Ubiquitination of plasma membrane ectophosphatase in bloodstream forms of Trypanosoma brucei.

Bloodstream forms of Trypanosoma brucei contain plasma-membrane-integral acidic ectophosphatase. Here, it is shown by N-terminal sequencing that the ectophosphatase found in ricin-binding material was modified by ubiquitin. Three different ubiquitinated species corresponding to single, double and triple ubiquitinated forms of the enzyme were identified. Immunofluorescence studies with live bloodstream-form parasites showed that the ectophosphatase was localized in the flagellar pocket-the sole site for endocytosis in trypanosomes. As ubiquitin modification of plasma membrane proteins serves as an internalization signal, it is suggested that ubiquitinated ectophosphatase is labelled for endocytosis.

Increased trypanolytic activity in sera of sleeping sickness patients after chemotherapy.

We tested sera from patients previously treated for human African trypanosomiasis, from patients infected with trypanosomes, and from individuals never diagnosed with African trypanosomiasis living in the Trypanosoma brucei gambiense sleeping sickness focus of Mbini in Equatorial Guinea for their trypanolytic activity against bloodstream forms of T. b. rhodesiense expressing a metacyclic and bloodstream variant surface glycoprotein (VSG). Nearly 80% of the sera from treated patients showed high trypanolytic activity against trypanosomes expressing a metacyclic VSG. The trypanolytic activity of part of these sera was mediated by IgM while that of the other part was antibody-independent. On the other hand, only 40% of the sera exhibited high trypanolytic activity against trypanosomes expressing a bloodstream VSG which also was almost completely abolished by heat-inactivation. In contrast, most sera from infected and negative individuals displayed only low to moderate trypanolytic activity against either trypanosomes expressing a metacyclic or a bloodstream VSG. These results suggest that trypanolytic activity of sera increases after African sleeping sickness and is directed against trypanosomes expressing metacyclic VSG.

Identification of a developmentally regulated iron superoxide dismutase of Trypanosoma brucei.

An iron superoxide dismutase (FeSOD) gene of the protozoan parasite Trypanosoma brucei has been cloned and its gene product functionally characterized. The gene encodes a protein of 198 residues which shows 80% identity with FeSODs from other trypanosomatids. Inhibitor studies with purified recombinant FeSOD expressed in Escherichia coli confirmed that the enzyme is an iron-containing SOD. The FeSOD is developmentally regulated in the parasite, expression being lowest in the cell-cycle-arrested, short stumpy bloodstream forms. Differential expression of the FeSOD protein contrasts with only minor quantitative changes in the FeSOD mRNA, indicating post-transcriptional regulation of the enzyme. As the level of FeSOD increases during differentiation of cell-cycle-arrested short stumpy into dividing procyclic forms, it is suggested that the enzyme is only required in proliferating stages of the parasite for the elimination of superoxide radicals which are released during the generation of the iron-tyrosyl free-radical centre in the small subunit of ribonucleotide reductase.

Trypanosoma evansi: demonstration of a transferrin receptor derived from expression site-associated genes 6 and 7.

In Trypanosoma brucei, uptake of host transferrin is mediated by a heterodimeric, glycosylphosphatidylinositol-anchored receptor derived from the 2 expression site-associated genes 6 and 7 (ESAG6 and ESAG7). By using specific antibodies, it is shown here that T. evansi, a trypanosome species transmitted mechanically by biting flies, also expresses a transferrin receptor composed of ESAG6 and ESAG7. The cellular uptake of transferrin in T. evansi is completely inhibited with anti-T. brucei (ESAG6/7 heterodimer) antibodies. The demonstration of a functional ESAG6/7 transferrin receptor in T. evansi supports further its close relationship to T. brucei.

Active site mapping, biochemical properties and subcellular localization of rhodesain, the major cysteine protease of Trypanosoma brucei rhodesiense.

Cysteine protease activity of African trypanosome parasites is a target for new chemotherapy using synthetic protease inhibitors. To support this effort and further characterize the enzyme, we expressed and purified rhodesain, the target protease of Trypanosoma brucei rhodesiense (MVAT4 strain), in reagent quantities from Pichia pastoris. Rhodesain was secreted as an active, mature protease. Site-directed mutagenesis of a cryptic glycosylation motif not previously identified allowed production of rhodesain suitable for crystallization. An invariable ER(A/V)FNAA motif in the pro-peptide sequence of rhodesain was identified as being unique to the genus Trypanosoma. Antibodies to rhodesain localized the protease in the lysosome and identified a 40-kDa protein in long slender forms of T. b. rhodesiense and all life-cycle stages of T. b. brucei. With the latter parasite, protease expression was five times greater in short stumpy trypanosomes than in the other stages. Radiolabeled active site-directed inhibitors identified brucipain as the major cysteine protease in T. b. brucei. Peptidomimetic vinyl sulfone and epoxide inhibitors designed to interact with the S2, S1 and S' subsites of the active site cleft revealed differences between rhodesain and the related trypanosome protease cruzain. Using fluorogenic dipeptidyl substrates, rhodesain and cruzain had acid pH optima, but unlike some mammalian cathepsins retained significant activity and stability up to pH 8.0, consistent with a possible extracellular function. S2 subsite mapping of rhodesain and cruzain with fluorogenic peptidyl substrates demonstrates that the presence of alanine rather than glutamate at S2 prevents rhodesain from cleaving substrates in which P2 is arginine.

In vitro effect of alkaloids on bloodstream forms of Trypanosoma brucei and T. congolense.

The effect of 34 alkaloids of the piperidine, pyridine, tropane, isoquinoline, indole, quinolizidine, quinoline, purine, and steroidal types on the growth of Trypanosoma brucei, T. congolense, and human HL-60 cells was investigated in vitro. Berbamine, berberine, cinchonidine, cinchonine, emetine, ergotamine, quinidine, quinine, and sanguinarine showed trypanocidal activities with ED(50) (50% effective dose) values below 10 microM. Berberine, emetine, and quinidine were the most active compounds found; their ED(50) values and minimum inhibitory concentrations were comparable to those of the antitrypanosomal drugs suramin and diminazene aceturate. However, most of these compounds were also cytotoxic. In the case of emetine, the ratio of cytotoxic/trypanocidal activity was only 3 while for quinidine it was 300 indicating that this alkaloid could be a candidate for further drug development. DNA intercalation in combination with protein biosynthesis inhibition, which is the major mode of action of the active alkaloids, could be responsible for the observed trypanocidal and cytotoxic effects.

Trypanosoma brucei CTP synthetase: a target for the treatment of African sleeping sickness.

The drugs in clinical use against African sleeping sickness are toxic, costly, or inefficient. We show that Trypanosoma brucei, which causes this disease, has very low levels of CTP, which are due to a limited capacity for de novo synthesis and the lack of salvage pathways. The CTP synthetase inhibitors 6-diazo-5-oxo-l-norleucine (DON) and alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin) reduced the parasite CTP levels even further and inhibited trypanosome proliferation in vitro and in T. brucei-infected mice. In mammalian cells, DON mainly inhibits de novo purine biosynthesis, a pathway lacking in trypanosomes. We could rescue DON-treated human and mouse fibroblasts by the addition of the purine base hypoxanthine to the growth medium. For treatment of sleeping sickness, we propose the use of CTP synthetase inhibitors alone or in combination with appropriate nucleosides or bases.

In vitro effect of essential oils and isolated mono- and sesquiterpenes on Leishmania major and Trypanosoma brucei.

The effect of different essential oils as well as of isolated mono- and sesquiterpenes on the viability of bloodstream forms of Trypanosoma brucei, promastigotes of Leishmania major and human HL-60 cells was evaluated using the Almar Blue assay. Of the 12 essential oils and 8 terpenes investigated, only three essential oils, Melissa officinalis (balmmint) oil, Thymus vulgaris (thyme) oil, and Melaleuca alternifolia (tea tree) oil were about 50-fold and 80-fold more toxic to bloodstream forms of T. brucei than to HL-60 cells, respectively. Terpinen-4-ol, the main compound of the Australian tea tree oil, was even 1000-fold more toxic to trypanosomes than to the human cells. On the other hand, none of the essential oils and terpenes tested were more toxic to promastigotes of L. major than to HL-60 cells.

Ribonucleotide reductase is regulated via the R2 subunit during the life cycle of Trypanosoma brucei.

We have examined the occurrence of the R1 and R2 subunits of ribonucleotide reductase during the life cycle of Trypanosoma brucei. Whereas the R1 protein is present throughout the life cycle, the R2 protein is not found in cell cycle-arrested short stumpy trypanosomes. RT-PCR/hybridization analysis revealed almost equal amounts of the R1 and R2 mRNAs in all life cycle stages of the parasite. The data indicate that ribonucleotide reductase of African trypanosomes is developmentally controlled by post-transcriptional regulation of the R2 subunit.

Studies on the recycling of the transferrin receptor in Trypanosoma brucei using an inducible gene expression system.

Uptake of host transferrin in bloodstream forms of Trypanosoma brucei is mediated by a heterodimeric, glycosylphosphatidylinositol-anchored receptor. After endocytosis, transferrin is delivered to lysosomes where it is proteolytically degraded. Whether the heterodimeric transferrin receptor is returned to mediate several cycles in ligand uptake is undefined. By using an inducible gene expression system we provide evidence for recycling of the transferrin receptor in bloodstream forms of T. brucei. The metabolic half-life of the transferrin receptor in bloodstream-form trypanosomes is determined to be 7 h which is comparable to the half-lives of recycling receptors in mammalian cells. The cycling time of the trypanosomal transferrin receptor is calculated to be 11 min. By means of the half-life and the cycling time, we calculated that each receptor is recycled 60 times before being degraded on average.

Identification and functional characterization of thioredoxin from Trypanosoma brucei brucei.

Trypanosomes and Leishmania, the causative agents of several tropical diseases, lack the glutathione/glutathione reductase system but have trypanothione/trypanothione reductase instead. The uniqueness of this thiol metabolism and the failure to detect thioredoxin reductases in these parasites have led to the suggestion that these protozoa lack a thioredoxin system. As presented here, this is not the case. A gene encoding thioredoxin has been cloned from Trypanosoma brucei, the causative agent of African sleeping sickness. The single copy gene, which encodes a protein of 107 amino acid residues, is expressed in all developmental stages of the parasite. The deduced protein sequence is 56% identical with a putative thioredoxin revealed by the genome project of Leishmania major. The relationship to other thioredoxins is low. T. brucei thioredoxin is unusual in having a calculated pI value of 8.5. The gene has been overexpressed in Escherichia coli. The recombinant protein is a substrate of human thioredoxin reductase with a K(m) value of 6 microM but is not reduced by trypanothione reductase. T. brucei thioredoxin catalyzes the reduction of insulin by dithioerythritol, and functions as an electron donor for T. brucei ribonucleotide reductase. The parasite protein is the first classical thioredoxin of the order Kinetoplastida characterized so far.

The transferrin receptor of Trypanosoma brucei.

Bloodstream forms of Trypanosoma brucei, the causative agent of sleeping sickness in humans, require transferrin for growth. Uptake of host transferrin is mediated by a heterodimeric glycosylphosphatidylinositol-anchored receptor. The trypanosomal transferrin receptor is homologous to the N-terminal domain of the variant surface glycoprotein (VSG) and bears no structural similarity with the human transferrin receptor. In this review, the structure, biochemical properties and function of the transferrin receptor of T. brucei are summarized and compared to the transferrin receptor of mammalian cells.

A simple colorimetric method to screen drug cytotoxicity against Leishmania using the dye Alamar Blue.

A quantitative colorimetric assay using the oxidation-reduction indicator Alamar Blue was developed to measure cytotoxicity of compounds against the protozoan parasite Leishmania. Absorbance increased linearly with the plating density of promastigotes of L. major MRHO/IR/76 vaccine strain up to at least 2.5 x 10(6) cells/ml when parasites were incubated for 72 h in the presence of 10% Alamar Blue. The 50% effective dose values of common drugs (amphotericin B, pentostam and paromomycin) obtained by this assay were in the same range as previously determined by other methods. The Alamar Blue assay permits a simple, reproducible and reliable method for screening antileishmanial drugs.

Cysteine proteinases of trypanosome parasites: novel targets for chemotherapy.

The protozoan parasites, Trypanosoma brucei and T. cruzi, that cause sleeping sickness in sub-Saharan Africa and Chagas' Disease in Latin America, respectively, exert significant morbidity and mortality in man. Combinations of toxicity and differential efficacy of current drugs provide an urgent need to develop novel, cheap and effective chemotherapies. Research over the last decade with cultured trypanosomes and mice experimentally infected with these parasites has demonstrated that trypanosome cysteine proteinases are valid targets for the rational design of new drugs. In particular, potent peptidyl and peptidomimetic inhibitors of brucipain (a.k.a. trypanopain-Tb) and cruzain (a.k.a. cruzipain), the respective cysteine proteinases of T. brucei and T. cruzi, have proved trypanocidal. Efforts are ongoing to develop more specific non-toxic inhibitors of various chemistries with improved biological half-lives and biovailability characteristics. Here, the biochemical and biological properties together with the history, current status and perceived directions on the development of specific inhibitors of trypanosome cysteine proteinases will be reviewed.

Detection of Trypanosoma brucei gambiense in sleeping sickness suspects by PCR amplification of expression-site-associated genes 6 and 7.

We have developed a sensitive and specific method to identify Trypanosoma brucei ssp. using PCR to amplify conserved expression-site-associated gene 6 and 7 DNA target sequences. Amplification of 10% of the DNA in a single trypanosome produced sufficient PCR product to be visible as a band in an agarose gel stained with ethidium bromide. We analysed 59 blood samples of serologically positive cases of sleeping sickness by PCR, and directed parasitological examination of tissue fluids. The PCR test detected 87% of the parasitologically positive cases, with a specificity of 97%. In 5 cases, the parasite was demonstrated by the PCR test 4-6 months prior to parasitological detection. This result shows the potential of the assay in early diagnosis of actual T. b. gambiense infections in apparently aparasitaemic sleeping sickness patients.

Iron-dependent regulation of transferrin receptor expression in Trypanosoma brucei.

Transferrin is an essential growth factor for African trypanosomes. Here we show that expression of the trypanosomal transferrin receptor, which bears no structural similarity with mammalian transferrin receptors, is regulated by iron availability. Iron depletion of bloodstream forms of Trypanosoma brucei with the iron chelator deferoxamine resulted in a 3-fold up-regulation of the transferrin receptor and a 3-fold increase of the transferrin uptake rate. The abundance of expression site associated gene product 6 (ESAG6) mRNA, which encodes one of the two subunits of the trypanosome transferrin receptor, is regulated 5-fold by a post-transcriptional mechanism. In mammalian cells the stability of transferrin receptor mRNA is controlled by iron regulatory proteins (IRPs) binding to iron-responsive elements (IREs) in the 3'-untranslated region (UTR). Therefore, the role of a T. brucei cytoplasmic aconitase (TbACO) that is highly related to mammalian IRP-1 was investigated. Iron regulation of the transferrin receptor was found to be unaffected in Deltaaco::NEO/Deltaaco::HYG null mutants generated by targeted disruption of the TbACO gene. Thus, the mechanism of post-transcriptional transferrin receptor regulation in trypanosomes appears to be distinct from the IRE/IRP paradigm. The transferrin uptake rate was also increased when trypanosomes were transferred from medium supplemented with foetal bovine serum to medium supplemented with sera from other vertebrates. Due to varying binding affinities of the trypanosomal transferrin receptor for transferrins of different species, serum change can result in iron starvation. Thus, regulation of transferrin receptor expression may be a fast compensatory mechanism upon transmission of the parasite to a new host species.

Transcription of 'inactive' expression sites in African trypanosomes leads to expression of multiple transferrin receptor RNAs in bloodstream forms.

African trypanosomes express a heterodimeric transferrin receptor that mediates iron uptake from the host bloodstream. The genes encoding the receptor, ESAG6 and ESAG7, are found at the beginning of VSG expression sites: these are telomeric, polycistronic transcription units that each terminate with a gene encoding a trypanosome variant surface glycoprotein, VSG. Approximately 20 of these VSG expression sites are found in the trypanosome genome, but only one VSG is expressed at a time. The conventional view is that one expression site promoter is extremely active whereas the others are either inactive or show very low, poorly processive activity, and that all transferrin receptor molecules are encoded by the active expression site. The 3'-end of the ESAG6 gene is more than 5 kb from the promoter. We show here that 20% of ESAG6 mRNA originates from the 'inactive' expression sites. We suggest that many expression site promoters in trypanosomes show low-level activity throughout the life cycle, and that transcription proceeds for at least 5 kb. This suggests a simplified model of VSG expression site control, whereby the only regulated event is the strong activation of a single expression site promoter in bloodstream forms.

Trypanosoma brucei: killing of bloodstream forms in vitro and in vivo by the cysteine proteinase inhibitor Z-phe-ala-CHN2.

Cysteine proteinases were tested for their suitability as targets for chemotherapy of sleeping sickness using the peptidyl inhibitor Z-Phe-Ala-diazomethyl ketone (Z-Phe-Ala-CHN2). In vitro, the inhibitory concentration of Z-Phe-Ala-CHN;2 required to reduce the growth rate by 50% was 400 times lower for culture-adapted bloodstream forms of Trypanosoma brucei than for a mouse myeloma cell line. At an inhibitor concentration of 10;M the parasites were lysed within 48 h of incubation. Parasitemia of mice infected with T. brucei decreased to undetectable levels for 3 days following treatment with 250 mg/kg Z-Phe-Ala-CHN2 on days 3 to 6 after infection. Although parasitemia returned thereafter to control levels, infected mice treated with the inhibitor survived approximately twice as long as those treated with placebo. Z-Phe-Ala-CHN2 inhibited proteinolysis in lysosomes in vitro and almost completely blocked cysteine proteinase activity in vivo. The results demonstrate the importance of cysteine proteinase activity for survival of T. brucei and suggest that such activity is an appropriate target for antitrypanosomal chemotherapy.