Cysteine eliminates the feeder cell requirement for cultivation of Trypanosoma brucei bloodstream forms in vitro.

In all previous studies, bloodstream forms of Trypanosoma brucei could be grown in vitro only when supported by a feeder layer of mammalian fibroblasts. We have axenically cultivated bloodstream T. brucei by adding L-cysteine at regular intervals and appropriate concentrations. The optimum cysteine concentration depends on cell density and is close to physiological serum levels. At concentrations greater than 24 mg/liter (2 X 10(-4) M), cysteine was acutely toxic to trypanosome concentrations of 3 X 10(7)/ml. Toxicity was prevented by addition of pyruvate or catalase, which neutralize H2O2 produced by cysteine autoxidation. In uptake studies using [35S]cysteine and [35S]cystine, T. brucei efficiently incorporated only cysteine. The Km for cysteine uptake was 4 X 10(-4) M. Cystine supported axenic growth if low concentrations of 2-mercaptoethanol were added at regular intervals.

Transfer of glycosyl-phosphatidylinositol membrane anchors to polypeptide acceptors in a cell-free system.

Glycosylinositol phospholipid (GPI) membrane anchors are the sole means of membrane attachment of a large number of cell surface proteins, including the variant surface glycoproteins (VSGs) of the parasitic protozoan, Trypanosoma brucei. Biosynthetic data suggest that GPI-anchored proteins are synthesized with carboxy-terminal extensions that are immediately replaced by GPI, suggesting the existence of preformed GPI species available for transfer to the nascent protein in the ER. Candidate precursor glycolipids having a linear sequence indistinguishable from the conserved core structure found on all GPI anchors, have been characterized in T. brucei. In this paper we describe the transfer of three GPI variants to endogenous VSG in vitro. GPI addition is not reduced by inhibitors of protein synthesis and does not require ATP or GTP, consistent with a transpeptidation mechanism.

Deletion of an immunodominant Trypanosoma cruzi surface glycoprotein disrupts flagellum-cell adhesion.

Null mutants of the Trypanosoma cruzi insect stage-specific glycoprotein GP72 were created by targeted gene replacement. Targeting plasmids were constructed in which the neomycin phosphotransferase and hygromycin phosphotransferase genes were flanked by GP72 sequences. These plasmids were sequentially transfected into T. cruzi epimastigotes by electroporation. Southern blot analyzes indicated that precise replacement of the two genes had occurred. No aberrant rearrangements occurred at the GP72 locus and no GP72 gene sequences had been translocated elsewhere in the genome. Western blots confirmed that GP72 is not expressed in these null mutants. The morphology of the mutants is dramatically different from wild-type. In both mutant and wild-type parasites, the flagellum emerges from the flagellar pocket. In the null mutant the normal attachment of the flagellum to the cell membrane of the parasite is lost.

Transport of fluorescent phospholipid analogues from the erythrocyte membrane to the parasite in Plasmodium falciparum-infected cells.

The asexual development of the human malaria parasite Plasmodium falciparum is largely intraerythrocytic. When 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazole-4-yl)amino]caproyl] phosphatidylcholine (NBD-PC) was incorporated into infected and uninfected erythrocyte membranes at 0 degrees C, it remained at the cell surface. At 10 degrees C, the lipid was rapidly internalized in infected erythrocytes at all stages of parasite growth. Our results indicate that the internalization of NDB-PC was not because of endocytosis but rapid transbilayer lipid flip-flop at the infected erythrocyte membrane, followed by monomer diffusion to the parasite. Internalization of the lipid was inhibited by (a) depleting cellular ATP levels; (b) pretreating the cells with N-ethyl maleimide or diethylpyrocarbonate; and (c) 10 mM L-alpha-glycerophosphorylcholine. The evidence suggests protein-mediated and energy dependent transmembrane movement of the PC analogue. The conditions for the internalization of another phospholipid analogue N-4-nitrobenzo-2-oxa-1,3-diazoledipalmitoyl phosphatidylethanolamine (N-NBD-PE) were distinct from that of NBD-PC and suggest the presence of additional mechanism(s) of parasite-mediated lipid transport in the infected host membrane. In spite of the lack of bulk, constitutive endocytosis at the red cell membrane, the uptake of Lucifer yellow by mature infected cells suggests that microdomains of pinocytotic activity are induced by the intracellular parasite. The results indicate the presence of parasite-induced mechanisms of lipid transport in infected erythrocyte membranes that modify host membrane properties and may have important implications on phospholipid asymmetry in these membranes.

Intracellular transport of a variant surface glycoprotein in Trypanosoma brucei.

Trypanosome variant surface glycoproteins (VSGs) have a novel glycan-phosphatidylinositol membrane anchor, which is cleavable by a phosphatidylinositol-specific phospholipase C. A similar structure serves to anchor some membrane proteins in mammalian cells. Using kinetic and ultrastructural approaches, we have addressed the question of whether this structure directs the protein to the cell surface by a different pathway from the classical one described in other cell types for plasma membrane and secreted glycoproteins. By immunogold labeling on thin cryosections we were able to show that, intracellularly, VSG is associated with the rough endoplasmic reticulum, all Golgi cisternae, and tubulovesicular elements and flattened cisternae, which form a network in the area adjacent to the trans side of the Golgi apparatus. Our data suggest that, although the glycan-phosphatidylinositol anchor is added in the endoplasmic reticulum, VSG is nevertheless subsequently transported along the classical intracellular route for glycoproteins, and is delivered to the flagellar pocket, where it is integrated into the surface coat. Treatment of trypanosomes with 1 microM monensin had no effect on VSG transport, although dilation of the trans-Golgi stacks and lysosomes occurred immediately. Incubation of trypanosomes at 20 degrees C, a treatment that arrests intracellular transport from the trans-Golgi region to the cell surface in mammalian cells, caused the accumulation of VSG molecules in structures of the trans-Golgi network, and retarded the incorporation of newly synthesized VSG into the surface coat.

Expression of a bacterial gene in a trypanosomatid protozoan.

A simple and reproducible assay for DNA-mediated transfection in the trypanosomatid protozoan Leptomonas seymouri has been developed. The assay is based on expression of the Escherichia coli chloramphenicol acetyl transferase (CAT) gene flanked by Leptomonas DNA fragments that are likely to contain necessary elements for gene expression in trypanosomes. After electroporation of cells in the presence of plasmid DNA, CAT activity was detected in crude cell lysates. No activity was detected when the orientation of the L. seymouri mini-exon sequence (placed upstream of the CAT gene) was reversed, or in additional control experiments. This system provides a method for defining transcriptional control elements in trypanosomes.

Stable expression of mosaic coats of variant surface glycoproteins in Trypanosoma brucei.

The paradigm of antigenic variation in parasites is the variant surface glycoprotein (VSG) of African trypanosomes. Only one VSG is expressed at any time, except for short periods during switching. The reasons for this pattern of expression and the consequences of expressing more than one VSG are unknown. Trypanosoma brucei was genetically manipulated to generate cell lines that expressed two VSGs simultaneously. These VSGs were produced in equal amounts and were homogeneously distributed on the trypanosome surface. The double-expressor cells had similar population doubling times and were as infective as wild-type cells. Thus, the simultaneous expression of two VSGs is not intrinsically harmful.

rRNA genes of Naegleria gruberi are carried exclusively on a 14-kilobase-pair plasmid.

An extrachromosomal DNA was discovered in Naegleria gruberi. The 3,000 to 5,000 copies per cell of this 14-kilobase-pair circular plasmid carry all the 18S, 28S, and 5.8S rRNA genes. The presence of the ribosomal DNA of an organism exclusively on a circular extrachromosomal element is without precedent, and Naegleria is only the third eucaryotic genus in which a nuclear plasmid DNA has been found.

DNA rearrangements associated with multiple consecutive directed antigenic switches in Trypanosoma brucei.

Changes in variant surface glycoprotein (Vsg) expression allow Trypanosoma brucei to elude the immune response. The expressed vsg is always located at the telomeric end of a polycistronic transcription unit known as an expression site (ES). Although there are many ESs, only one is active at any particular time. The mechanisms regulating ES transcription and switching are unknown. Chromosome rearrangements within or upstream of the ES have been described to occur in occasional switch events, but no changes have been consistently associated with switching. We inserted the drug resistance genes neo and ble, conferring resistance to G418 and phleomycin, respectively, 1 kb downstream of "silent" ES promoters. This demonstrated that short-range transcription could be achieved from a silent ES promoter. From one initial transformant clone, panels of independent consecutive on-off-on switch clones were generated and analyzed. The first activation of the neo-targeted ES was always associated with deletion of the upstream tandem promoter in this ES, but no further rearrangements were detected in consecutive off-on switches of this ES. On the other hand, direct analysis of ES promoters showed that deletions and duplications occurred elsewhere. Activation of a ble-tagged 300-kb chromosome could not be achieved, but phleomycin-resistant clones could be obtained. One such clone arose from recombination between three ESs. Taken together, our experiments suggest that ES switching may occur after a period of chromosomal interactivity that may or may not leave tangible evidence in the form of detectable sequence changes.

Purification and properties of nucleic acids from an unusual cytoplasmic organelle in the flagellate protozoan Crithidia oncopelti.

A simple, rapid method for preparing bipolar bodies from sonicated (rithidia oncopelti cells, with a yield of 2-5%, is described. Apart from 2-4% contamination with unbroken cells the fraction was considered pure with respect to contamination by other nucleic acid-containing organelles, as judged by light and electron microscopy. A light satellite DNA, f bouyant density 1.695 g/ml in neutral CsCl, and derived from the bipolar body, had a Tm of 81.6 degrees C in0.15 M NaCl/0.015 M sodium citrate (pH 7.0) and a kinetic complexity of 2.7 with 109. The bipolar body fraction also contained ribonucleoprotein particles with and s20,w of 67 S, in contrast to cytoplasmic ribosomes (87 S). Bipolar body ribosomes contained rRNA components which migraged coincidentally with Escherichia coli rRNA (molecular weights 1.07 with 10-6 and 0.56 with 10-6) on polyacrylamide gel electrophoresis. Cytoplasmic ribosomes contained rRNAs of molecular weights 1.30 with 10-6 and 0.83 with 10-6. Bipolar body rRNA accounted for up to 10% of the rRNA extracted from cells. The properties of these bipolar body nucleic acids provide good evidence for the bacterial nature of this subcellular component.

The isolation of plasmids containing DNA complementary to messenger RNA for variant surface glycoproteins of Trypanosoma brucei.

We have isolated poly(A)+ RNA from four antigenic variants (117, 118, 121, 221) of one clone of Trypanosoma brucei. Translation of these poly(A)+ RNAs in a rabbit reticulocyte lysate gave rise to proteins that could be precipitated with antisera against homologous variant surface glycoprotein, the protein responsible for antigenic variation in trypanosomes. From the electrophoretic mobility of these in vitro products in sodium dodecyl sulphate (SDS) gels we infer that variant surface glycoproteins (VSGs) are made as pre-proteins, which require trimming to yield mature VSGs. The total translation products from the four poly(A)+ RNAs produced a complex set of bands on SDS gels, which only differed in the region where the variant pre-glycoproteins migrated. The only detectable variation in the messenger RNA populations of these variants is, therefore, in the messenger RNA for variant pre-glycoproteins. We have made duplex DNA copies of these poly(A)+ RNAs, linked the complementary DNA to plasmid pBR322 by GC tailing and cloned this recombinant DNA in Escherichia coli. Colony hybridization with complementary DNA made on poly(A)+ RNA showed that 7--10% of the colonies contained DNA that hybridized only with the homologous probe. Plasmid DNA was isolated from ten such colonies (two or three of each variant complementary DNA), bound to diazobenzyloxymethyl-cellulose (DBM) paper and used to select complementary messenger RNA from total poly(A)+ RNA by hybridization. In eight cases the RNA recovered from the filter gave variant pre-glycoprotein as the predominant product of in vitro translation. Poly(A)+ RNA from each of the variants only hydridized to the homologous complementary DNA in filter hybridizations. Each trypanosome variant, therefore, contains no detectable messenger RNAs for the three heterologous variant-specific glycoproteins tested. We conclude from this lack of cross-hybridization that antigenic diversity in trypanosomes, unlike antibody diversity in mammals, does not involve the linkage of a repertoire of genes for the variable N-terminal half to a single gene for the C-terminal half of the VSGs.

Rapid isolation of DNA from trypanosomatid protozoa using a simple 'mini-prep' procedure.

Although several methods for isolating genomic DNA from trypanosomatid protozoa exist, all are time-consuming and cumbersome. Faster, simpler and efficient protocols for preparation of DNA from these protozoa are needed to ease the screening of mutants and transfectants. We describe the use of a bacterial lysis method to isolate chromosomal DNA from a wide range of trypanosomatids. The method is based on the finding reported by He et al., who noticed that phenol/chloroform treatment of Escherichia coli cells in the presence of LiCl and Triton X-100 solubilizes plasmid DNA, while precipitating unwanted chromosomal DNA and denatured cellular proteins. In applying this lysis method to the isolation of episomal DNA from transfected trypanosomatids, we found that, unlike bacterial genomic DNA, chromosomal DNA of trypanosomatids was soluble in the phenol/chloroform/Triton/LiCl mixture. This observation prompted us to use the bacterial lysis method as a routine protocol for extraction of DNA from trypanosomatids.

Glycopeptides from variant surface glycoproteins of Trypanosoma Brucei. C-terminal location of antigenically cross-reacting carbohydrate moieties.

Glycopeptides have been purified after trypsin or pronase cleavage of variant surface glycoproteins from five antigenic variants of one clone of Trypanosoma brucei. Each peptide has been characterised by its amino acid and sugar composition and partial or complete amino acid sequence. The peptides were assayed in an indirect precipitation radioimmunoassay for inhibition of precipitation of one variant surface glycoprotein by a heterologous antiserum raised against another variant. Two classes of glycopeptide were obtained, those which showed complete inhibition and those which had no inhibitory activity. The non-cross-reacting glycopeptides were all derived from internal sites in the polypeptide chain and had only mannose and glucosamine as the constituents of the glycosyl group with one exception which also contained galactose. The carbohydrate moieties involved in the immunological cross-reaction were found to be attached at or very close to the C-terminus of the protein and contained galactose, mannose and glucosamine. Considerable variation in the size and composition of these glycosyl moieties occurred between the variant glycoproteins and, in two cases, within individual glycoproteins. The immunological cross-reactivity was constant despite this oligosaccharide heterogeneity. There was significant amino acid sequence homology between glycopeptides from certain variants in contrast to the absence of homology observed previously for the N-terminal sequence of intact surface glycoproteins. Some C-terminal glycopeptide sequences suggest the occurrence of proteolytic processing subsequent to glycosylation and prior to variant surface glycoprotein isolation. The terminal amino acid (Asx or Ser) of all isolated variant surface glycoproteins was glycosylated.

Effects of 3' untranslated and intergenic regions on gene expression in Trypanosoma cruzi.

The effects of 3' untranslated regions (UTR) and intergenic regions (INT), from various Trypanosoma cruzi stage-specific and constitutive genes, on the expression of the reporter firefly luciferase gene (luc), were studied using stable episomal transformation. The 3' UTR influenced luciferase expression by changing the steady-state level and/or the translation efficiency of luc mRNA. Glycoprotein 72 gene (gp72), glycoprotein 85 gene (gp85) or amastin gene (ama) 3' UTR decreased the luc mRNA level 6- to 14-fold, compared to the glyceraldehyde 1-phosphate dehydrogenase gene (gapdh) 3' UTR, in epimastigotes. Luciferase activity decreased in parallel with the luc mRNA level in transformants utilizing the gp85 or ama 3' UTR, whereas luc mRNA containing the gp72 3' UTR showed approximately 5-fold higher translation efficiency than luc mRNA containing a minimal 3' UTR. In amastigotes, the inhibitory effect of the ama 3' UTR observed in other life cycle stages was abolished and luciferase expression was stimulated 16-fold. The overall stage-specific difference mediated by the ama 3' UTR, between epimastigotes and amastigotes, was approximately 100-fold. INT, which was expected to influence polyadenylation efficiency, of gapdh, gp72, or heat shock protein 60 gene inserted after gapdh 3' UTR increased luc mRNA 2- to 8-fold, whereas gp85 INT slightly decreased luc mRNA. By separating effects attributable to the 3' UTR and INT, this study shows the effects of 3' UTR on RNA levels and translational efficiency in T. cruzi.

Functional complementation of glycoprotein 72 in a Trypanosoma cruzi glycoprotein 72 null mutant.

To investigate the role of a developmentally regulated 72-kDa surface antigen of Trypanosoma cruzi (GP72), a GP72 null mutant was previously produced [Cooper et al., 1993, J. Cell Biol. 122, 149-156]. Abnormal morphology of epimastigote and metacyclic trypomastigote stages of the GP72 null mutant suggested that GP72 is associated with flagellum-cell adhesion [Cooper et al., 1993, J. Cell Biol. 122, 149-156; De Jesus et al., J. Cell Sci., in press]. In the present study, functional complementation of the GP72 null mutant was accomplished by transformation with two independent episomal vectors expressing GP72 and phleomycin or tunicamycin resistance genes. A correlation between gene copy number, RNA level, expression of GP72, and morphological phenotypes was demonstrated. Disparities were observed between gene copy number and RNA level and between the apparent level of GP72 polypeptide and the carbohydrate epitope recognized by monoclonal antibody WIC29.26. Restoration of morphology reflects recovery of the carbohydrate epitope, suggesting that the novel glycosylation of GP72 is the limiting step in the expression of its function.

In situ analysis of a variant surface glycoprotein expression-site promoter region in Trypanosoma brucei.

In Trypanosoma brucei, the active variant surface glycoprotein genes (vsg) are located at telomeric expression sites (ES), whose expression is highly regulated during the life cycle. In the procyclic form, all ESs are repressed. In the bloodstream form, where antigenic variation occurs, only one of approximately 20 ESs is active at a given time. We have investigated chromatin structure and DNA sequence around the ES promoter to identify cis-acting regulatory regions. A marker gene, inserted 1 kb downstream of the ES promoter, was used as a specific probe to map the position of nuclease hypersensitive sites. A prominent hypersensitive site was detected within the core promoter. This site was present in both active and inactive ES promoters, suggesting that a protein complex is bound to the promoter irrespective of its transcriptional state. However, none of the regions showed differential nuclease sensitivity between active and inactive transcriptional states. A systematic deletion analysis of the sequences surrounding the active ES promoter in situ confirmed the absence of cis-regulatory elements. We find that only 70 bp within the ES promoter are necessary to support ES regulation. Analysis of the reporter activities in an inactive bloodstream-form ES revealed the existence of an intermediate promoter activity in some clones, but we never observed full activation of more than one ES. The vsg mRNA from this intermediate ES was expressed less efficiently.

An 85-kilodalton surface antigen gene family of Trypanosoma cruzi encodes polypeptides homologous to bacterial neuraminidases.

We have determined the sequence of a cDNA (Tt34c1) encoding a Trypanosoma cruzi trypomastigote stage-specific 85-kDa surface glycoprotein (gp85). Within the peptide sequence of Tt34c1 are two 8-amino acid motifs, Ser-X-Asp-X-Gly-X-Thr-Trp, that are characteristic of bacterial neuraminidases. Analysis of the Tt34c1 sequence predicts the presence of an amino-terminal signal sequence and a hydrophobic carboxy-terminus that is probably replaced by a glycosyl phosphatidylinositol membrane anchor. Gp85 is encoded by an extensive multigene family that is distributed throughout the genome and can be divided into subsets on the basis of oligonucleotide hybridisation patterns. By sequencing products of polymerase chain reaction (PCR) amplification of the 5' end of trypomastigote gp85 mRNA we show that multiple copies of the gene family are transcribed simultaneously in a trypanosome population. Comparison of the sequence of the PCR clones and another gp85 cDNA showed a highly conserved region 5' of the first methionine extending 180 nt into the coding sequence. Insertions and point mutations were observable outside these homologous sequences demonstrating the variant nature of the gp85 mRNAs.

Analysis of Trypanosoma brucei vsg expression site switching in vitro.

Trypanosoma brucei can undergo antigenic variation by switching between distinct telomeric variant surface glycoprotein gene (vsg) expression sites (ESs) or by replacing the active vsg. DNA rearrangements have often been associated with ES switching, but it is unclear if such rearrangements are necessary or whether ES inactivation always accompanies ES activation. To explore these issues, we derived ten independent clones, from the same parent, that had undergone a similar vsg activation event. This was achieved in the absence of an immune response, in vitro, using cells with selectable markers integrated into an ES. Nine of the ten clones had undergone ES switching. Such heritable changes in transcription state occurred at a frequency of approximately 6 x 10(-7). Comparison of switched and un-switched clones highlighted the dynamic nature of T. brucei telomeres, but changes in telomere length were not specifically associated with ES switching. Mapping within and beyond the ESs revealed no detectable DNA rearrangements, indicating that rearrangements are not necessary for ES activation/inactivation. Examination of individual cells indicated that ES activation consistently accompanied inactivation of the previously active ES. In some cases, however, we found cells that appeared to have efficiently established the switched state but which subsequently, at a frequency of approximately 2 x 10(-3), generated cells expressing both pre- and post-switch vsgs. These results show that ES activation/inactivation is usually a coupled process but that cells can inherit a propensity to uncouple these events.

Cloning and transcriptional analysis of a variant surface glycoprotein gene expression site in Trypanosoma brucei.

The variant surface glycoprotein (VSG) gene expression site in Trypanosoma brucei variant 117a has been mapped to a point about 40 kb upstream from the VSG gene. Sequences upstream from the previously identified [Cully, D.F. et al. (1985) Cell 42, 173-182] expression site associated gene (ESAG-I) have been cloned and a stable 1.3 kb transcript has been localized immediately 5' to ESAG-I. This transcript is in the same orientation and approximately as abundant as the ESAG-I message. A highly conserved region, of which at least 15 copies are present in the genome, has been identified further upstream. A stable transcript corresponding to this region was not detected in variant 117a, but a 1.7 kb transcript was detected in variant 221a. In isolated nuclei, representative sequences from the 117a, expression site were transcribed unidirectionally at similar rates, and transcription was insensitive to alpha-amanitin.

Targeted disruption of expression site-associated gene-1 in bloodstream-form Trypanosoma brucei.

Each variant surface glycoprotein (Vsg) expression site (ES) in bloodstream-form Trypanosoma brucei is a polycistronic transcription unit containing several distinct expression site-associated genes (esag), in addition to a single vsg gene. esag1 genes from different ESs encode a highly polymorphic family of membrane-associated glycoproteins, whose function is unknown. In the hope of producing a phenotype that could indicate a function, we disrupted the esag1 genes in two ESs by targeted insertion of a hygromycin phosphotransferase gene. Our failure to produce an obvious phenotype prompted us to search for other esag1 transcripts. RNA from the mutant trypanosomes hybridized with an esag1-specific oligonucleotide. Cloning and sequencing of mRNA from both mutant and wild-type cells showed that several esag1 family members were expressed, each at a much lower level than the esag1 transcript from the active ES in wild-type trypanosomes. Long-range DNA mapping showed that these additional esag1 genes, some of which contained premature translation-termination codons, most probably originate from chromosomal-internal genes and pseudogenes. We have therefore been unable to determine whether esag1 is an essential gene, or what function it fulfils, or whether any competent Esag1 protein is expressed in the mutant trypanosomes.