The major cell surface glycoprotein procyclin is a receptor for induction of a novel form of cell death in African trypanosomes in vitro.

Bloodstream forms (BSF) and procyclic culture forms (PCF) of African trypanosomes were incubated with a variety of lectins in vitro. Cessation of cell division and profound morphological changes were seen in procyclic forms but not in BSF after incubation with concanavalin A (Con A), wheat germ agglutinin and Ricinus communis agglutinin. These lectins caused the trypanosomes to cease division, become round and increase dramatically in size, the latter being partially attributable to the formation of what appeared to be a large 'vacuole-like structure' or an expanded flagellar pocket. Con A was used in all further experiments. Spectrophotometric quantitation of extracted DNA and flow cytometry using the DNA intercalating dye propidium iodide showed that the DNA content of Con A-treated trypanosomes increased dramatically when compared to untreated parasites. Examination of these cells by fluorescence microscopy showed that many of the Con A-treated cells were multinucleate whereas the kinetoplasts were mostly present as single copies, indicating a disequilibrium between nuclear and kinetoplast replication. Immunofluorescence experiments using monoclonal antibodies (mAb) specific for paraflagellar rod proteins and for kinetoplastid membrane protein-11 (KMP-11), showed that the Con A-treated parasites had begun to duplicate the flagellum but that this had only proceeded along part of the length of the cells, suggesting that the cell division process was initiated but that cytokinesis was subsequently inhibited. Tunicamycin-treated wild-type trypanosomes and mutant trypanosomes expressing both high levels of non-glycosylated procyclins and procyclin isoforms with truncated N-linked sugars were resistant to the effects of Con A, suggesting that N-linked carbohydrates on the procyclin surface coat were the ligands for Con A binding. This was supported by data obtained using mutant parasites created by deletion of all three EP procyclin isoforms, two of which contain N-glycosylation sites, by homologous recombination. The knockout mutants showed reduced binding of fluorescein-labelled Con A as determined by flow cytometry and were resistant to the effects of Con A. Taken together the results show that Con A induces multinucleation, a disequilibrium between nuclear and kinetoplast replication and a unique form of cell death in procyclic African trypanosomes and that the ligands for Con A binding are carbohydrates on the EP forms of procyclin. The possible significance of these findings for the life cycle of the trypanosomes in the tsetse fly vector is discussed.

Molecular characterization of the kinetoplastid membrane protein-11 from African trypanosomes.

The kinetoplastid membrane protein-11 molecule was purified from Trypanosoma brucei rhodesiense and an internal peptide sequence was obtained. This sequence information was used with cosmid library screening and polymerase chain reaction amplifications of both genomic DNA and cDNA to obtain the entire DNA sequence of the encoding gene and the corresponding translated amino acid sequence of 92 residues. The sequence showed 18% divergence from the corresponding molecule of the related kinetoplastid Leishmania donovani, including one key amino acid at position 45 which may be of functional relevance. The protein had a calculated molecular mass of 11078 Da, a pI of 6.0 and an overall net charge of -2 at physiological pH. The secondary structure of the molecule was predicted to consist of two amphipathic helices connected by a random-coil segment, and suggests that it would interact with lipid bilayers in the trypanosome cell membrane. Northern and Southern blot analyses showed that the trypanosome kinetoplastid membrane protein-11 molecule was translated from a single transcript and was transcribed from a single gene copy, thus making this molecule an attractive target for knockout mutagenesis.

Kinetoplastid membrane protein-11 (KMP-11) is differentially expressed during the life cycle of African trypanosomes and is found in a wide variety of kinetoplastid parasites.

An abundant 11-kDa membrane protein was purified from African trypanosomes by organic solvent extraction and octyl-Sepharose chromatography. This protein cross-reacts with monoclonal antibodies originally generated against the lipophosphoglycan-associated protein of Leishmania donovani. Immunoblot analysis showed that the 11-kDa molecule was present in a variety of species of kinetoplastids. It was found in several species and subspecies of African trypanosomes and was present in low amounts in bloodstream forms and in larger amounts in procyclic, epimastigote and metacyclic life cycle stages. Expression of the 11-kDa molecule rapidly increased during transformation from bloodstream forms to procyclic forms, paralleling expression of the major surface glycoprotein of Trypanosoma congolense, the glutamic acid/alanine-rich protein, an analogue of T. brucei procyclin. The molecule was present in procyclic trypanosome membranes at approximately 2 x 10(5)-1 x 10(6) molecules per cell, suggesting it may have an important role in parasite membrane organization and function. Amino-acid analysis of the trypanosome 11-kDa protein showed it had a different composition than that of its leishmania counterpart. Its wide distribution in kinetoplastids and its membrane disposition suggest a name for this class of molecules: kinetoplastid membrane protein-11 (KMP-11).

The kinetoplastid membrane protein 11 of Leishmania donovani and African trypanosomes is a potent stimulator of T-lymphocyte proliferation.

Kinetoplastid membrane protein 11 (KMP-11) from Leishmania donovani is an abundant 11-kDa surface membrane glycoprotein. Lymph node cells from mice of six different H-2 haplotypes immunized with KMP-11 or with L. donovani promastigotes were stimulated to proliferate in vitro KMP-11. Primed purified T cells required antigen presentation since they were not stimulated unless KMP-11-pulsed or L. donovani-infected macrophages were added. Promastigotes of a wide variety of Leishmania species and procyclic forms of African trypanosomes stimulated proliferation of KMP-11-primed or L. donovani promastigote-primed lymph node cells. All of the Leishmania promastigotes and African trypanosomes tested contained an 11-kDa protein, as detected by immunoblotting with KMP-11-specific monoclonal antibodies. The widespread distribution of the 11-kDa (KMP-11) molecules and their ability to stimulate strong T-lymphocyte proliferation in a non-H-restricted fashion suggest that they may be important molecules for induction of cell-mediated immune responses.

Identification and characterization of an acidic major surface glycoprotein from procyclic stage Trypanosoma congolense.

Monoclonal antibodies (mAbs) were derived against the procyclic culture form of Trypanosoma congolense and 14 were selected which bound to the surface of living procyclics in immunofluorescence assays. These antibodies bound to procyclics and epimastigotes of T. congolense (both savannah-type and Kilifi-type) and procyclics of Trypanosoma simiae, but not to procyclics of other species of trypanosomes, to bloodstream forms of several species of trypanosomes or to Leishmania, and were thus life cycle stage- and subgenus-specific. Fluorescence-activated cell sorter analysis with these antibodies showed that the kinetics of expression of the surface antigen during transformation from bloodstream to procyclic forms was similar to that of procyclin or procyclic acidic repetitive protein (PARP) of T. brucei spp. appearing at the cell surface as early as 8 h after initiating transformation. All fourteen antibodies detected broad bands of 40-44 and 28-32 kDa in immunoblot analysis of whole procyclic lysates and were specific for carbohydrate epitopes. The antigen was purified by cation-exchange chromatography and gel electrophoresis, and was shown to be an acidic glycoprotein. Amino acid microanalysis of the purified antigen showed an abundance of glutamic acid/glutamine and alanine. Sequences of peptides produced by cyanogen bromide cleavage matched amino acid sequences predicted by the nucleotide sequence of a gene described in the accompanying paper by Bayne et al. [26]. No sequence similarity to T. brucei procyclin/PARP or to any other protein was found. However, its stage and subgenus specificity, surface disposition, immunodominance, acidity and kinetics of expression during transformation from bloodstream to procyclic forms indicate that the molecule is an analog of procyclin/PARP described in T. brucei spp.

Identification of basic fibroblast growth factor-like proteins in African trypanosomes and Leishmania.

Basic fibroblast growth factor (bFGF) is a multifunctional, heparin-binding, mitogenic polypeptide found in all tissues or cells of multicellular organisms so far examined. Here we report that Trypanosoma brucei rhodesiense procyclic culture forms (PCF) and Leishmania donovani promastigotes grown in serum-containing and serum-free medium, contained peptides of 15-34 kDa which bound heparin-sepharose with high affinity and which reacted in immunoblots with several preparations of antibodies specific for bovine brain bFGF. Similar peptides were not detectable in foetal bovine serum. Immunofluorescence studies showed bFGF-like molecules to have a cytoplasmic distribution in both species growing in serum-free media. A nuclear and/or perinuclear distribution of immunoreactivity was also observed in parasites which had been grown in the presence of serum. The data indicate that both species of parasites synthesize their own bFGF-like molecules. Association of an ubiquitous growth factor with parasitic protozoa may play an important role in parasite multiplication and in host-parasite interactions.

The immunochemical structure and surface arrangement of Leishmania donovani lipophosphoglycan determined using monoclonal antibodies.

Using intact Leishmania donovani promastigotes or purified L. donovani lipophosphoglycan (LPG) as immunogens, we have derived four LPG-specific monoclonal antibodies (MAbs). Two of these MAbs recognize an epitope consisting of the repeating phosphorylated galactose beta-1,4-mannose disaccharide portion of the molecule and cross-reacted with LPG from Leishmania major. These MAbs bound to the surface of living promastigotes of both species. The two other MAbs bound to the phosphosaccharide core structure of LPG and did not bind to the surface of living parasites, presumably due to masking of the core region. Experiments using all four MAbs with an LPG-deficient promastigote mutant indicated that both the repeat epitope and phosphosaccharide core were present in these cells, suggesting that incomplete assembly was responsible for the absence of intact LPG.

Expression of Trypanosoma brucei procyclin as a fusion protein in Escherichia coli.

Procyclin, a glycoprotein surface antigen of procyclic forms of Trypanosoma brucei, was expressed in Escherichia coli as a cro-beta-galactosidase fusion protein. Antibodies produced in rabbits immunised with gel-purified fusion protein bound to the surface of living procyclic culture forms in indirect immunofluorescence assays and were able to immunoprecipitate procyclin from lysates of trypanosomes biosynthetically labelled with tritiated proline. In addition, the antibodies recognised synthetic peptides corresponding to three different regions of the procyclin molecule, including a glutamic acid-proline dipeptide repeat. The results indicate that T. brucei procyclin expressed as a fusion protein is immunogenic and antigenically intact. In contrast, no rabbit antibodies could be produced against a 16-amino-acid synthetic peptide consisting of the dipeptide repeat, even when the peptide was coupled to carrier proteins.

Procyclin gene expression and loss of the variant surface glycoprotein during differentiation of Trypanosoma brucei.

In the mammalian host, the unicellular flagellate Trypanosoma brucei is covered by a dense surface coat that consists of a single species of macromolecule, the membrane form of the variant surface glycoprotein (mfVSG). After uptake by the insect vector, the tsetse fly, bloodstream-form trypanosomes differentiate to procyclic forms in the fly midgut. Differentiation is characterized by the loss of the mfVSG coat and the acquisition of a new surface glycoprotein, procyclin. In this study, the change in surface glycoprotein composition during differentiation was investigated in vitro. After triggering differentiation, a rapid increase in procyclin-specific mRNA was observed. In contrast, there was a lag of several hours before procyclin could be detected. Procyclin was incorporated and uniformly distributed in the surface coat. The VSG coat was subsequently shed. For a single cell, it took 12-16 h to express a maximum level of procyclin at the surface while the loss of the VSG coat required approximately 4 h. The data are discussed in terms of the possible molecular arrangement of mfVSG and procyclin at the cell surface. Molecular modeling data suggest that a (Asp-Pro)2 (Glu-Pro)22-29 repeat in procyclin assumes a cylindrical shape 14-18 nm in length and 0.9 nm in diameter. This extended shape would enable procyclin to interdigitate between the mfVSG molecules during differentiation, exposing epitopes beyond the 12-15-nm-thick VSG coat.

Procyclin: an unusual immunodominant glycoprotein surface antigen from the procyclic stage of African trypanosomes.

An immunodominant species-specific surface glycoprotein antigen was purified from procyclic culture forms of Trypanosoma brucei rhodesiense using lectin affinity chromatography and a monoclonal antibody immunoadsorbent. The purified molecule appears on a 10% polyacrylamide gel as a wide, dark silver staining band having an apparent molecular mass of between 30 and 40 kDa, identical to that revealed by immunoblotting using anti-procyclic lysates. The molecule, which we have named procyclin, was shown by immunofluorescence and immunoelectron microscopy to be exposed on the surface of procyclic trypanosomes. Gas-phase protein microsequencing and micro-amino acid analysis revealed an unusual acidic polypeptide with an amino-terminal amino acid sequence which matched portions of previously published sequences predicted from two different cDNAs obtained using mRNA from procyclic trypanosomes. The procyclin molecules contained a large glutamic acid-proline repeat and the form we isolated was highly water soluble. Ten different monoclonal antibodies were used in ELISA with synthetic peptides to localize parasite surface epitopes to various portions of procyclin. The results showed that surface epitopes were spread throughout most of the procyclin molecule, including the glutamic acid-proline repeat portion. Procyclin is distributed over the surface of both culture form and tsetse fly midgut form procyclic trypanosomes, is developmentally regulated and is immunologically species-specific.

Use of procyclic trypanosomes for detection of antibodies in sera from vervet monkeys infected with Trypanosoma rhodesiense: an immunodiagnostic test for African sleeping sickness.

Uncoated procyclic culture forms of African trypanosomes were used in immunofluorescence and simple agglutination assays to detect antibodies in the sera of vervet monkeys infected with T. b. rhodesiense. Antibodies to procyclic surface antigens were found in sera from animals with active, untreated infections or sera taken soon after treatment with trypanocidal drugs. The antibodies were detectable within 7 days of infection. No specific antibodies were detected in sera prior to infection or long after drug cure. The results indicate that antigens expressed on the surface of procyclic culture forms of T. brucei spp. are useful for the detection of antibodies produced in response to infection with T. b. rhodesiense and may allow the development of a simple immunodiagnostic test for African sleeping sickness. In addition, the use of a form of the trypanosome of a different differentiation state from the infecting organism illustrates the utility of this approach for detection of antibodies to common antigens.

Procyclic tsetse fly midgut forms and culture forms of African trypanosomes share stage- and species-specific surface antigens identified by monoclonal antibodies.

Procyclic culture form (PCF) trypanosomes were established from a bloodstream form population of cloned Trypanosoma brucei rhodesiense and were used to immunize mice for hybridoma production. Indirect immunofluorescence was used to select 10 hybridomas which secreted antibodies that bound to the surface of homologous living PCF. The antibodies reacted with PCF of several clones of T.b. brucei, T.b. gambiense, and T.b. rhodesiense, but not with PCF of T. congolense or T. vivax, or with promastigotes of several species of Leishmania parasites. The antigens were not detectable in ethanol/acetic acid-fixed bloodstream forms or in lysates of bloodstream forms of any of the T. brucei subspecies, and are thus species-specific and stage-specific markers. Selected monoclonal antibodies bound to procyclic trypanosomes taken directly from the midgut of infected tsetse flies, and to immature epimastigote forms in salivary probes, and may therefore be useful in epidemiologic investigations.