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 brucei brucei: in vitro production of metacyclic forms.

An in vitro method has been established to obtain metacyclic form populations of Trypanosoma brucei brucei. Trypanosome populations containing more than 98% of metacyclic forms were obtained from cultures which were: 1) initiated with bloodstream forms in primary cultures in the presence of Microtus montanus embryonic fibroblast-like cells (feeder cell layers); 2) maintained in glucose-free Eagle's minimum essential medium supplemented with 10 mM L-proline, 2 mM L-glutamine and 20% (v/v) fetal bovine serum at 27 degrees C without medium change for five days; 3) subcultured in the absence of the feeder cell layers but in the presence of Cytodex 3 beads; 4) maintained for an additional nine days with medium changes on days 5, 8 and 11; and 5) harvested on day 14 by means of diethylaminoethyl cellulose column chromatography prior to the appearance of other infective forms. Most of the trypanosomes obtained under these conditions were morphologically similar to metacyclic forms derived from tsetse fly vectors, coated with variable surface glycoprotein and were infective for mice. In the primary cultures procyclic forms, epimastigotes and metacyclic forms appeared by day 8. When the duration of the subculture was prolonged to 17 days or more at 27 degrees C, the metacyclic forms decreased in number while short trypomastigotes, long slender epimastigotes, and long slender trypomastigotes increased in number. These forms in such long-term cultures also appeared in diethylaminoethyl cellulose-isolated populations along with metacyclic forms.     

Purification of metacyclic forms of Trypanosoma cruzi by Percoll discontinuous gradient centrifugation

A method for the purification of metacyclic forms of Trypanosoma cruzi has been developed. Metacyclic forms obtained in modified Grace medium were separated from the epimastigote forms by Percoll discontinuous density gradient centrifugation. Four different osmotic pressures were applied: 160 +/- 10, 260 +/- 10, 310 +/- 10 and 510 +/- 10 mosmol/kg H2O. At 160 mosmol/kg H2O, 100% of the metacyclic forms with a 21.3% yield were found in the interphase 1.120/1.125 g/ml, while 92.7% of the metacyclic forms with a 73.7% yield were found in the interphase 1.115/1.120 g/ml. At 310 mosmol/kg H2O, 100% of the metacyclic forms in the interphase 1.135/1.140 g/ml with a 36.8% yield were obtained. Metacyclic forms purified in this way do not show alterations in their capacity to infect cultures of HeLa cells.     

Disulfide bond as a structural determinant of prion protein membrane insertion

Conversion of the normal soluble form of prion protein, PrP (PrP^C), to proteinase K-resistant form (PrP^Sc) is a common molecular etiology of prion diseases. Proteinase K-resistance is attributed to a drastic conformational change from α-helix to β-sheet and subsequent fibril formation. Compelling evidence suggests that membranes play a role in the conformational conversion of PrP. However, biophysical mechanisms underlying the conformational changes of PrP and membrane binding are still elusive. Recently, we demonstrated that the putative transmembrane domain (TMD; residues 111–135) of Syrian hamster PrP penetrates into the membrane upon the reduction of the conserved disulfide bond of PrP. To understand the mechanism underlying the membrane insertion of the TMD, here we explored changes in conformation and membrane binding abilities of PrP using wild type and cysteine-free mutant. We show that the reduction of the disulfide bond of PrP removes motional restriction of the TMD, which might, in turn, expose the TMD into solvent. The released TMD then penetrates into the membrane. We suggest that the disulfide bond regulates the membrane binding mode of PrP by controlling the motional freedom of the TMD.     

Crossed-immunoelectrophoretic analyses of Trypanosoma cruzi epimastigote, metacyclic, and bloodstream forms

Sonicated suspensions of epimastigote, metacyclic, or bloodstream forms of Trypanosoma cruzi were emulsified in Freund's complete adjuvant. Rabbits immunized with epimastigotes or metacyclics received five intramuscular (i.m.) injections of 1 x 10(9) sonicated trypanosomes at weekly intervals. Immunization with bloodstream forms included three i.m. injections of 5 x 10(7) and six injections of 2 x 10(8) sonicated trypanosomes. Selected antisera from these rabbits were employed in crossed immunoelectrophoretic studies against the homologous or heterologous extracts of sonicated trypanosomes. Extracts of epimastigote, metacyclic, and trypomastigotes produced 31, 29, and 11 precipitin peaks respectively against the homologous rabbit antisera. Tandem, crossed-immunoelectrophoresis of these extracts against antiepimastigote or antimetacyclic sera revealed that epimastigotes or metacyclics may each have at least four antigens that did not appear to be shared by the other, whereas each of these forms may have at least eight or nine antigens that were not detected with extracts from trypomastigotes. Cross-absorptions of antiepimastigote or antimetacyclic sera with live trypanosomes caused marked reductions in the numbers of precipitin peaks formed against the homologous extracts, but cross-absorptions with sonicated suspensions of epimastigotes or metacyclics showed that epimastigotes or metacyclics each have at least two antigens that were not detected in extracts of the other. Differentiation appeared to be accompanied by antigenic change. More antigens appear to be shared by epimastigotes and metacyclic forms than by trypomastigotes and epimastigotes or metacyclics.     

Morphological comparison of axenic amastigogenesis of trypomastigotes and metacyclic forms of Trypanosoma cruzi

Amastigogenesis occurs first when metacyclic trypomastigotes from triatomine urine differentiate into amastigotes inside mammalian host cells and a secondary process when tissue-derived trypomastigotes invade new cells and differentiate newly to amastigotes. Using scanning electron microscopy, we compared the morphological patterns manifested by trypomastigotes and metacyclic forms of Trypanosoma cruzi during their axenic-transformation to amastigotes in acidic medium at 37 C. We show here that in culture MEMTAU medium, secondary and primary axenic amastigogenesis display different morphologies. As already described, we also observed a high differentiation rate of trypomastigotes into amastigotes. Conversely, the transformation rate of in vitro-induced-metacyclic trypomastigotes to amastigotes was significantly slower and displayed distinct patterns of transformation that seem environment-dependent. Morphological comparisons of extracelullar and intracellular amastigotes showed marked similarities, albeit some differences were also detected. SDS-PAGE analyses of protein and glycoprotein from primary and axenic extracelullar amastigotes showed similarities in glycopeptide profiles, but variations between their proteins demonstrated differences in their respective macromolecular constitutions. The data indicate that primary and axenic secondary amastigogenesis of T. cruzi may be the result of different developmental processes and suggest that the respective intracellular mechanisms driving amastigogenesis may not be the same.     

Characterisation of a cysteine protease from bloodstream forms of Trypanosoma congolense.

A cysteine protease (trypanopain-Tc) with cathepsin-L-like properties has been purified from Trypanosoma congolense. The enzyme has an apparent molecular mass of 31-32 kDa by SDS/PAGE and 66 kDa by gel chromatography. It has a pI 7.4 and a high affinity for concanavalin A. Trypanopain-Tc catalyses the limited proteolysis of a variety of protein substrates such as fibrinogen, serum albumin and trypanosome variant-surface glycoprotein. It has minimal or no activity against casein or elastin. A variety of peptidyl amidomethylcoumarins and peptidyl diazomethanes were used to test the specificity of trypanopain-Tc. The better substrates had Arg or Lys in P1 and hydrophobic amino acids in P2 and P3. The best substrate found for trypanopain-Tc was Z-Phe-Arg-NHMec (Z, benzyloxycarbonyl; NHMec, 7-amido-4-methylcoumarin). The kinetic constants for the hydrolysis of Z-Phe-Arg-NHMec were kcat = 17.4 s-1, Km = 4.4 microM, kcat/Km = 4.0 microM-1.s-1, which are very similar to those of cathepsin L with this substrate. The specific substrates for cathepsin B (Z-Arg-Arg-NHMec) and cathepsin H (Arg-NHMec) were not hydrolysed by trypanopain-Tc under the conditions tested. The pH optimum of trypanopain-Tc against Z-Phe-Arg-NHMec was pH 6.0 but it showed a broad peak of activity extending well into the alkaline region. The enzyme was activated by low-molecular-mass thiol compounds and inhibited by cystatin, L-trans-epoxysuccinyl-4-guanidinobutane (E-64) and a variety of peptidyl diazomethanes. The most effective diazomethane inhibitors (Z-Leu-Leu-Met-CHN2, Z-Leu-Met-CHN2 and Z-Leu-Lys-CHN2, were inhibitory at nanomolar concentrations and were trypanocidal in vitro after 24-48 h incubation in greater than or equal to 20 microM [inhibitor]. However, it is not clear whether the trypanocidal activity of these inhibitors is a consequence of the inhibition of trypanopains or of some other essential proteolytic activities within the parasites.     

In vitro growth inhibition of bloodstream forms of Trypanosoma brucei and Trypanosoma congolense by iron chelators

African trypanosomes exert significant morbidity and mortality in man and livestock. Only a few drugs are available for the treatment of trypanosome infections and therefore, the development of new anti-trypanosomal agents is required. Previously it has been shown that bloodstream-form trypanosomes are sensitive to the iron chelator deferoxamine. In this study the effect of 13 iron chelators on the growth of Trypanosoma brucei, T. congolense and human HL-60 cells was tested in vitro. With the exception of 2 compounds, all chelators exhibited anti-trypanosomal activities, with 50% inhibitory concentration (IC₅₀) values ranging between 2.1 – 220 μM. However, the iron chelators also displayed cytotoxicity towards human HL-60 cells and therefore, only less favourable selectivity indices compared to commercially available drugs. Interfering with iron metabolism may be a new strategy in the treatment of trypanosome infections. More specifically, lipophilic iron-chelating agents may serve as lead compounds for novel anti-trypanosomal drug development.     

Cross-reactivity of vector-borne metacyclic forms of Trypanosoma cruzi with mammalian and culture stages

Metacyclic forms of Trypanosoma cruzi isolated from the hindgut of infected insect vectors (Rhodnius prolixus) were found to be immunologically cross-reactive with cultured epimastigote, amastigote, and metacyclic stages of the parasite as well as with bloodstream trypomastigote forms by direct agglutination and indirect immunofluorescence techniques. Sera specific for each of these forms of the parasite systematically yielded maximal antibody titers when measured against the homologous antigen, indicating that antigenic determinants are shared by all of the developmental forms used in this work. Supporting this conclusion were the significant reductions in anti-insect-derived metacyclic antibody titer caused by absorption with any of the other life stages of T. cruzi. These results are relevant to the potential use of laboratory-grown forms of T. cruzi in vaccination against a natural infection with this parasite.     

In vitro production of metacyclic forms of Trypanosoma musculi and their infectivity in CBA mice

Bloodstream forms of Trypanosoma musculi, cultured in Schneider's drosophila medium at room temperature, multiply and differentiate through a series of developmental stages into infective metacyclic trypomastigotes in 10 days. Oral inoculation with these culture forms into CBA mice produced a parasitemia similar to that produced by intraperitoneal infection with bloodstream forms except for a three-day longer prepatent period. Attempts to induce parasitemia with bloodstream forms given orally were unsuccessful.     

Trypanosoma congolense bloodstream forms evade complement lysis in vitro by shedding of immune complexes

In the presence of antibodies against the variant surface glycoprotein (VSG) and guinea pig complement, Trypanosoma congolense bloodstream forms were lysed. Parasites, which had been preincubated with antibodies at 37 degrees C before addition of complement, escaped from complement lysis in a time- and temperature-dependent process. Preincubation caused removal of the antibodies from the cell surface by formation of filopodia and accumulation of the immune complexes between aggregated cells. Addition of secondary antibodies or of complement component C1q did not enhance this effect. In order to eliminate effects due to cell aggregation, single living trypanosomes, which had been immobilized by attachment to formvar-coated glass slides, were incubated under equivalent conditions. Immunofluorescence showed that in these experiments, anti-VSG antibodies were neither capped nor shed from the surface unless coincubation with secondary antibodies or C1q was performed. Fixation of the cells after incubation with anti-VSG prevented patching and capping of the antibodies. Removal of immune complexes apparently required no secondary cross-linker: removal from the surface of T. congolense obviously occurred during cell aggregation. This mechanism could therefore be of significance also in vivo.     

Trypanosoma brucei: in vitro propagation of metacyclic forms derived from the salivary glands of Glossina morsitans

1 Metacyclic forms of Trypanosoma brucei obtained from the salivary glands of the tsetse fly, Glossina morsitans have been cultured for the first time in their infective forms for more than 200 days in continuous culture. The parasites were grown at 25 C and 30 C on a bovine embryonic spleen (BESP) feeder layer in buffered RPMI 1640 medium supplemented with 20% heat-inactivated bovine fetal serum (BFS) and 5% lactalbumin hydrolysate. Initial growth rate was enhanced when normal, noninfected, salivary glands were added to the cultures. The parasites thus cultured appeared like slender or intermediate blood stream forms which were infective to rats and mice. Addition of rat anti-T. brucei specific antiserum to the cultures caused agglutination of the parasites and rendered them noninfective. This study opens up new areas of investigating sleeping sickness. The cultured metacyclic parasites have the potential of being applied as antigens for controlling African trypanosomiasis.     

Azaanthraquinone inhibits respiration and in vitro growth of long slender bloodstream forms of Trypanosoma congolense

An ethanolic extract of Mitracarpus scaber was found to possess in vitro and in vivo trypanocidal activity against Trypanosoma congolense. At a dosage of 50 mg kg(-1) day(-1) in normal saline for 5 days, the extract cured Balbc mice infected with T. congolense without any relapse. The isolated active component benz(g)isoquinoline 5,10 dione (Azaanthraquinone) (AQ) purified from the extract was found to inhibit glucose-dependent cellular respiration and glycerol-3-phosphate-dependent mitochondrial O(2) assimilation of the long bloodstream forms of Trypanosoma congolense. On account of the pattern of inhibition, the target could be the mitochondrial electron transport system composed of glyceraldehyde 3-phosphate dehydrogenase (G3PDH). The azaanthraquinone specifically inhibited the reduced coenzyme Q(1)-dependent O(2) uptake of the mitochondria with respect to ubiquinone. The susceptible site could be due to ubiquinone redox system which links the two enzyme activities.     

The variant surface glycoproteins of Trypanosoma equiperdum. Identification of a phosphorylated glycopeptide as the cross-reacting antigenic determinant

The cross-reacting antigenic determinant in the variant surface glycoproteins (VSGs) of Trypanosoma equiperdum was studied by testing the ability of VSG glycopeptides to bind heterologous anti-VSG sera. VSG glycopeptide purification revealed the presence of 3 oligosaccharide sidechains on the mature VSG. These consist of two sidechains containing only mannose and glucosamine and a third containing galactose and mannose (in a 5:1 ratio) as well as phosphorous and ethanolamine. This phosphorylated fragment completely blocked the binding of VSG to heterologous anti-VSG and therefore contained the cross-reacting determinants.     

Characterization of the cross-reacting determinant (CRD) of the glycosyl-phosphatidylinositol membrane anchor of Trypanosoma brucei variant surface glycoprotein

The cross-reacting determinant (CRD epitope) of the glycosyl-phosphatidylinositol (GPI) membrane anchor of Trypanosoma brucei variant surface glycoprotein has been analysed by selective chemical and enzymic modification of the isolated GPI structure combined with the use of a competitive ELISA inhibition assay for the detection of CRD epitopes. The data show that the CRD consists of at least three overlapping epitopes involving different regions of the molecule including the inositol 1,2-cyclic phosphate, the non-N-acetylated-glucosamine residue and the galactose branch. Although the presence of all three of these structural features is required for quantitative binding of anti-CRD antibodies in ELISA and Western blotting, the Western blot reaction obtained in the presence of any one epitope is still significant. The use of anti-CRD antibodies for the detection of GPI anchors is discussed.     

MS and NMR analysis of the cross-reacting determinant glycan from Trypanosoma brucei brucei MITat 1.6 variant specific glycoprotein

The cross-reacting determinant glycan from Trypanosoma brucei brucei MITat 1.6 is known to contain galactose, mannose and non-acetylated glucosamine. The structural elucidation of this oligosaccharide has been impeded by an unusual non-glycosidic linkage to the peptide chain and a glycosidic linkage to inositol phosphate on either side of the oligosaccharide. Using two different approaches for the isolation of the glycan, namely hydrolysis to give the oligosaccharide directly or pronase digestion to yield the glycan-containing C-terminal glycophosphopeptide, the structure of this glycan was elucidated by mass spectrometry and 1H-NMR spectroscopy. There was evidence of heterogeneity in the glycan residue.     

Biochemical Diversity in the Trypanosoma congolense Trans-sialidase Family

Trans-sialidases are key enzymes in the life cycle of African trypanosomes in both, mammalian host and insect vector and have been associated with the disease trypanosomiasis, namely sleeping sickness and nagana. Besides the previously reported TconTS1, we have identified three additional active trans-sialidases, TconTS2, TconTS3 and TconTS4, and three trans-sialidase like genes in Trypanosoma congolense. At least TconTS1, TconTS2 and TconTS4 are found in the bloodstream of infected animals. We have characterised the enzymatic properties of recombinant proteins expressed in eukaryotic fibroblasts using fetuin as model blood glycoprotein donor substrate. One of the recombinant trans-sialidases, TconTS2, had the highest specific activity reported thus far with very low sialidase activity. The active trans-sialidases share all the amino acids critical for the catalytic reaction with few variations in the predicted binding site for the leaving or acceptor glycan. However, these differences cannot explain the orders of magnitudes between their transfer activities, which must be due to other unidentified structural features of the proteins or substrates selectivity. Interestingly, the phylogenetic relationships between the lectin domains correlate with their specific trans-sialylation activities. This raises the question whether and how the lectin domains regulate the trans-sialidase reaction. The identification and enzymatic characterisation of the trans-sialidase family in T. congolense will contribute significantly towards the understanding of the roles of these enzymes in the pathogenesis of Animal African Trypanosomiasis.     

Morphological evidence by scanning electron microscopy of excretion of metacyclic forms of Trypanosoma cruzi in vector's urine

Comparison by scanning electron microscopy (SEM) of Trypanosoma cruzi flagellates attached to the cuticle of the rectal gland of infected Dipetalogaster maxima nymphs, showed marked differences before and after feeding. Before feeding numerous metacyclic trypomastigotes were observed among the abundant epimastigotes that formed the carpet of flagellates. On the other hand, in insects that were allowed to urinate for 24 hours after a meal, the metacyclics were scarce, indicating that they had been detached by the urine flow. An asymmetric type of cell division, probably originating both an epi- and a trypomastigote, was occasionally observed. The occurrence of swellings at different levels of the flagella of epimastigotes suggests that secondary sites of attachment may be common.