Trypanosoma congolense: Surface glycoproteins of two early bloodstream variants: III. Immunochemical characterization

A radioimmunoassay (RIA) for the variant-specific glycoproteins (VSG-1 and VSG-2) of two sequentially appearing variants of Trypanosoma congolense has been devised. When the isoelectrically focused VSG-1 components (VSG-1a, VSG-1b, and VSG-1c) are used as inhibitors of the VSG-1-anti-VSG-1 interaction, the RIA inhibition curves resemble each other, although minor differences in the high-affinity region of the curves can be detected. The heterologous antigen (VSG-2) does not inhibit the VSG-1-anti-VSG-1 interaction except at very high concentrations, indicating there is little cross-reactivity between highly purified VSG-1 and VSG-2. Nevertheless, heterologous antiserum, directed against VSG-2, will inhibit the VSG-1 -anti-VSG-1 interaction, and this property is shared to a significant degree by rabbit antiserum directed against an unrelated antigen. We have interpreted these findings as suggesting that: (1) there may be a constant region common to both VSG proteins, and (2) the constant region of the immunoglobulin molecule may also bind VSG proteins. Preliminary experiments show that the VSG-1 molecule augments binding of the Clq component of complement to the Fc region of immunoglobulin G.     

Trypanosoma congolense: surface glycoproteins of two early bloodstream variants. I. Production of a relapsing infection in rodents

A strain of Trypanosoma congolense has been cloned, passaged through the tsetse fly, and subsequently recloned. Relapsing infections have been induced in two rats by syringe passage of the cloned trypanosomes. The variant-specific glycoprotein of the initial cloned variant (VSG-1) and those from the two different variants produced in the two relapsing infections (VSG-2 and VSG-3) may be distinguished from each other by their isoelectric-focusing patterns. In this experimental system, cloned T. congolense, like Trypanosoma brucei, undergoes antigenic variation; the conversion of the VSG-1 into the VSG-2 isoelectric-focusing spectrotype was followed. These VSGs may be the products of sequentially expressed genes.     

Trypanosoma congolense: Surface glycoproteins of two early bloodstream variants: II. Purification and partial chemical characterization

Two sequential variant-specific glycoproteins have been purified from two variants of Trypanosoma congolense expressed during a relapsing infection. Isolation of the two glycoproteins, termed VSG-1 and VSG-2, respectively, employed glycerol lysis followed by purification on concanavalin A, Sephadex G-25, and gradient-eluted DE-52 columns. Partially purified VSG proteins were immunologically cross-reactive, but highly purified VSGs showed no cross-reactivity under the conditions employed. Both VSG-1 and VSG-2 consisted of a triplet of polypeptides. Although each member of a triplet subset could be distinguished by isoelectric focusing, all three gave identical N-terminal amino acid sequences and nearly identical tryptic peptide maps. The members of the VSG-1 polypeptide subset differed from those of the VSG-2 subset both with regard to N-terminal amino acid sequence and in tryptic peptide map patterns. Comparison of N-terminal sequences of VSG-1 and VSG-2 did, however, show that the sequences could be aligned to give a modest degree of amino acid homology (27%). This alignment also produced a minimum in the number of two-base changes, suggesting that the observed homology is not a coincidence and that these two proteins may well have arisen by gene duplication followed by retention of multiple point mutations.     

Antigenic variation and the surface glycoproteins of Trypanosoma congolense

Two Trypanosoma congolense variant-specific glycoproteins, which are expressed sequentially during a relapsing infection, have been purified. The proteins, termed VSG-1 and VSG-2, both have a molecular weight of 53,000 as determined by SDS polyacrylamide electrophoresis. When either antigen is electrophoresed through a pH gradient on an isoelectric focusing (IEF) gel, it gives a characteristic spectrotype of three bands. The IEF components of each VSG are antigenically similar to each other but not identical. The components of VSG-1 are immunologically distinct from the components of VSG-2, as shown by lack of cross-reactivity. The three spectrotypes may reflect microheterogeneity in amino acid sequence among the components. Both VSG-1 and VSG-2 are selectively cleaved by trypsin near their carboxy-terminal ends, indicating the existence of a possible common VSG region. Significant homology in the aminoterminal amino acid sequences of VSG-1 and VSG-2 suggests that sequentially reduplicated genes are sequentially expressed by trypanosomes during relapsing infections.     

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.     

Molecular characterization of two invariant surface glycoproteins specific for the bloodstream stage of Trypanosoma brucei.

In the accompanying paper (Ziegelbauer, K., and Overath, P. (1992) J. Biol. Chem. 267, 10791-10796), two invariant surface glycoproteins, ISG65 and ISG75, were identified in the mammalian stage of the parasitic protozoan, Trypanosoma brucei. In this study, the genes coding for these proteins have been isolated. Their nucleotide sequence suggests no relationship to other known genes and predicts polypeptides with NH2-terminal signal sequences, hydrophilic extracellular domains, single trans-membrane alpha-helices, and short cytoplasmic domains. ISG65 and ISG75 are expressed in bloodstream forms (70,000 and 50,000 molecules/cell, respectively) but not in the insect midgut stage. They can be detected in all T. brucei brucei variant clones investigated. Both polypeptides are distributed over the entire surface of the parasite.     

Identification of invariant surface glycoproteins in the bloodstream stage of Trypanosoma brucei.

Surface proteins of the mammalian stage of the parasitic protozoan, Trypanosoma brucei, were biotinylated with sulfosuccinimidyl 6-(biotinamido) hexanoate. Since the predominant protein labeled by this reagent is the membrane form of the variant surface glycoprotein (mfVSG), a procedure was developed to convert mfVSG to its soluble form by the endogenous glycosylphosphatidylinositol-specific phospholipase C while retaining other biotinylated surface proteins in a membrane-bound state. From these membranes, three novel glycoproteins of 60, 65, and 75 kDa could be isolated by a combination of Triton X-114 phase separation and precipitations by streptavidin and concanavalin A coupled to solid supports. These polypeptides were detected in trypanosomes expressing different mfVSGs and are thus considered to be invariant. In a variant clone in which the mfVSG is trypsin-sensitive, the invariant surface glycoproteins of 65 and 75 kDa, designated ISG65 and ISG75, respectively, were proteolytically degraded with similar kinetics as the mfVSG. Neither ISG65 nor ISG75 could be detected in procyclic trypanosomes, the stage of the parasite characteristic for the insect midgut. Gene cloning reported in the accompanying paper (Ziegelbauer, K., Multhaup, G., and Overath, P. (1992) J. Biol. Chem. 267, 10797-10803) suggests that ISG65 and ISG75 are transmembrane proteins.     

Minichromosomal variable surface glycoprotein genes and molecular karyotypes of Trypanosoma (Nannomonas) congolense

Employing orthogonal-field-alternation gel electrophoresis (OFAGE), we have separated chromosome-sized DNA molecules from Trypanosoma (Nannomonas) congolense clones, the clones being derived from several distinct antigenic repertoires. Trypanosome clones that belong to a specific antigenic repertoire appear to have a chromosome pattern characteristic of that particular repertoire. Hybridization of the separated chromosomes with cloned DNA fragments encoding variable surface glycoproteins revealed the presence of two different T.(N.) congolense variable surface glycoprotein genes on mini-chromosomes (mc) and the modes by which these genes may be activated: one by duplicative and the other by non-duplicative activation.     

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.     

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.     

Trypanosoma congolense: mechanical removal of the surface coat in vitro

By shaking suspensions of Trypanosoma congolense in isotonic buffer the surface coat could be separated from the cell body. The release of radioactivity from trypanosomes, selectively labeled in the surface coat by diazoniobenzenesulfonate, was used to follow the kinetics of coat detachment. The proteins in the supernatants of shaken trypanosomes were analyzed by sodium dodecyl sulfate—polyacrylamide gel electrophoresis. The shaking conditions had to be carefully controlled to avoid complete rupture of trypanosomes. Otherwise the coat protein was rapidly degraded by endogenous proteases. The influence of several parameters on the yield of coat release and the degree of degradation of the coat protein was investigated, including the ratio of trypanosome suspension volume to shaking vessel volume, vessel surface, temperature, shaking frequency, and preincubation of the trypanosomes at 0 C. By combining these parameters an optimal scheme was developed which allowed the separation of more than 90% of the coat protein from T. congolense, the detached protein showing no degradation at all. These results could be confirmed by electron microscopy of shaken and unshaken trypanosomes.     

Trypanosoma brucei rhodesiense bloodstream forms: surface ricin-binding glycoproteins are localized exclusively in the flagellar pocket and the flagellar adhesion zone

Specific binding of fluoresceinated succinyl-concanavalin A, wheat germ agglutinin, and ricin to untreated and trypsinized bloodstream forms of Trypanosoma brucei rhodesiense was quantitated by flow cytofluorimetry, and sites of lectin binding were identified by fluorescence microscopy. All three lectins only bound to the flagellar pocket of untreated parasites. When parasites were trypsinized to remove the variant surface glycoprotein coat, new lectin binding sites were exposed, and specific binding of all three lectins increased significantly. New specific binding sites for succinyl-concanavalin A and wheat germ agglutinin were present along both the free flagellum and flagellar adhesion zone and were uniformly distributed on the parasite surface. However, ricin did not bind uniformly on the surface and did not stain the free flagellum of trypsinized cells. Ricin only bound to the flagellar adhesion zone of trypsinized cells and of cells that had been treated with formaldehyde prior to staining. Electron microscopy of cells exposed to ricin-colloidal gold complexes revealed that that ricin binding was restricted to the anterior membrane of the flagellar pocket of untrypsinized cells and to this portion of the flagellar pocket and the cell body membrane in the flagellar adhesion zone of trypsinized cells. Evidence that these membranes constitute a functionally important membrane microdomain is reviewed.     

Immunological evidence of a common structure between Paramecium surface antigens and Trypanosoma variant surface glycoproteins

The surface antigens (SAgs) of Paramecium and the variant surface antigens (VSGs) of Trypanosoma can be purified in two distinct molecular forms: a soluble form (solubilized in dilute ethanolic solution in the case of Paramecium, or in water for Trypanosoma) and a membranal form, amphiphile (solubilized in SDS). In trypanosomes, the enzymatic conversion of the membrane form into the soluble form is accompanied by the unmasking of a particular immunological determinant, called cross-reacting determinant (CRD), which is located in the COOH-terminal phospho-ethanolamine glycopeptide. We demonstrate immunological homologies between Paramecium SAgs and Trypanosoma VSGs. A determinant corresponding to the CRD of VSGs is borne by the ethanol-soluble form of the SAgs and by two cross-reacting light chains also present in ethanolic cellular extracts (together with the soluble form), and not by the membranal form of SAgs. Furthermore, we show that the membranal form of Paramecium SAgs can be converted into soluble form and that this enzymatic conversion also yields cross-reacting light chains. We also demonstrate that the membranal form is the physiological form in paramecia stably expressing a given SAg.     

Expression of bloodstream variant surface glycoproteins in procyclic stage Trypanosoma brucei: role of GPI anchors in secretion.

Using transformed procyclic trypanosomes, the synthesis, intracellular transport and secretion of wild-type and mutant variant surface glycoprotein (VSG) is characterized. We find no impediment to the expression of this bloodstream stage protein in insect stage cells. VSG receives a procyclic-type phosphatidylinositol-specific phospholipase C-resistant glycosyl phosphatidylinositol (GPI) anchor, dimerizes and is N-glycosylated. It is transported to the plasma membrane with rapid kinetics (t(1/2) approximately 1 h) and then released by a cell surface zinc-dependent metalloendoprotease activity, a possible homolog of leishmanial gp63. Deletion of the C-terminal GPI addition signal generates a soluble form of VSG that is exported with greatly reduced kinetics (t(1/2) approximately 5 h). Fusion of the procyclic acidic repetitive protein (PARP) GPI anchor signal to the C-terminus of the truncated VSG reporter restores both GPI addition and transport competence, suggesting that GPI anchors play a critical role in the folding and/or forward transport of newly synthesized VSG. The VSG-PARP fusion is also processed near the C-terminus by events that do not involve N-linked oligosaccharides and which are consistent with GPI side chain modification. This unexpected result suggests that GPI processing may be influenced by adjacent peptide sequence or conformation.     

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.     

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.