secondly, a group which is indistinguishable from animal isolates in West Africa; and thirdly, a single stock which is indistinguishable from East African T. b. rhodesiense. In addition, we observe that T. b. rhodesiense stocks from East Africa are indistinguishable from animal isolates from the same area. Finally, we show that a group of T. b. rhodesiense stocks, isolated from a 1978 sleeping sickness outbreak in Zambia, are probably derived from a single parasite strain, and that this strain is distinct from T. b. rhodesiense parasites from Kenya and Uganda.

Transformation of Monomorphic and Pleomorphic Trypanosoma brucei

African trypanosomes, such as Trypanosoma brucei, are protozoan parasites of mammals that were first described over 100 hundred years ago. They have long been the subjects of biological investigation, which has yielded insights into a number of fundamental, as well as novel, cellular processes in all organisms. In the last decade or so, genetic manipulation of trypanosomes has become possible through DNA transformation, allowing yet more detailed analysis of the biology of the parasite. One facet of this is that DNA transformation has itself been used as an assay for recombination and will undoubtedly lead to further genetic approaches to examine this process. Here we describe protocols for DNA transformation of Trypanosoma brucei, including two different life cycle stages and two different strain types that are distinguished by morphological and developmental criteria. We consider the application of transformation to recombination, as well as the uses of transforming the different life cycle stages and strain types.

A developmentally regulated cysteine proteinase gene of Leishmania mexicana

We have isolated a gene encoding a previously unreported class of trypanosomatid cysteine proteinase (CP) from the protozoan parasite Leishmania mexicana. The single‐copy gene (Imcpa) has several unusual features that distinguish it from CP genes cloned from the related species Trypanosoma brucei and Trypanosoma cruzi. These include a shorter C‐terminal extension of only 10 amino acids and a three‐amino‐acid insertion, GlyValMet, close to the predicted N‐terminus of the mature protein. Northern blot analysis showed that the gene is expressed in all life‐cycle stages but at higher levels in the amastigote stage in the mammal and in stationary phase promastigote cultures which contain the infective meta‐cyclic form of the parasite. A precursor protein of 38 kDa was detected in amastigotes and stationary phase promastigotes with antisera specific to the LmCPa pro‐region, but was barely detectable in early log‐phase promastigotes. Anti‐central domain antisera recognized the 38 kDa precursor and 24 and 27 kDa proteins. The major CPs of L. mexicana amastigotes, previously designated types A, B and C, were not detected with the antisera, suggesting that the gene codes for a previously uncharacterized CP in L. mexicana. The 24 kDa protein detected by the antiserum has no activity towards gelatin but apparently hydrolyses the peptide substrate BzPhe‐ValArgAMC. The relative levels of the 24 and 27 kDa proteins vary between the different life‐cycle stages. The results indicate that expression of this CP is regulated at both the RNA and protein level.

NUP-1 Is a Large Coiled-Coil Nucleoskeletal Protein in Trypanosomes with Lamin-Like Functions

NUP1, the first example of a nuclear lamin analog in nonmetazoans, performs roles similar to those of lamins in maintaining the structure and organization of the nucleus in Trypanosoma brucei.

A major surface antigen of procyclic stage Trypanosoma congolense

Five monoclonal antibodies (mAb) were raised that bound to the surface of procyclic stage Trypanosoma congolense with high intensity in immunofluorescence. Immunoblot analysis of trypanosome lysates using 3 of these mAb revealed a diffuse SDS-PAGE band of 36-40 kDa. The purified antigen did not react with Coomassie Blue or silver stains, but did stain blue with Stains-all, indicating acidity. For the one mAb tested, the epitope was periodate-sensitive and therefore probably glycan. Although this antigen shares properties with procyclin/PARP, which forms a surface coat on procyclic Trypanosoma brucei, a search in T. congolense for homologues of a procyclin/PARP gene revealed only non-coding sequence of partial similarity. Using a differential screen, a procyclic stage T. congolense cDNA clone was isolated that encoded a putative 256-amino acid protein containing 2 peptides chemically sequenced independently by Beecroft et al. [36]. The protein, termed glutamate and alanine-rich protein (GARP), has potential hydrophobic leader and tail sequences (the latter with potential for replacement by a glycosyl phosphoinositol anchor) and no potential N-linked glycosylation sites. It has no significant sequence homology with known proteins. Antibodies against a translational fusion of GARP bound specifically in Western blots to a band very similar to that detected by the mAb and also to the purified antigen. Immunogold electron microscopy revealed a dense packing of the antigen on the cell surface. It appears that procyclic T. brucei and T. congolense have major surface proteins with structural analogy, but with no sequence homology.