The identification of circular extrachromosomal DNA in the nuclear genome of Trypanosoma brucei.

Nuclear extrachromosomal DNA elements have been identified in several kinetoplastids such as Leishmania and Trypanosoma cruzi, but never in Trypanosoma brucei. They can occur naturally or arise spontaneously as the result of sublethal drug exposure of parasites. In most cases, they are represented as circular elements and are mitotically unstable. In this study we describe the presence of circular DNA in the nucleus of Trypanosoma brucei. This novel type of DNA was termed NR-element (NlaIII repeat element). In contrast to drug-induced episomes in other kinetoplastids, the T. brucei extrachromosomal NR-element is not generated by drug selection. Furthermore, the element is stable during mitosis over many generations. Restriction analysis of tagged NR-element DNA, unusual migration patterns during pulsed field gel electrophoresis (PFGE) and CsCl/ethidium bromide equilibrium centrifugation demonstrates that the NR-element represents circular DNA. Whereas it has been found in all field isolates of the parasites we analysed, it is not detectable in some laboratory strains notably the genome reference strain 927. The DNA sequence of this element is related to a 29 bp repeat present in the subtelomeric region of VSG-bearing chromosomes of T. brucei. It has been suggested that this subtelomeric region is part of a transition zone on chromosomes separating the relatively stable telomeric repeats from the recombinationaly active region downstream of VSG genes. Therefore, we discuss a functional connection between the occurrence of this circular DNA and subtelomeric recombination events in T. brucei.

Innate and acquired resistance to African trypanosomiasis.

The review discusses the current field status of human and bovine trypanosomiases, and focuses on the molecular basis of innate and acquired control of African trypanosomes in people, cattle, and Cape buffalo.

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.

Programmed cell death in procyclic form Trypanosoma brucei rhodesiense --identification of differentially expressed genes during con A induced death.

Trypanosoma brucei rhodesiense can be induced to undergo apoptosis after stimulation with Con A. As cell death in these parasites is associated with de novo gene expression we have applied a differential display technique, Randomly Amplified Differential Expressed Sequence-Polymerase Chain Reaction (RADES-PCR) to the study of gene expression during Con A induced cell death in these organisms. Twenty-two differentially displayed products have been cloned and sequenced. These represent the first endogenous genes to be identified as implicated in cellular death in trypanosomatids (the most primitive eukaryote in which apoptosis has been described). Evidence for an ancestral death machinery, 'proto-apoptosis' in single celled organisms is discussed.

Expression, purification, crystallization and preliminary X-ray analysis of cyclophilin A from the bovine parasite Trypanosoma brucei brucei.

Cyclophilin A from the bovine parasite Trypanosoma brucei brucei has been cloned, expressed in Escherichia coli, purified and crystallized in the presence of cyclosporin A using ammonium sulfate as a precipitant. The crystals belong to the orthorhombic crystal system with unit-cell dimensions of a = 118.61, b = 210.15 and c = 153.21 A. A data set complete to 2.7 A has been collected using rotating-anode radiation, however the crystals diffract to at least 2.1 A resolution using synchrotron radiation.

Prohibitin and RACK homologues are up-regulated in trypanosomes induced to undergo apoptosis and in naturally occurring terminally differentiated forms.

Two genes have been identified as up-regulated late during ConA-induced apoptosis in procyclic form Trypanosoma brucei rhodesiense. The first represents a homologue of prohibitin, a proto-oncogene originally described in mammals and subsequently in yeast, which is involved in cell-cycle control and senescence. The Trypanosoma prohibitin homologue appears to contain within it a putative death domain. The second gene, homologous to a family of regulatory proteins which are receptors for activated protein kinase C (RACKs), is also shown to be up-regulated in terminally differentiated bloodstream form trypanosomes. These are the first endogenous genes to be identified as up-regulated in programmed cell death (PCD) in unicellular organisms.

Programmed cell death in procyclic Trypanosoma brucei rhodesiense is associated with differential expression of mRNAs.

Procyclic Trypanosoma brucei rhodesiense have a cell death mechanism which can be activated by an external signal, the lectin ConA, in vitro. ConA has been shown to cause profound changes in cellular morphology and induce fragmentation of nuclear DNA in T.b. rhodesiense which are characteristic of apoptosis, a form of programmed cell death (PCD) in other eukaryotic cells. RNA analysis of trypanosomes induced to undergo PCD revealed that RNA remains intact up to 48 h into the process, a time when nuclear DNA fragmentation has already started. Using the randomly amplified differentially expressed sequences polymerase chain reaction method, ConA-induced cell death in T.b. rhodesiense is shown to be associated with differential expression of mRNAs, including up regulation of mRNAs late in the death process. The results demonstrate that trypanosomes actively participate in their own destruction through a PCD process and confirm that cell death in trypanosomes is associated with de novo gene expression.

Frequency of diminazene-resistant trypanosomes in populations of Trypanosoma congolense arising in infected animals following treatment with diminazene aceturate.

The frequency of trypanosomes resistant to diminazene aceturate at a dose of 25 mg/kg of body weight was investigated for populations of Trypanosoma congolense IL 3274 which reappeared in infected mice after intraperitoneal treatment with diminazene aceturate at the same dosage. At inoculum sizes of 10(2), 10(3), 10(4), 10(5), and 10(6) trypanosomes per mouse, the relapse populations were used to initiate infections in five groups of 100 mice each by the intravenous route. Immediately after infection, 50 mice in each group were treated intraperitoneally with diminazene aceturate at the aforementioned dosage; the other 50 mice functioned as untreated controls. Thereafter, all animals were monitored for 100 days for the presence of trypanosomes. In each group, trypanosomes were detected in 50 of 50 control mice, indicating 100% infectivity for all five inoculum sizes. In contrast, in the groups of 50 mice infected with 10(2), 10(3), 10(4), 10(5) and 10(6) trypanosomes and treated with diminazene aceturate, trypanosomes were detected in 4, 11, 13, 28, and 39 of 50 mice, respectively. By logistic regression, a good fit was found between the number of mice identified as parasitemic and the inoculum sizes. Maximum likelihood estimates for the proportions of trypanosomes resistant to diminazene aceturate at 25 mg/kg of body weight for the inoculum of 10(2), 10(3), 10(4), 10(5), and 10(6) organisms were 8.335 x 10(-4), 2.485 x 10(-4), 3.02 x 10(-5), 8.3 x 10(-6), and 1.6 x 10(-6), respectively. These finding indicate that the majority of the relapse trypanosomes were susceptible the the drug dosage used for selecting the population and that, surprisingly, the calculated proportion of organisms which survived drug exposure varied inversely with the inoculum size. Further experiments with mice indicated that the inverse relationship did not result from alterations in the pharmacokinetics of the drug with different inoculum sizes. The data therefore suggest that parasite inoculum size and drug dosage are important factors in estimating the apparent frequency of diminazene-resistant trypanosomes in populations of T. congolense occurring in vivo.

Apparent rarity of diminazene-resistant trypanosomes in goats infected with a diminazene-resistant population of Trypanosoma congolense.

Experiments were carried out in goats to determine the frequency with which diminazene-resistant trypanosomes occur in parasite populations before and after the intramuscular treatment of the goats with diminazene aceturate. Trypanosoma congolense IL 3274, a diminazene-resistant clone, was used to initiate infections in three groups of five goats. The goats in the first group were treated with diminazene aceturate at a dose of 7.0 mg kg-1 bodyweight within 10 seconds of infection; one of the goats was cured. All of the second group, which received no treatment, became parasitaemic. The third group of goats received the same dose of drug as the first group but three days after all of them were first detected parasitaemic; trypanosomes reappeared in all the five goats. When this third group was treated, the frequency of trypanosomes resistant to the drug dosage was estimated to be less than 1 in 10(3). The parasites which reappeared after the treatment of these animals were used to infect two additional groups of five goats intravenously. The goats in one group were treated with the same dose of drug as before, within 10 seconds of infection and were all cured. In contrast, the five goats in the second, untreated, group became parasitaemic. Finally, when the goats in which the infections had relapsed were retreated with diminazene aceturate at the same dose rate, the level of parasitaemia temporarily decreased by at least 10(3) trypanosomes ml-1. These findings suggest that diminazene-resistant T congolense occur at low levels in trypanosome populations despite attempts to select for a population resistant to the dose of drug used.

Three genes and two isozymes: gene conversion and the compartmentalization and expression of the phosphoglycerate kinases of Trypanosoma (Nannomonas) congolense.

The glycosome, a microbody organelle found only in kinetoplastid protozoa, compartmentalizes the first six enzymes of glycolysis. In order to better understand the regulation and targeting of glycolytic enzymes in trypanosomes, we have cloned and analyzed the three genes of the phosphoglycerate kinase (PGK) complex of Trypanosoma (Nannomonas) congolense. The organization of the genes within the complex is similar to that of Trypanosoma brucei brucei. The nucleotide and amino-acid sequences, including those of the novel high-molecular-weight 56PGK, show substantial cross-species similarity. However, the two downstream genes, c1PGK and c2PGK, encode identical isozymes in T. congolense, while they encode distinct glycosomal and cytoplasmic isozymes in T. brucei. Western analysis also indicated that there are only two isozymes in T. congolense and that these are constitutively expressed. Differential digitonin solubilization of the trypanosomes indicated that 56PGK is primarily localized to the glycosome, as expected, and that c1/c2PGK is cytoplasmic. Northern analysis demonstrates that while 56PGK is constitutively expressed, c1PGK and c2PGK mRNAs are differentially expressed in the T. congolense developmental stages. This work demonstrates that T. congolense has only one PGK isozyme, 56PGK, that is predominantly localized in glycosomes.

Response of diminazene-resistant and diminazene-susceptible Trypanosoma congolense to treatment with diminazene when occurring as a mixed infection in goats.

A study was carried out to determine whether a drug-resistant trypanosome population could influence the survival of a drug-sensitive population in mixed infections in goats. To identify both populations during the course of a mixed infection, a system for distinguishing them was developed; using a nucleotide sequence of a cDNA that was derived from Trypanosoma congolense ILNat 3.3 (IL 1616), a pair of 20-mer primers was designed which, in a PCR, amplified a 900-bp sequence from the diminazene-sensitive trypanosome, T. congolense IL 1180, but not the diminazene-resistant trypanosome, T. congolense IL 3247. The PCR technique detected 100 pg of IL 1180 DNA when mixed with 25 ng of total genomic DNA of IL 3274, as determined by gel electrophoresis and ethidium bromide-staining of the PCR products. Using the 900-bp PCR product as a 32P-labelled probe on Southern blots, the sensitivity was increased 100-fold. Three groups of five goats each were infected with IL 1180 (group A), IL 3274 (group B) or both clones simultaneously (group C), and treated with diminazene aceturate at a dose of 7.0 mg/kg body weight following detection of trypanosomes. Three other groups of three goats each were similarly infected and kept as untreated controls. All group A animals were cured, while all in group B and four animals in group C relapsed. Trypanosomes were harvested from all animals at regular intervals up to 60 days post treatment. Using the PCR techniques, IL 1180 DNA could not be detected in any post-treatment trypanosome DNA sample. It therefore appeared, on the basis of the sensitivity of the DNA detection systems used, that IL 1180 is unable to survive treatment with diminazene aceturate when mixed with IL 3274 in goats.

The use of arbitrary primers and the RADES method for the rapid identification of developmentally regulated genes in trypanosomes.

Biological processes, such as the cell-division cycle, differentiation and development, are driven by changes in gene expression. Short oligodeoxyribonucleotide primers (10-mers) of arbitrary sequence are currently used in the polymerase chain reaction (PCR) to generate genomic fingerprints (RAPDs) for the characterisation and differentiation of organisms and for mapping loci of interest. Since the products of such reactions are generally less than 1 kb in size, the use of arbitrary primers on cDNA should generate RAPDs which are characteristic of expressed genes. To assess this possibility, two model systems were employed; one in which actively dividing Trypanosoma brucei brucei bloodstream forms differentiate to non-dividing forms, and the second in which non-dividing metacyclic forms of T. congolense differentiate to actively dividing bloodstream forms. In the technique herein, mRNA from each differentiated form was reverse transcribed into cDNA which was then used as the template in the PCR. The resultant products were examined by agarose-gel electrophoresis. As few as 10(3) trypanosomes were sufficient for the generation of a RAPD print after first amplifying the total cDNA through exploitation of the fixed 3' and 5' ends of trypanosome nuclear mRNAs. Differences in RAPD patterns between the differentiated forms examined were mainly due to differences in gene expression. The technique can rapidly identify genes expressed at very low levels and which are up- or down-regulated in the different forms examined. PCR products of interest are easily purified from the agarose gels for direct cloning and complete sequence determination due to their relatively small size (0.1-1 kb).

Stage-specific differential polyadenylation of mini-exon derived RNA in African trypanosomes.

The differentiation of African trypanosomes through several distinct stages in their mammalian host and insect vector correlates with differential expression of some of the parasites' genes. In the search for genes from Trypanosoma brucei brucei involved in switching from the actively dividing long slender to the non-dividing short stumpy bloodstream forms, we have isolated cDNA clones which hybridise specifically to mRNA from short stumpy forms. All clones that were characterised contained similar sequences. Northern blot analysis showed that: (i) RNA transcripts which hybridise to these clones are barely detectable in the poly(A)+ fraction of RNA from long slender bloodstream forms and absent in procyclic culture forms, but are abundant in the poly(A)+ fraction of RNA from short stumpy forms; (ii) the RNA transcripts are abundant in the poly(A)- fraction of RNA from all life cycle stages of the trypanosomes, without significant differences and, (iii) three transcripts of 160, 280 and 400 nucleotides in size are detected in the poly(A)+ fraction of RNA, whereas only a single size-class of transcript of between 140 and 160 nucleotides is detectable in the poly(A)- fraction. Sequence analysis revealed that these clones correspond to mini-exon derived RNA containing a poly(A) tail at their 3' end. The polyadenylation of the transcript is a post-transcriptional event since sequences from genomic DNA could not be amplified in the polymerase chain reaction when mini-exon and oligo(dT) nucleotide sequences were used as primers. The differences in size of the transcripts detected can be accounted for by variations in the poly(A) tail length.

In vivo UV-cross-linking hybridization: a powerful technique for isolating RNA binding proteins. Application to trypanosome mini-exon derived RNA.

Differential gene expression in cells achieved, in part, through direct RNA-protein interactions. Methods for the identification of RNA binding proteins require cross-linking of proteins to RNA by chemicals or ultraviolet (UV) light followed by chromatography or density-gradient centrifugation (7,11,16). We have developed a simplified method for the rapid and efficient identification of potential regulatory RNA binding proteins. In this method, irradiation of cells with UV light induces cross-links between RNA and proteins in close contact (7,11). Boiling of extracts from irradiated cells in the presence of sodium dodecyl sulfate dissociates any non-specific RNA-protein interactions (11). After analysis of the cell extracts by SDS-PAGE, followed by Western blotting onto a nitrocellulose membrane and washing of the filter, we have found that only RNA molecules that are covalently bound to proteins are retained on the filter. Hybridization of this Western blot with an appropriate nucleic acid probe allows detection of bands of RNA-protein complexes. Antisera against the binding proteins are raised by immunizing mice with a region of the nitrocellulose membrane containing the bands of RNA-protein complexes. Using this approach we have found that in African trypanosomes, mini-exon derived RNA transcripts form complexes with cytoplasmic binding proteins in different life cycle stages of the parasite. Evidence for the specificity of mini-exon derived RNA-protein interactions is shown using in vitro UV-cross-linking analysis in which only in vitro generated sense (but not antisense) mini-exon derived RNA transcripts form complexes with cytoplasmic proteins.

Use of an in vivo system to determine the G418 resistance phenotype of bloodstream-form Trypanosoma brucei brucei transfectants.

We determined the level of susceptibility of Trypanosoma brucei brucei to the aminoglycoside G418 in vivo and demonstrated that it is possible to select for G418-resistant transfected T. brucei brucei bloodstream parasites in a mouse host by inoculating the drug intraperitoneally at doses between 40 and 80 mg/kg of body weight daily for 3 days. The ability to select for transfectants in vivo offers new possibilities for studies on genetic recombination in these parasites.

Derivation and characterization of a quinapyramine-resistant clone of Trypanosoma congolense.

Over a period of 208 days a quinapyramine-resistant population was derived in vivo from a quinapyramine-susceptible clone of Trypanosoma congolense: IL 1180. While the dose of quinapyramine sulfate required to cure 50% of mice infected with the parental clone was 0.23 mg/kg of body weight, the 50% curative dose for the resistant derivative, IL 1180/Stabilate 12, was greater than 9.6 mg/kg. This approximately 40-fold increase in resistance to quinapyramine was shown to be associated with an 8-fold increase in resistance to isometamidium, a 28-fold increase in resistance to homidium, and a 5.5-fold increase in resistance to diminazene. Cross-resistance to homidium and diminazene was also demonstrated in goats. Two clones derived from the drug-resistant derivative underwent cyclical development in Glossina morsitans centralis, producing mature infection rates of 39.6 and 23.9%. Thus, induction of resistance to quinapyramine in T. congolense IL 1180 was associated with cross-resistance to isometamidium, homidium, and diminazene and did not compromise the population's ability to undergo full cyclical development in tsetse flies.

DNA fingerprinting of Trypanosoma vivax isolates rapidly identifies intraspecific relationships.

Using the polymerase chain reaction and arbitrarily selected oligonucleotide primers of 10 or 11 bases, we have amplified DNA sequences from Trypanosoma vivax parasites isolated from South America and Africa. On the basis of polymorphisms in the DNA fingerprints generated by three of the primers, the parasites could be separated into two major groups, one comprising T. vivax isolates from Kenya and the second including all the other T. vivax parasites (from Colombia, The Gambia, Nigeria and Uganda). One of these three primers (ILo 525) also gave isolate-specific DNA fingerprints for the parasites tested, which will allow the use of this technique both in the species identification and discrimination of T. vivax parasites.