The use of transgenic Trypanosoma brucei to identify compounds inducing the differentiation of bloodstream forms to procyclic forms.

The expression of procyclins is the earliest known marker of differentiation of bloodstream forms of Trypanosoma brucei to procyclic forms. We have generated transgenic bloodstream and procyclic forms in which the coding region of one procyclin gene was replaced by E. coli beta-glucuronidase (GUS). GUS activity can be monitored in a simple one-step colour reaction in microtitre plates; this assay is potentially suitable for large-scale screening for compounds that influence differentiation. GUS was stage-specifically expressed in procyclic forms and its synthesis occurred in parallel with that of procyclin when bloodstream forms were triggered to differentiate by the addition of cis-aconitate. GUS could also be induced by brief treatment with the proteases trypsin, pronase or thermolysin, but not with pepsin or thrombin. Interestingly, a combination of one of the active proteases with cis-aconitate resulted in increased GUS activity relative to either trigger alone. In contrast to cis-aconitate, protease treatment resulted in considerable cell death. Experiments with the pleomorphic strain AnTat 1.1 showed that long slender bloodstream forms were rapidly killed by proteases, whereas stumpy forms were largely resistant. Stumpy forms treated with trypsin differentiated synchronously and expressed procyclin with faster kinetics than when they were triggered by cis-aconitate. As predicted by the GUS assay, differentiation was even more rapid when both inducers were used simultaneously, with all cells expressing maximal levels of procyclin within 3 h.

Contributions of the procyclin 3' untranslated region and coding region to the regulation of expression in bloodstream forms of Trypanosoma brucei.

When bloodstream forms of Trypanosoma brucei differentiate into procyclic forms they rapidly synthesise a new surface coat composed of procyclins. Procyclin genes are transcribed in bloodstream forms at approximately one-tenth of the rate in procyclic forms, but little, if any, mRNA can be detected, indicating that further down-regulation must occur post-transcriptionally. We have examined the role of the 297 bp procyclin 3' untranslated region (UTR) in regulating expression in bloodstream forms and have identified three discrete elements: a dominant, negative element between positions 101 and 173, and two positive elements. When chloramphenicol acetyl transferase (CAT) was used as the reporter gene, deletion of the negative element caused a approximately 6-fold increase in the level of steady state mRNA and > 30-fold increase in CAT activity, suggesting that both RNA stability and translation were affected. Similar results were obtained with glutamic acid/alanine-rich protein (GARP), the T. congolense analogue of procyclin, indicating that the 3' UTR acts independently of the coding region. In contrast, when trypanosomes were stably transformed with a construct in which the procyclin coding region was linked to a truncated form of the 3' UTR which lacked the negative element, they expressed high levels of mRNA, but no protein could be detected in cell lysates or culture supernatants. These results imply that the procyclin coding region exerts yet another layer of control which prevents inappropriate expression of the protein in the mammalian host.

Elements in the 3' untranslated region of procyclin mRNA regulate expression in insect forms of Trypanosoma brucei by modulating RNA stability and translation.

Procyclins are the major surface glycoproteins of insect forms of Trypanosoma brucei. We have previously shown that a conserved 16-mer in the 3' untranslated region (UTR) of procyclin transcripts functions as a positive element in procyclic-form trypanosomes. A systematic analysis of the entire 297-base 3' UTR has now revealed additional elements which are involved in posttranscriptional regulation: a positive element which requires the first 40 bases of the 3' UTR and at least one negative element between nucleotides 101 and 173 (the LII domain). Deletion of either positive element resulted in a >8-fold reduction in the amount of protein but only an approximately 2-fold decrease in the steady-state level of mRNA, suggesting that regulation also occurred at the level of translation. In contrast, deletion of LII caused a threefold increase in the steady-state levels of both the mRNA and protein. LII-16-mer double deletions also gave high levels of expression, suggesting that the 16-mer functions as an antirepressor of the negative element rather than as an independent activator. All three elements have an effect on RNA turnover. When either positive element was deleted, the half-life (t(1/2)) of the mRNA was reduced from approximately 50 min (the t(1/2) of the wild-type 3' UTR) to < 15 min, whereas removal of the LII element resulted in an increased t(1/2) of approximately 100 min. We present a model of posttranscriptional regulation in which the negative domain is counteracted by two positive elements which shield it from nucleases and/or translational repressors.

Survival of Trypanosoma brucei in the tsetse fly is enhanced by the expression of specific forms of procyclin.

African trypanosomes are not passively transmitted, but they undergo several rounds of differentiation and proliferation within their intermediate host, the tsetse fly. At each stage, the survival and successful replication of the parasites improve their chances of continuing the life cycle, but little is known about specific molecules that contribute to these processes. Procyclins are the major surface glycoproteins of the insect forms of Trypanosoma brucei. Six genes encode proteins with extensive glutamic acid-proline dipeptide repeats (EP in the single-letter amino acid code), and two genes encode proteins with an internal pentapeptide repeat (GPEET). To study the function of procyclins, we have generated mutants that have no EP genes and only one copy of GPEET. This last gene could not be replaced by EP procyclins, and could only be deleted once a second GPEET copy was introduced into another locus. The EP knockouts are morphologically indistinguishable from the parental strain, but their ability to establish a heavy infection in the insect midgut is severely compromised; this phenotype can be reversed by the reintroduction of a single, highly expressed EP gene. These results suggest that the two types of procyclin have different roles, and that the EP form, while not required in culture, is important for survival in the fly.

Mini-exon gene variation in human pathogenic Leishmania species.

We have used polymerase chain reaction to amplify the mini-exon gene repeat from 18 Leishmania strains. DNA sequence analysis of the cloned products reveals high conservation of both the exon and intron (i.e. transcribed region). In contrast, variation is evident in both the length and primary sequence of the non-transcribed spacers. Dermotropic species of the New World subgenus Leishmania possess a 0.3-kb gene that differs from the 0.25-kb gene of New World dermotropic species of the subgenus Viannia. The Old/New World viscerotropic species and Old World dermotropic species possess a 0.4-kb mini-exon gene. However, the genes from the viscerotropic and dermotropic groups may be distinguished on the basis of sequence differences in the non-transcribed spacer. Comparative analysis of the -86 to -1 region from all species has been used to measure relatedness within the genus. In general, all the observed differences correlate with the four major groups of Leishmania (New World dermotropic Leishmania, New World dermotropic Viannia, Old World dermotropic Leishmania and viscerotropic Leishmania). Two of the three repeats cloned from L. donovani show short deletions. The missing sequence is flanked by direct, 7-bp repeats suggesting that the sequences may have been deleted by homologous recombination. Such rearrangements could account for the diversity detected in the non-transcribed spacers of the mini-exon genes.

Identification of a gp63 surface glycoprotein in Leishmania tarentolae.

The promastigote stage of most if not all Leishmania species possesses an abundant surface glycoprotein of 63 kDa (gp63) that has protease activity. We show that the lizard parasite Leishmania tarentolae appears to lack the surface protease activity. L. tarentolae does, however, possess an approximately 63-kDa molecule that is antigenically cross-reactive with the L. major gp63. Additionally, the genome of L. tarentolae contains sequences that hybridise at high stringency to a L. major gp63 gene probe.

Two simple media for biochemical experimentation with cultured procyclic Trypanosoma brucei.

Two simple media are presented which are particularly well suited for biochemical experimentation with procyclic culture forms of Trypanosoma brucei. ME-83 is a simple semi-defined medium which supports active cell growth, and from which individual components can conveniently be deleted or replaced. HHP-84 is a fully defined minimal medium, which allows vigorous cell motility over extended times, but in which cell proliferation is not occurring.