Trypanosoma congolense procyclins: unmasking cryptic major surface glycoproteins in procyclic forms

In the tsetse fly, the protozoan parasite Trypanosoma congolense is covered by a dense layer of glycosylphosphatidylinositol (GPI)-anchored molecules. These include a protease-resistant surface molecule (PRS), which is expressed by procyclic forms early in infection, and a glutamic acid- and alanine-rich protein (GARP), which appears at later stages. Since neither of these surface antigens is expressed at intermediate stages, we investigated whether a GPI-anchored protein of 50 to 58 kDa, previously detected in procyclic culture forms, might constitute the coat of these parasites. We therefore partially purified the protein from T. congolense Kilifi procyclic forms, obtained an N-terminal amino acid sequence, and identified its gene. Detailed analyses showed that the mature protein consists almost exclusively of 13 heptapeptide repeats (EPGENGT). The protein is densely N glycosylated, with up to 13 high-mannose oligosaccharides ranging from Man(5)GlcNAc(2) to Man(9)GlcNAc(2) linked to the peptide repeats. The lipid moiety of the glycosylphosphatidylinositol is composed of sn-1-stearoyl-2-lyso-glycerol-3-HPO(4)-1-(2-O-acyl)-d-myo-inositol. Heavily glycosylated proteins with similar repeats were subsequently identified in T. congolense Savannah procyclic forms. Collectively, this group of proteins was named T. congolense procyclins to reflect their relationship to the EP and GPEET procyclins of T. brucei. Using an antiserum raised against the EPGENGT repeat, we show that T. congolense procyclins are expressed continuously in the fly midgut and thus form the surface coat of cells that are negative for both PRS and GARP.     

Trypanosoma simiae and Trypanosoma congolense: surface glycoconjugates of procyclic forms-the same coats on different hangers?

Organic solvent extraction, reverse-phase high performance liquid chromatography and enzyme-linked immunosorbent assay with surface binding monoclonal antibodies were used to isolate membrane molecules of procyclic culture forms of Trypanosoma simiae and Trypanosoma congolense. Gel electrophoresis of the purified molecules revealed two predominant molecular species from each parasite that were broadly similar yet showed different apparent molecular masses and staining characteristics. The molecules were shown to be glycosylphosphatidylinositol-lipid anchored glycoconjugates, rich in carbohydrates. Each moiety displayed surface-disposed carbohydrate epitopes that were recognized on the surface of both species of trypanosomes by monoclonal antibodies specific for procyclic parasites of the subgenus Nannomonas. The epitopes were previously shown to be displayed on the glutamic acid-alanine rich protein of T. congolense yet neither this protein, nor its encoding gene is present in T. simiae. The results indicate that although T. congolense and T. simiae share common carbohydrate surface epitopes, these are displayed on biochemically different molecules. We speculate that the surface disposed carbohydrate structures are involved in parasite-tsetse interactions since these species have the same developmental cycles in the insect vector.     

Immunisation of cattle with cysteine proteinases of Trypanosoma congolense: targetting the disease rather than the parasite

In order to test the hypothesis that trypanosome cysteine proteinases (CPs) contribute to pathology of trypanosomosis, cattle were immunised with CP1 and/or CP2, the major CPs of Trypanosoma congolense, and subsequently challenged with T. congolense. Immunisation had no effect on the establishment of infection and the development of acute anaemia. However, immunised cattle, unlike control cattle, maintained or gained weight during infection. Their haematocrit and leukocyte counts showed a tendency to recovery after 2-3 months of infection. Cattle immunised with CP2 mounted early and prominent IgG responses to CPs and to the variable surface glycoprotein following challenge. Thus trypanosome CPs may play a role in anaemia and immunosuppression; conversely, anti-CP antibody may modulate the trypanosome-induced pathology.     

Characterization of a novel alanine-rich protein located in surface microdomains in Trypanosoma brucei

Heterologous expression in COS cells followed by orientation-specific polymerase chain reaction to select and amplify cDNAs encoding surface proteins in Trypanosoma brucei resulted in the isolation of a cDNA ( approximately 1.4 kilobase) which encodes an acidic, alanine-rich polypeptide that is expressed only in bloodstream forms of the parasite and has been termed bloodstream stage alanine-rich protein (BARP). Analysis of the amino acid sequence predicted the presence of a typical NH(2)-terminal leader sequence as well as a COOH-terminal hydrophobic extension with the potential to be replaced by a glycosylphosphatidylinositol anchor. A search of existing protein sequences revealed partial homology between BARP and the major surface antigen of procyclic forms of Trypanosoma congolense. BARP migrated as a complex, heterogeneous series of bands on Western blots with an apparent molecular mass ( approximately 50-70 kDa) significantly higher than predicted from the amino acid sequence ( approximately 26 kDa). Confocal microscopy demonstrated that BARP was present in small discrete spots that were distributed over the entire cellular surface. Detergent extraction experiments revealed that BARP was recovered in the detergent-insoluble, glycolipid-enriched fraction. These data suggested that BARP may be sequestered in lipid rafts.     

Detection and identification of Trypanosoma of African livestock through a single PCR based on internal transcribed spacer 1 of rDNA

Primers hybridising with the rDNA cistron have previously been evaluated for PCR diagnosis specific for kinetoplastids, and shown to detect and differentiate the Trypanosoma brucei complex and Trypanosoma cruzi. Kin1 and Kin2 primers, amplifying internal transcribed spacer 1, were subsequently evaluated for the diagnosis of African livestock trypanosomosis. Based on the size of the PCR products obtained, Kin primers allowed detection and identification of three Trypanosoma congolense types (savannah, forest and Kenya Coast), with distinction among themselves and from the subgenus Trypanozoon (T. brucei spp., Trypanosoma evansi and Trypanosoma equiperdum), Trypanosoma vivax, Trypanosoma simiae and Trypanosoma theileri. These primers were shown to be suitable for the sensitive and type-specific diagnosis of African livestock trypanosome isolates through a single PCR even in the case of multi-taxa samples. With field samples (buffy-coat from cattle blood) sensitivity was close to the sensitivity observed in single reactions with the classical specific primers for the Trypanozoon subgenus and T. congolense-type savannah, but was lower for detection of T. vivax. Additional reaction, improvement of DNA preparation, and/or new primers design are necessary to improve the sensitivity for detection of T. vivax in field samples. However, these primers are suitable for isolate typing through a single PCR.     

Alternative oxidase (AOX) genes of African trypanosomes: phylogeny and evolution of AOX and plastid terminal oxidase families

To clarify evolution and phylogenetic relationships of trypanosome alternative oxidase (AOX) molecules, AOX genes (cDNAs) of the African trypanosomes, Trypanosoma congolense and Trypanosoma evansi, were cloned by PCR. Both AOXs possess conserved consensus motifs (-E-, -EXXH-). The putative amino acid sequence of the AOX of T. evansi was exactly the same as that of T. brucei. A protein phylogeny of trypanosome AOXs revealed that three genetically and pathogenically distinct strains of T. congolense are closely related to each other. When all known AOX sequences collected from current databases were analyzed, the common ancestor of these three Trypanosoma species shared a sister-group position to T. brucei/T. evansi. Monophyly of Trypanosoma spp. was clearly supported (100% bootstrap value) with Trypanosoma vivax placed at the most basal position of the Trypanosoma clade. Monophyly of other eukaryotic lineages, terrestrial plants + red algae, Metazoa, diatoms, Alveolata, oomycetes, green algae, and Fungi, was reconstructed in the best AOX tree obtained from maximum likelihood analysis, although some of these clades were not strongly supported. The terrestrial plants + red algae clade showed the closest affinity with an alpha-proteobacterium, Novosphingobium aromaticivorans, and the common ancestor of these lineages, was separated from other eukaryotes. Although the root of the AOX subtree was not clearly determined, subsequent phylogenetic analysis of the composite tree for AOX and plastid terminal oxidase (PTOX) demonstrated that PTOX and related cyanobacterial sequences are of a monophyletic origin and their common ancestor is linked to AOX sequences.     

Cleavage of trypanosome surface glycoproteins by alkaline trypsin-like enzyme(s) in the midgut of Glossina morsitans

EP and GPEET procyclin, the major surface glycoproteins of procyclic forms of Trypanosoma brucei, are truncated by proteases in the midgut of the tsetse fly Glossina morsitans morsitans. We show that soluble extracts from the midguts of teneral flies contain trypsin-like enzymes that cleave the N-terminal domains from living culture-derived parasites. The same extract shows little activity against a variant surface glycoprotein on living bloodstream form T. brucei (MITat 1.2) and none against glutamic acid/alanine-rich protein, a major surface glycoprotein of Trypanosoma congolense insect forms although both these proteins contain potential trypsin cleavage sites. Gel filtration of tsetse midgut extract revealed three peaks of tryptic activity against procyclins. Trypsin alone would be sufficient to account for the cleavage of GPEET at a single arginine residue in the fly. In contrast, the processing of EP at multiple sites would require additional enzymes that might only be induced or activated during feeding or infection. Unexpectedly, the pH optima for both the procyclin cleavage reaction and digestion of the trypsin-specific synthetic substrate Chromozym-TRY were extremely alkaline (pH 10). Direct measurements were made of the pH within different compartments of the tsetse digestive tract. We conclude that the gut pH of teneral flies, from the proventriculus to the hindgut, is alkaline, in contradiction to previous measurements indicating that it was mildly acidic. When tsetse flies were analysed 48 h after their first bloodmeal, a pH gradient from the proventriculus (pH 10.6+/-0.6) to the posterior midgut (pH 7.9+/-0.4) was observed.     

Evolutionary Insights from Bat Trypanosomes: Morphological, Developmental and Phylogenetic Evidence of a New Species, Trypanosoma (Schizotrypanum) erneyi sp. nov., in African Bats Closely Related to Trypanosoma (Schizotrypanum) cruzi and Allied Species

Parasites of the genus Trypanosoma are common in bats and those of the subgenus Schizotrypanum are restricted to bats throughout the world, with the exception of Trypanosoma (Schizotrypanum) cruzi that also infects other mammals and is restricted to the American Continent. We have characterized trypanosome isolates from Molossidae bats captured in Mozambique, Africa. Morphology and behaviour in culture, supported by phylogenetic inferences using SSU (small subunit) rRNA, gGAPDH (glycosomal glyceraldehyde 3-phosphate dehydrogenase) and Cyt b (cytochrome b) genes, allowed to classify the isolates as a new Schizotrypanum species named Trypanosoma (Schizotrypanum) erneyi sp. nov. This is the first report of a Schizotrypanum species from African bats cultured, characterized morphologically and biologically, and positioned in phylogenetic trees. The unprecedented finding of a new species of the subgenus Schizotrypanum from Africa that is closest related to the America-restricted Trypanosoma (Schizotrypanum) cruzi marinkellei and T. cruzi provides new insights into the origin and evolutionary history of T. cruzi and closely related bat trypanosomes. Altogether, data from our study support the hypothesis of an ancestor trypanosome parasite of bats evolving to infect other mammals, even humans, and adapted to transmission by triatomine bugs in the evolutionary history of T. cruzi in the New World.     

Selection of susceptible and refractory lines of Glossina morsitans centralis for Trypanosoma congolense infection and their susceptibility to different pathogenic Trypanosoma species

Abstract .In a single generation of selection, two lines of Glossina morsitans centralis were established that differed significantly in susceptibility to Trypanosoma congolense clone IL 1180. Reciprocal crosses demonstrated that susceptibility was a maternally inherited trait. Differences between the lines, to all phases of the trypanosome infection, were maintained for eight generations, whereas differences in susceptibility to midgut infections were maintained for twenty-eight generations. Thereafter, the lines did not differ in susceptibility to Trypanosoma congolense IL 1180. Susceptibility to infections with Trypanosoma congolense IL 1180 was only a weak predictor of susceptibility to T. congolense clones IL 13-E3 and K60/1, as well as clone T. brucei brucei STIB 247-L. However, the susceptible and refractory lines displayed these phenotypes when tested with Trypanosoma vivax, indicating that the factors that affect susceptibility to trypanosomes are expressed both within and outside the midgut.     

Trying to predict and explain the presence of African trypanosomes in tsetse flies.

Trypanosome infections identified by polymerase chain reaction on field-caught tsetse flies from various locations were analyzed with respect to factors intrinsic and extrinsic to the trypanosome-tsetse association. These factors were then simultaneously analyzed using artificial neural networks (ANNs) and the important factors were identified to predict and explain the presence of trypanosomes in tsetse. Among 4 trypanosome subgroups (Trypanosoma brucei s.l., T. congolense of the 'savannah' and of the 'riverine-forest' types, and T. simiae), the presence of the 2 types of T. congolense was predictable in more than 80% of cases, suggesting that the model incorporated some of the key variables. These 2 types of T. congolense were significantly associated in tsetse. Among all the examined factors, it was the presence of T. congolense savannah type that best explained the presence of T. congolense riverine forest type. One possible biological mechanism would be 'hitchhiking,' as previously suspected for other parasites. The model could be improved by adding other important variables to the trypanosome tsetse associations.     

Does isometamidium chloride treatment protect tsetse flies from trypanosome infections during SIT campaigns?

African animal trypanosomosis is a major pathological constraint to cattle breeding across 10 million km2 of sub-Saharan West African countries infested by tsetse flies, their cyclic vectors. The release of sterile males (sterile insect technique [SIT]) is a potentially important control technique aimed at eliminating the vectors. Prior to release, tsetse are generally treated with isometamidium chloride, a trypanocide, to prevent them from transmitting parasites. The present study investigated the preventive action of isometamidium chloride (0.5 mg/L) on the subsequent susceptibility of tsetse released into the wild. A total of 1755 Glossina palpalis gambiensis Vanderplank and 744 Glossina tachinoides Westwood were released, of which 50 and 48, respectively, were recaptured 22-43 days after release. Their probosces were analysed by polymerase chain reaction to identify mature infections with three trypanosome species (Trypanosoma vivax, Trypanosoma brucei sensu lato and Trypanosoma congolense savannah type). Two mature infections with T. vivax and four with T. congolense were detected, indicating that the use of this treatment regimen in an SIT campaign would not totally prevent sterile males from transmitting trypanosomes.     

Identification of an antigen specific to Trypanosoma congolense by using monoclonal antibodies

Using indirect immunofluorescence assays on acetone-fixed smears of a series of different parasites, we have shown that two monoclonal antibodies bind specifically to Trypanosoma congolense organisms. The antibodies bind to both bloodstream trypomastigotes and procyclic culture forms of the parasite and are thus not stage specific. Immunoprecipitation and immunoblot analysis showed that both monoclonal reagents bound a protein of approximately 31,000 m.w. This antigen appeared to be located on the plasma membrane of T. congolense, but the epitope was not exposed on the surface of living bloodstream or procyclic organisms. The antigen was detectable on acetone-fixed organisms or in trypanosome lysates in enzyme-linked immunosorbent assays and may therefore by useful as a species-specific marker in field assays for epidemiologic and clinical investigations.     

Trypanosoma (Duttonefla) vivax

Although Trypanosoma vivax was first discovered in 1905 (Ref. 1), the fact that most stocks of this parasite are restricted to ruminant hosts has retarded investigation of this species compared with the experimentally more amenable T. brucei and T. congolense. The veterinary importance of T. vivax (Box 1) and a recent report suggesting that T. vivax may have an even more extended range than previously thought (Box 2) prompts an evaluation of the current knowledge of the biology of this trypanosome.     

Structural characterization and epitope mapping of the glutamic acid/alanine-rich protein from Trypanosoma congolense: defining assembly on the parasite cell surface

Trypanosoma congolense is an African trypanosome that causes serious disease in cattle in Sub-Saharan Africa. The four major life cycle stages of T. congolense can be grown in vitro, which has led to the identification of several cell-surface molecules expressed on the parasite during its transit through the tsetse vector. One of these, glutamic acid/alanine-rich protein (GARP), is the first expressed on procyclic forms in the tsetse midgut and is of particular interest because it replaces the major surface coat molecule of bloodstream forms, the variant surface glycoprotein (VSG) that protects the parasite membrane, and is involved in antigenic variation. Unlike VSG, however, the function of GARP is not known, which necessarily limits our understanding of parasite survival in the tsetse. Toward establishing the function of GARP, we report its three-dimensional structure solved by iodide phasing to a resolution of 1.65 Å. An extended helical bundle structure displays an unexpected and significant degree of homology to the core structure of VSG, the only other major surface molecule of trypanosomes to be structurally characterized. Immunofluorescence microscopy and immunoaffinity-tandem mass spectrometry were used in conjunction with monoclonal antibodies to map both non-surface-disposed and surface epitopes. Collectively, these studies enabled us to derive a model describing the orientation and assembly of GARP on the surface of trypanosomes. The data presented here suggest the possible structure-function relationships involved in replacement of the bloodstream form VSG by GARP as trypanosomes differentiate in the tsetse vector after a blood meal.     

Feeding behaviour of Glossina pallidipes and g. morsitans centralis on Boran cattle infected with trypanosoma congolense or T. vivax under laboratory conditions

In field studies, tsetse flies (Diptera: Glossinidae) feed more successfully on cattle infected with Trypanosoma congolense Broden (Kinetoplastida: Trypanosomatidae) than on cattle infected with T. vivax Ziemann or uninfected cattle. Here we describe the first laboratory investigation of this phenomenon. In the first experiment, caged Glossina pallidipes Austen were fed for 1 and 5 min on a Boran steer infected with T. congolense clone IL 1180 and on an uninfected steer. Feeding success was recorded in this way five times over several weeks. The same protocol was subsequently used in three additional experiments with the following combinations: G. pallidipes and a steer infected with T. vivax stock IL 3913, G. morsitans centralis Machado and a steer infected with T. congolense, and G. morsitans centralis and a steer infected with T. vivax. The four experiments were replicated once, making eight experiments in total. In three experiments there was increased tsetse feeding success, measured at 1 min, after a steer became infected (T. congolense, two experiments and T. vivax, one experiment). Analysis of all data combined found no significant differences in tsetse feeding success on the different groups of cattle prior to infection, but after infection tsetse feeding success was significantly greater on the infected cattle (P< 0.001). Trypanosoma congolense infection led to a greater increase in tsetse feeding success than T. vivax infection. The increase in feeding success was not related to changes in the level of anaemia, skin surface temperature or parasitaemia. A possible explanation is the effects of trypanosome infection on cutaneous vasodilation and/or blood clotting in infected cattle. When allowed to feed for 5 min, nearly all tsetse engorged successfully and effects of cattle infection on feeding success were not found.     

The taxonomic position and evolutionary relationships of Trypanosoma rangeli.

This paper presents a re-evaluation of the taxonomic position and evolutionary relationships of Trypanosoma (Herpetosoma) rangeli based on the phylogenetic analysis of ssrRNA sequences of 64 Trypanosoma species and comparison of mini-exon sequences. All five isolates of T. rangeli grouped together in a clade containing Trypanosoma (Schizotrypanum) cruzi and a range of closely related trypanosome species from bats [Trypanosoma (Schizotrypanum) dionisii, Trypanosoma (Schizotrypanum) vespertilionis] and other South American mammals [Trypanosoma (Herpetosoma) leeuwenhoeki, Trypanosoma (Megatrypanum) minasense, Trypanosoma (Megatrypanum) conorhini] and an as yet unidentified species of trypanosome from an Australian kangaroo. Significantly T. rangeli failed to group with (a) species of subgenus Herpetosoma, other than those which are probably synonyms of T. rangeli, or (b) species transmitted via the salivarian route, although either of these outcomes would have been more consistent with the current taxonomic and biological status of T. rangeli. We propose that use of the names Herpetosoma and Megatrypanum should be discontinued, since these subgenera are clearly polyphyletic and lack evolutionary and taxonomic relevance. We hypothesise that T. rangeli and T. cruzi represent a group of mammalian trypanosomes which completed their early evolution and diversification in South America.     

New epidemiological features on animal trypanosomiasis by molecular analysis in the pastoral zone of Sideradougou, Burkina Faso

A multidisciplinary work was undertaken in the agropastoral zone of Sidéradougou, Burkina Faso to try to elucidate the key factors determining the presence of tsetse flies. In this study the PCR was used to characterize trypanosomes infecting the vector ( Glossina tachinoides and Glossina palpalis gambiensis ) and the host, i.e. cattle. A 2-year survey involved dissecting 2211 tsetse of the two Glossina species. A total of 298 parasitologically infected tsetse were analysed by PCR. Trypanosoma vivax was the most frequently identified trypanosome followed by the savannah type of T. congolense and, to a lesser extent, the riverine forest type of T. congolense , and by T. brucei . No cases of T. simiae were found. From the 107 identified infections in cattle, the taxa were the same, but T. congolense savannah type was more frequent, whereas T. vivax and T. congolense riverine forest types were found less frequently. A correlation was found between midgut infection rates of tsetse, nonidentified infections and reptile bloodmeals. These rates were higher in G.p. gambiensis , and in the western part of the study area. T. vivax infections were related to cattle bloodmeals, and were more frequent in G. tachinoides and in the eastern study area. The PCR results combined with bloodmeal analysis helped us to establish the relationships between the vector and the host, to assess the trypanosome challenge in the two parts of the area, to elucidate the differences between the two types of T. congolense , and to suspect that most midgut infections were originating from reptilian trypanosomes.     

Tsetse immunity and the transmission of trypanosomiasis

Cyclical transmission of African trypanosomes - Trypanosoma congolense and subspecies of T. brucei - depends on their uptake by and development within their tsetse fly vectors. Tsetse susceptibility to such trypanosome infection seems to be controlled by maternally inherited rickettsia-like organisms (RLOs) (Fig. 1) and it now seems that the RLOs may exert this effect by controlling midgut lectins in the fly. Ian Maudlin and Susan Welburn explain the latest findings.