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.     

Lectin signalling of maturation of T.congolense infections in tsetse

The process of maturation of Trypanosoma congolense Broden in tsetse has been shown to be initiated by lectin secreted in the fly midgut. In the present study the duration of lectin signal required to induce maturation was determined by the sequential addition or removal of a specific lectin inhibitor (D+glucosamine) to the diet of infected male Glossina morsitans Westwood. An established midgut infection of T.congolense was found to require, at most, 72 h exposure to midgut lectin to begin the process of maturation. Longer exposure to midgut lectin increased the frequency of maturation, suggesting clonal variation in response to lectin stimulation occurs within trypanosome stocks. It is suggested that this variation corresponds to differences in lectin binding sites on the trypanosome surface. Midgut trypanosomes retained their ability to mature throughout their life in the fly; when lectin activity in the midgut was inhibited, the trypanosomes remained as procyclic forms but when this inhibition was removed maturation was able to proceed. This indicates that the process of maturation is dependent upon a signal from the fly and is not predetermined by the trypanosomes undergoing a fixed number of division cycles. The possible role of lectins in the maturation of trypanosomes in vitro is discussed.     

Fish Trypanosomes: Their Position in Kinetoplastid Phylogeny and Variability as Determined from 12S rRNA Kinetoplast Sequences

Fish trypanosomes have traditionally been classified according to the host species from which they were isolated, each isolate being regarded as a distinct species. To test the soundness of this practice, the genetic variabilities of the kinetoplast 12S rRNA-encoding genes of different fish trypanosomes isolates were compared. The DNAs were extracted from trypanosomes cloned from blood samples of 15 donors representing ten different fish species in four orders from waters of three major river systems of Central and Northern Europe. Comparison with other trypanosomatid sequences revealed that the fish trypanosomes form a monophyletic group with Trypanosoma brucei as a sister group. Pairwise comparisons of genetic distances yielded a wide range of continuous variation with no indication of any discontinuities attributable to barriers to gene flow. The genetic distances did not correlate with either the identity of the host species or geography. The host specificity of fish trypanosomes appears to be limited.     

The relative significance of mechanisms of antigenic variation in African trypanosomes

The large number of genes involved in antigenic variation in African trypanosomes has been the focus of a wide literature that describes an almost bewildering array of mechanisms for their differential activation. To the outsider searching for an underlying strategy for antigenic variation, this can appear as a rather disordered and confusing picture. Here, David Barry argues that an understanding of which mechanisms are significant, which ones are primarily inconsequential and which ones perhaps even arise from overdependence on laboratory models, might be achieved by turning attention to trypanosomes that have not undergone adaptation in laboratory conditions. Application of such an approach has led to a proposal for a main mechanism for antigenic variation.     

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.     

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.     

Malaria and trypanosome transmission: different parasites, same rules?

African trypanosomes produce different specialized stages for within-host replication and between-host transmission and therefore face a resource allocation trade-off between maintaining the current infection (survival) and investment into transmission (reproduction). Evolutionary theory predicts the resolution of this trade-off will significantly affect virulence and infectiousness. The application of life history theory to malaria parasites has provided novel insight into their strategies for survival and reproduction; how this framework can now be applied to trypanosomes is discussed. Specifically, predictions for how parasites trade-off investment in survival and transmission in response to variation in the within-host environment are outlined. An evolutionary approach has the power to explain why patterns of investment vary between strains and during infections, giving important insights into parasite biology.     

Surface proteins,ERAD and antigenic variation in Trypanosoma brucei

Variant surface glycoprotein (VSG) is central to antigenic variation in African trypanosomes. Although much prior work documents that VSG is efficiently synthesized and exported to the cell surface, it was recently claimed that 2–3 fold more is synthesized than required, the excess being eliminated by ER‐Associated Degradation (ERAD) (Field et al., 2010 ). We now reinvestigate VSG turnover and find no evidence for rapid degradation, consistent with a model whereby VSG synthesis is precisely regulated to match requirements for a functional surface coat on each daughter cell. However, using a mutated version of the ESAG7 subunit of the transferrin receptor (E7:Ty) we confirm functional ERAD in trypanosomes. E7:Ty fails to assemble into transferrin receptors and accumulates in the ER, consistent with retention of misfolded protein, and its turnover is selectively rescued by the proteasomal inhibitor MG132. We also show that ER accumulation of E7:Ty does not induce an unfolded protein response. These data, along with the presence of ERAD orthologues in the Trypanosoma brucei genome, confirm ERAD in trypanosomes. We discuss scenarios in which ERAD could be critical to bloodstream parasites, and how these may have contributed to the evolution of antigenic variation in trypanosomes.     

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 ecology of antigenic variation

A detailed molecular analysis using recombinant DNA technologies is extremely important to our understanding of the phenomena of antigenic variation in the African trypanosomes; however, by itself, it may not completely explain antigenic variation as it occurs in vivo. Several laboratories have demonstrated the ability of one variant population to replace another in vivo as well as the presence of heterogeneous populations of trypanosomes within an individual animal. These two phenomena do not permit us to explain antigen variation solely on the basis of the molecular regulation of variant antigen expression. In addition to studies in molecular biology, it will be necessary to define clearly the differences in growth rates of variant populations and the role of competition between these variants in a single anatomical site. It will also be necessary to determine the influence of various physiological environments on growth rates and the competition between the different variants of a single repertoire. It is concluded that the phenomenon of antigenic variation is a complex problem in ecology and population dynamics as well as molecular regulation. This paper is designated to examine a variety of the ecological parameters presumably involved in antigenic variation.     

The Ecology of Antigenic Variation1,2

A detailed molecular analysis using recombinant DNA technologies is extremely important to our understanding of the phenomena of antigenic variation in the African trypanosomes; however, by itself, it may not completely explain antigenic variation as it occurs in vivo. Several laboratories have demonstrated the ability of one variant population to replace another in vivo as well as the presence of heterogeneous populations of trypanosomes within an individual animal. These two phenomena do not permit us to explain antigen variation solely on the basis of the molecular regulation of variant antigen expression. In addition to studies in molecular biology, it will be necessary to define clearly the differences in growth rates of variant populations and the role of competition between these variants in a single anatomical site. It will also be necessary to determine the influence of various physiological environments on growth rates and the competition between the different variants of a single repertoire. It is concluded that the phenomenon of antigenic variation is a complex problem in ecology and population dynamics as well as molecular regulation. This paper is designated to examine a variety of the ecological parameters presumably involved in antigenic variation.     

Trypanosoma lewisi Infections in Normal Rats and in Rats Treated with Dexamethasone

SYNOPSIS. Administration of dexamethasone to rats infected with Trypanosoma lewisi resulted in the development of exceedingly large populations of trypanosomes which were fatal to their hosts. The elevated levels of parasitemia in treated rats early in infections were thought not to be a result of an increased reproductive rate. However, trypanosomes in treated rats 2 days postinfection did have a higher coefficient of variation in total length and a greater percentage of dividing forms than those observed from infected rats which were not given the drug. The course of infection may be markedly altered not only in intensity but also in length by this corticosteroid. It is suggested that dexamethasone administered at the levels recorded to rats infected with T. lewisi inhibits the production of ablastin and trypanocidal antibodies.     

Neisserial surface variation: how and why?

Neisseria gonorrhoeae exhibits striking variability in several of its surface components (pili, Opa proteins and lipooligosaccharide) in vivo and in vitro. Such flagrant variation of this mucosal pathogen's surface components contrasts sharply with changes in single surface components of blood-borne trypanosomes and borreliae. Despite these differences, similar molecular events are sometimes involved.     

Rapid change of the repertoire of variant surface glycoprotein genes in trypanosomes by gene duplication and deletion

To study the evolution of the variant surface glycoprotein (VSG) repertoire of trypanosomes we have analysed the DNA region surrounding the VSG 118 gene in different trypanosome strains. We find a remarkable degree of variation in this area. Downstream from the 118 gene a 5.7 X 10(3) base-pair DNA segment containing a potential VSG gene has been quadruplicated in strain 427 of Trypanosoma brucei, but not in most other strains analysed. The VSG 1.1000 gene, located immediately upstream from the 118 gene in one trypanosome strain, has been cleanly deleted in another. Our results are most easily explained by multiple unequal cross-overs between sister chromatids and are the first indication that sister chromatid exchange occurs in trypanosomes.     

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.     

A Phylogenetic Lineage of Closely Related Trypanosomes (Trypanosomatidae,Kinetoplastida) of Anurans and Sand Flies (Psychodidae,Diptera) Sharing the Same Ecotopes in Brazilian Amazonia1

ABSTRACT. Analysis of the phylogenetic relationships among trypanosomes from vertebrates and invertebrates disclosed a new lineage of trypanosomes circulating among anurans and sand flies that share the same ecotopes in Brazilian Amazonia. This assemblage of closely related trypanosomes was determined by comparing whole SSU rDNA sequences of anuran trypanosomes from the Brazilian biomes of Amazonia, the Pantanal, and the Atlantic Forest and from Europe, North America, and Africa, and from trypanosomes of sand flies from Amazonia. Phylogenetic trees based on maximum likelihood and parsimony corroborated the positioning of all new anuran trypanosomes in the aquatic clade but did not support the monophyly of anuran trypanosomes. However, all analyses always supported four major clades (An01‐04) of anuran trypanosomes. Clade An04 is composed of trypanosomes from exotic anurans. Isolates in clades An01 and An02 were from Brazilian frogs and toads captured in the three biomes studied, Amazonia, the Pantanal and the Atlantic Forest. Clade An01 contains mostly isolates from Hylidae whereas clade An02 comprises mostly isolates from Bufonidae; and clade An03 contains trypanosomes from sand flies and anurans of Bufonidae, Leptodactylidae, and Leiuperidae exclusively from Amazonia. To our knowledge, this is the first study describing morphological and growth features, and molecular phylogenetic affiliation of trypanosomes from anurans and phlebotomines, incriminating these flies as invertebrate hosts and probably also as important vectors of Amazonian terrestrial anuran trypanosomes.     

DNA breaks as triggers for antigenic variation in African trypanosomes

The DNA repair machinery has been co-opted for antigenic variation in African trypanosomes. New work directly demonstrates that a double-strand break initiates a switch in the expressed variant surface coat.