Trypanosoma brucei brucei Research Papers (original) (raw)

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- Immunology, Kinetics, Immunology of the Gut, Innate immunity

Oligopeptidases are emerging as important pathogenic factors and therapeutic targets in trypanosome infections. We describe here the purification, cloning, and biochemical analysis of a new oligopeptidase from two pathogenic African trypanosomes. This oligopeptidase, which we have called tropolysin (encoded by the trn gene), represents an evolutionarily distant member of the M3A subfamily of metallopeptidases, ancestral to thimet oligopeptidase, neurolysin, and saccharolysin. The trn gene was present as a single copy per haploid genome, was expressed in both the mammalian and insect stages of the parasite life cycle, and encoded an 84 kDa protein. Both purified and hyperexpressed tropolysin hydrolyzed bradykinin-derived fluorogenic peptide substrates at restricted sites, with an alkaline pH optimum, and were activated by dithiothreitol and reduced glutathione and by divalent metal cations, in the order Zn 2+ > Co 2+ > Mn 2+ . Under oxidizing conditions, tropolysin reversibly formed inactive multimers. Tropolysin exhibited a preference for acidic amino acid side chains in P 4 , hydrophobic side chains in P 3 , and hydrophobic or large uncharged side chains in P 1 , P 1 ′, and P 3 ′, while the S 2 ′ site was unselective. Highly charged residues were not tolerated in P 1 ′. Tropolysin was responsible for the bulk of the kinin-degrading activity in trypanosome lysates, potently (k cat ≈119 s -1 ) inactivated the vasoactive kinins bradykinin and kallidin, and generated angiotensin(1-7) from angiotensin I. This hydrolysis both abolished the capacity of bradykinin to stimulate the bradykinin B 2 receptor and abrogated bradykinin prohypotensive properties in vivo, raising the possibility that tropolysin may play a role in the dysregulated kinin metabolism observed in the plasma of trypanosome-infected hosts.

- by Vitor Oliveira
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- Biochemistry, Africa, Enzyme Inhibitors, Phylogeny

Two subspecies of Trypanosoma brucei s.l. co-exist within the animal populations of Eastern Africa; T. b. brucei a parasite which only infects livestock and wildlife and T. b. rhodesiense a zoonotic parasite which infects domestic livestock, wildlife, and which in humans, results in the disease known as Human African Trypanosomiasis (HAT) or sleeping sickness. In order to assess the risk posed to humans from HAT it is necessary to identify animals harbouring potentially human infective parasites. The multiplex PCR method described here permits differentiation of human and non-human infective parasites T. b. rhodesiense and T. b. brucei based on the presence or absence of the SRA gene (specific for East African T. b. rhodesiense), inclusion of GPI-PLC as an internal control indicates whether sufficient genomic material is present for detection of a single copy T. brucei gene in the PCR reaction.

- by Susan Welburn
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- Microbiology, Epidemiology, Zoonoses, DNA

Anderson, SA, Carter, V., and Parsons, M. 1998.Trypanosoma brucei:Molecular cloning and stage-regulated expression of a malate dehydrogenase localized to the mitochondrion.Experimental Parasitology89,6370. African trypanosomes exhibit... more

- by Marilyn Parsons
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- Microbiology, Mitochondria, Energy Metabolism, Organelles
- by Kwabena Bosompem
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- Microbiology, Zoology, Ghana, Sheep

Here, we characterise the cells arrested after the induction of VSG RNAi. We were able to rescue the VSG221 RNAi induced cell-cycle arrest through expression of a second different VSG (VSG117 which is not recognised by the VSG221 RNAi)... more

- by Eva Gluenz and +1
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- Electron Microscopy, Cell Cycle, Multidisciplinary, RNA interference

Imidazoline lead compound with improved activity in 1 a murine model of late stage T. b. brucei infection is not cross-resistant with 2 diamidines 3 4 2 26 27 28 29 30 Abbreviations: 31 BBB, blood-brain barrier 32 BSA, bovine serum albumine 33 clogP, calculated log of octanol-water partition coefficient 34 CNS, central nervous system 35 HAPT, high affinity pentamidine transporter 36 HAT, human African trypanosomiasis 37 hCMEC/D3, human cerebral microvessel endothelial cell line 38 LAPT, low affinity pentamidine transporter 39 LY, lucifer yellow 40 HSA, human serum albumin 41 P-gp, P-glycoprotein 42 SPR, surface plasmon resonance 43 WT, wild type 44 45 46 4 ABSTRACT 47

- by Fabienne Glacial
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- Microbiology, Medical Microbiology, Mice, Trypanosoma brucei brucei

Disruption of glycosylphosphatidylinositol biosynthesis is genetically and chemically validated as a drug target against the protozoan parasite Trypanosoma brucei, the causative agent of African sleeping sickness. The N-acetylglucosamine-phosphatidylinositol de-N-acetylase (deNAc) is a zinc metalloenzyme responsible for the second step of glycosylphosphatidylinositol biosynthesis. We recently reported the synthesis of eight deoxy-2-C-branched monosaccharides containing carboxylic acid, hydroxamic acid, or N-hydroxyurea substituents at the C2 position that may act as zinc-binding groups. Here, we describe the synthesis of a glucocyclitol-phospholipid incorporating a hydroxamic acid moiety and report the biochemical evaluation of the monosaccharides and the glucocyclitol-phospholipid as inhibitors of the trypanosome deNAc in the cell-free system and against recombinant enzyme. Monosaccharides with carboxylic acid or hydroxamic acid substituents were found to be the inhibitors of the trypanosome deNAc with IC 50 values 0.1-1.5 mM, and the glucocyclitol-phospholipid was found to be a dual inhibitor of the deNAc and the a1-4-mannose transferase with an apparent IC 50 = 19 ± 0.5 lM.

- by Michael Urbaniak
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- Phospholipids, Enzyme Inhibitors, Drug Design, Zinc

A small zinc-binding group (ZBG) library of deoxy-2-C-branched-monosaccharides, for example, 1,5-anhydroglucitols, consisting of either monodentate ligand binding carboxylic acids or bidentate ligand binding hydroxamic acids, were prepared to assess the zinc affinity of the putative metalloenzyme 2-acetamido-2-deoxy-α-D-glucopyranosyl-(1→6)-phosphatidylinositol de-Nacetylase (EC 3.5.1.89) of glycosylphosphatidylinositol biosynthesis. The N-ureido thioglucoside was also synthesised and added to the ZBG library because a previous N-ureido analogue, synthesised by us, had inhibitory activity against the aforementioned de-N-acetylase, presumably via the N-ureido motif.

Mammalian telomeres form large duplex loops (t-loops) that may sequester chromosome ends by invasion of the 3¢ TTAGGG overhang into the duplex TTAGGG repeat array. Here we document t-loops in Trypanosoma brucei, a kinetoplastid protozoan with abundant telomeres due to the presence of many minichromosomes. These telomeres contained 10±20 kb duplex TTAGGG repeats and a 3¢ TTAGGG overhang. Electron microscopy of psoralen/UV crosslinked DNA revealed t-loops in enriched telomeric restriction fragments and at the ends of isolated minichromosomes. In mammals, t-loops are large (up to 25 kb), often comprising most of the telomere. Despite similar telomere lengths, trypanosome t-loops were much smaller (~1 kb), indicating that t-loop sizes are regulated. Coating of non-cross-linked minichromosomes with Escherichia coli single-strand binding protein (SSB) often revealed 3¢ overhangs at both telomeres and several cross-linked minichromosomes had t-loops at both ends. These results suggest that t-loops and their prerequisite 3¢ tails can be formed on the products of both leading and lagging strand synthesis. We conclude that t-loops are a conserved feature of eukaryotic telomeres.

- by George Cross
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- Electron Microscopy, Biological Sciences, Trypanosoma brucei brucei, Telomere
- by Michael Urbaniak
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- Enzyme Inhibitors, Biological Sciences, Enzyme, CHEMICAL SCIENCES

Dirie, M.F., Wallbanks, K.R., Aden, A.A., Bornstein, S. and Ibrahim, M.D., 1989. Camel trypanosomiasis and its vectors in Somalia. Vet. Parasitol., 32: 285-291.

- by Abdi Aden
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- Microbiology, Somalia, Veterinary, Veterinary Parasitology

The polymerase chain reaction was used to characterize the trypanosomes infecting Glossina morsitans submorsitans and G. tachinoides in the game ranch of Nazinga, Burkina Faso, situated near an agropastoral zone. Dissection of 435 tsetse flies, and PCR analysis of 166 infected flies were conducted to assess the epidemiological situation. Trypanosomes of the Nannomonas subgenus were the most abundant in the two tsetse species (80.4% and 73.7% of identified infections in G. m. submorsitans and G. tachinoides respectively). T. 6i6ax and T. brucei infection rates were comparable between the two tsetse species. Mature infection pattern identified by PCR differed from overall infections, mainly because T. simiae infections did not mature, whereas T. 6i6ax represented the predominant taxon. Parasitological and PCR results showed some discrepancies ; possibly some typical Duttonella strains could not be recognized by the sets of primers used. The technologies used in this work helped to determine the high trypanosomosis risk in this area. : S 0 0 0 1 -7 0 6 X ( 9 8 ) 0 0 0 8 0 -1 T. Lefrançois et al. / Acta Tropica 72 (1999) 65-77 66

- by G. Cuny
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- Biological Sciences, Burkina Faso, Blood, Polymerase Chain Reaction

African trypanosomes escape the host immune response through a periodical change of their surface coat made of one major type of protein, the variant surface glycoprotein. From a repertoire of a thousand variant surface glycoprotein genes available, only one is expressed at a time, and this takes place in a specialised expression site itself selected from a collection of an estimated 20±30 sites. As the specialised expression sites are long polycistronic transcription units, the variant surface glycoprotein is co-transcribed with several other genes termed expression site-associated genes. How do the trypanosomes only use a single specialised expression site at a time? Why are there two dozen specialised expression sites? What are the functions of the other genes of these transcription units? We review the currently available answers to these questions. q

- by Luc Vanhamme
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- Microbiology, Zoology, Immune response, Trypanosoma brucei

Aim of the study: To evaluate in vitro the antiprotozoal and cytotoxic activies of 80% methanol extract from 45 medicinal plants collected in Sankuru (Democratic Republic of Congo) against Trypanosoma brucei brucei, Trypanosoma cruzi and the chloroquine-sensitive Ghanaian strain of Plasmodium falciparum, and MRC-5 cell lines respectively. Material and methods: Differents extracts were obtained by maceration of each plant part used with 80% methanol for 24 h. The mixture was filtered and evaporated in vacuo to give corresponding dried extract. The activity against Trypanosoma brucei brucei and Trypanosoma cruzi were performed in 96 well tissue plates each containing 10 l aqueous plant extract dilutions (100 to 0.01 g/ml) with 10 l of the parasite suspension cultured in Hirumi medium supplemented with 10% foetal calf serum, a solution of 2% penicillin/streptomycin (2% P/S) After 4 days incubation with Almar blueâ solution, fluorescence was measured at 500 nm emission and 530 nm excitation and results expressed as percentage reduction in parasite compared to control wells. The antiplasmodial activity of was assessed in vitro against the chloroquine-sensitive Ghanaian strain of Plasmodium falciparum cultured in RPMI-1640 medium by the lactate deshydrogenase assay in the presence of plant extracts (50 to 0.01 g/ml). Cell-lines MRC-5 were cultured in MEM medium supplemented with 20 mM l-glutamine, 16.5 mM NaHCO 3 , 5% foetal calf serum and 2% P/S solution. After 4 h incubation, cell proliferation/viability was spectrophotomecally assessed at 540 nm after addition of MTT. In each assay, the IC 50 value for each sample was derived by the drug concentration-response curves. Results: The extracts from Alcornea cordifolia leaves, Momordica charantia whole plant, Omphalocarpum glomerata, root bark and Piptadia africanum stem bark showed good antiprotozoal activity against Trypanosoma brucei brucei with IC 50 values from 0.7 to 7 g/ml. Only Piptadenia africanum extract showed a pronounced antiprotozoal activity against Trypanosoma cruzi (IC 50 = 4.0 ± 06 g/ml). The extracts from Alchornea cordifolia, Polyathia swaveleons stem bark, Sapium cornutum stem bark and Triclisia giletii stem bark exhibited a pronounced antiplasmodial activity against P. falciparum Ghanaian strain with IC 50 values ranging from 0.5 to 3.0 g/ml. Piptadenia africanum extract was the most cytotoxic sample (CC 50 = 0.25 g/ml) with poor selectivity against all selected protozoa (SI < 10) while other active extracts did not show a significant cytotoxic effect against MCR-5 cell-lines with good selectivity according to the case. Conclusion: These active plant extracts are selected for extensive studies leading to the isolation of active constituents.

A series of novel 4-thiazolidinoneepyrazoline conjugates have been synthesized and tested for anti-Trypanosoma brucei activity. Screening data allowed us to identify five thiazolidinoneepyrazoline hybrids, which possess promising trypanocidal activity, with IC 50 1.2 mM. The highest active thiazolidinoneepyrazoline conjugates 3c and 6b (IC 50 values of 0.6 mM and 0.7 mM, respectively) were 6-times more potent antitrypanosomal agents than nifurtimox. In addition, these compounds, as well as 6d and 6e had selectivity index higher than 50, and were more selective than nifurtimox. SAR study included substituent variations at the pyrazoline moiety, modifications of N3 position of the thiazolidinone portion, elongation of the linker between the heterocycles, as well as rhodanineeisorhodanine isomerism. It was also shown that methyl or aryl substitution at the thiazolidinone N3-position is crucial for trypanocidal activity.

- by Roman Lesyk
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- Organic Chemistry, Cell line, Drug Design, Rats
- by Rudolf Lucas
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- Immunology, Macrophages, Mice, Host-parasite interactions

Quinols have been developed as a class of potential anti-cancer compounds. They are thought to act as double Michael acceptors, forming two covalent bonds to their target protein(s). Quinols have also been shown to have activity against the parasite Trypanosoma brucei, the causative organism of human African trypanosomiasis, but they demonstrated little selectivity over mammalian MRC5 cells in a counterscreen. In this paper, we report screening of further examples of quinols against T. brucei. We were able to derive an SAR, but the compounds demonstrated little selectivity over MRC5 cells. In an approach to increase selectivity, we attached melamine and benzamidine motifs to the quinols, because these moieties are known to be selectively concentrated in the parasite by transporter proteins. In general these transporter motif-containing analogues showed increased selectivity; however they also showed reduced levels of potency against T. brucei.

- by Irene Hallyburton
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- Organic Chemistry, Cell line, Drug Delivery Systems, Trypanosoma brucei brucei

Eight extracts from seven selected Cameroonian medicinal plants, traditionally used to treat malaria and other protozoal diseases, were tested in vitro for their antiprotozoal activities against Plasmodium falciparum K1 chloroquine-resistant strain, Leishmania donovani, Trypanosoma cruzi and Trypanosoma brucei rhodesiense, protozoa responsible for malaria, visceral leishmaniasis, Chagas disease and African trypanosomiasis, respectively. The most active extract against Plasmodium falciparum K1 strain and Trypanosoma brucei rhodesiense was the methanolic extract of Albizia zygia (Fabaceae) stem bark with IC 50 values of 1.0 g/ml and 0.2 g/ml, respectively. Five extracts showed IC 50 values below 5 g/ml against Leishmania donovani, with the methanolic seed extract of Harungana madagascarensis showing the highest activity, but only the methanolic extract of Albizia zygia showed activity against Trypanosoma cruzi. Cytotoxicity and selectivity indexes were estimated for the most active extracts. The best ratio of cytotoxicity to antiplasmodial activity (SI a = 14) was established for the methanolic leaf extract of Symphonia globulifera (Clusiaceae), while the methanolic stem bark extract of Albizia zygia showed the best ratio of cytotoxicity to antitrypanosomal activity (SI b = 22.5).

- by Etienne Tsamo
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- Complementary and Alternative Medicine, Plant Biology, Cameroon, Ethnopharmacology

Drug resistance in pathogenic trypanosomes threatens successful control of fatal sleeping sickness in man and hinders economic livestock production in sub-Saharan Africa. We report on the occurrence and development of drug resistance, and discuss the genetic basis of such resistance in Trypanosoma brucei. Understanding these mechanisms at the molecular level will enable improved management of existing drugs and provide valuable clues to the development of new trypanocides.

- by Enock Matovu
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- Genetics, Microbiology, Immunology, Medical Microbiology

Here, we describe a series of readily obtainable benzophenone derivatives with antimalarial and antitrypanosomal activity. The most active compounds display submicromolar activity against Plasmodium falciparum. Micromolar activity is obtained against Trypanosoma brucei. Main problem of the compounds is low selectivity. However, there are indications that separation of antimalarial and cytotoxic activity might by possible. In addition, some compounds inhibit human ABC transporter with nanomolar activity.

- by Martin Schlitzer
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- Organic Chemistry, Malaria, Drug development, Multidrug Resistance
- by Peter Kennedy
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- Immunology, Cytokines, Neuroimmunology, Brain
- by andrew sinyangwe
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- Microbiology, Geography, Drug Use, Veterinary Parasitology

High resolution Fourier transform mass spectrometry (HRFTMS) and nuclear magnetic resonance (NMR) spectroscopy were employed as complementary metabolomic tools to dereplicate the chemical profile of the new and antitrypanosomally active... more

- by Robert Brasseur and +1
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- Science, Multidisciplinary, Ion Channels, Apolipoproteins

In trypanosomes, the parasite-specific thiol trypanothione [T(SH) 2 ] fulfills various functions, the best established being detoxification of H 2 O 2 and organic hydroperoxides and ribonucleotide reduction. Recently, a trypanothione synthetase (Tb-TryS) gene from Trypanosoma brucei was isolated and the heterologously expressed Tb-TryS catalyzed the entire synthesis of T(SH) 2 from glutathione (GSH) and spermidine in vitro. To confirm the in situ function of the complex Tb-TryS activities and to evaluate the importance of T(SH) 2 metabolism in T. brucei, TryS suppression by double-stranded RNA interference was performed. Knockdown of TryS led to depletion of both T(SH) 2 and glutathionylspermidine (Gsp) and accumulation of GSH, while concomitantly impairment of viability and arrest of proliferation were observed. TryS-downregulated cells displayed a significantly increased sensitivity to H 2 O 2 and tert.butyl hydroperoxide. These data verify the hypothesis that in T. brucei, a single enzyme synthesizes the spermidineconjugated thiols (Gsp and T(SH) 2 ) and further confirms the significance of trypanothione in the defense against oxidative stress and the maintenance of viability and proliferation in unstressed parasites. D 2004 Elsevier Inc. All rights reserved.

- by Heinrich Lünsdorf and +1
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- Free Radicals, Oxidative Stress, RNA interference, PCR

We have constructed artificial linear mini-chromosomes for the parasitic protozoan Trypanosoma brucei. These chromosomes exist at ∼2 copies per cell, are indefinitely stable under selection but are lost from 50% of the transformed population in ∼7 generations when grown in the absence of selective pressure. Consistent with results obtained earlier with natural chromosomes in T.brucei, the telomeres on these artificial chromosomes grow, adding ∼1-1.5 telomeric repeats per generation. The activity of a procyclic acidic repetitive protein (parp) gene promoter on these elements is unaffected by its proximity to a telomere, implying the lack of a telomere-proximal position effect (TPE) in procyclic trypanosomes. Among other things, these autonomously replicating dispensable genetic elements will provide a defined system for the study of nuclear DNA replication, karyotypic plasticity and other aspects of chromosomal behavior in this ancient eukaryotic lineage.

- by PRADEEP PATNAIK
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- Genetics, Biological Sciences, Environmental Sciences, Nucleic Acids

Gut samples prepared from laboratory-reared tsetse flies and applied in dots onto nitrocellulose (NC) membrane were found to stain the membrane with differing coloration and intensity. The stains were, predominantly, either reddish to brown or blackish-brown to black and occasionally greenish to almost colourless, depending on the stage of digestion of the bloodmeal in the fly. NC membrane strips applied with tsetse gut samples from T. brucei infected and uninfected control flies were tested with the standard antigen detection dot enzyme-linked immunoassay (dot-ELBA), using a T. brucei specific monoclonal antibody (MoAb) and horseradish peroxidase goat anti-mouse conjugate. The stains in both infected and uninfected sample dots persisted through the assay. Furthermore, the staining intensity of some assayed uninfected sample dots were enhanced as a result of non-specific reactivity, making it difficult to distinguish between the infected and uninfected flies. This necessitated the development of a simple technique by which the non-specific stains and reactions could be removed. Sample 'dotted' NC membrane strips were destained by incubation with 5% hydrogen peroxide (H,O,) diluted in 5% skimmed milk in Tris buffer, pH 8.0. After washing, the destained strips were tested in the dot-ELISA. This method gave satisfactory reproducible results, since the most intense stains could be removed, and it had no effect on trypanosome antigens detected by a panel of four T. brucei species-specific, three T. uiuax species-specific, four T. congolense species-specific and four Nannomonas subgenus-specific MoAbs. Using the destaining process in a modified dot-ELISA, 86 out of 95 (90.5%) of Glossina morsitans centralis flies experimentally infected with T. brucei, were identified. The destaining method was also used successfully to decolorize NC membrane bound tsetse faecal material.

- by Kwabena Bosompem
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- Microbiology, Parasitology, Immunology, Medical Microbiology
- by Phelix Majiwa
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- Multidisciplinary, Nature, Recombinant DNA Technology, Nucleic acid hybridization
- by Jose Requena and +1
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- Biological Sciences, DNA, Environmental Sciences, Nucleic Acids

Four xanthones isolated from the roots of Andrographis paniculata were tested in vitro for antiprotozoal activity against Trypanosoma brucei brucei, Trypanosoma cruzi and Leishmania infantum. Compound TDR13011 (1,2-dihydroxy-6,8-dimethoxy-xanthone) showed good activity against T. b. brucei and L. infantum with a 50% inhibitory concentration (IC 50 ) of 4.6 μ μ μ μ μg/ml and 8 μ μ μ μ μg/ml respectively. Xanthones from the roots of Andrographis paniculata exhibited promising anti-protozoal activity and these compounds could be chemically modified to obtain a more potent product.

- by Virendra Dua
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- Biological Sciences, Phytotherapy, Plant Roots, CHEMICAL SCIENCES
- by David Andreu and +2
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- Biomedical Engineering, Controlled release, Mice, Microbial genetic and drug resistance

Reproductive processes within the current Ugandan epidemic of sleeping sickness are investigated. Genotype frequencies derived from isoenzyme patterns in 44 stocks of Trypanosoma brucei s.l. collected in 1988 from Tororo, southeast Uganda are analysed by single and multiple loci methods. In the single locus method, the hypothesis of random mating is tested by agreement with Hardy-Weinberg equilibrium. The multiple loci method uses a contingency table approach to detect nonrandom associations between pairs of loci; this equates to the detection of disequilibrium. The results do not support the concept of a randomly mating population of T. brucei within the current epidemic. Results from the epidemic data set are discussed in relation to the broader problem of genetic exchange in Trypanozoon. The existence of a process which allows the exchange of genetic material during the life cycle of salivarian trypanosomes was considered theoretically plausible for many years (Baker 1977) prior to its original demonstration in the laboratory (Jenni et al. 1986). Before this, Gibson et al. (1980) had suggested the existence of homozygous and heterozygous isoenzymes at two loci in field samples of trypanosomes. Tait (1980), using Hardy-Weinberg (H-W) analysis, demonstrated that trypanosomes in tsetse flies were probably diploid and could undergo random mating and recombination. The apparent ease with which salivarian trypanosomes mate in the laboratory, in flies presented with mixed infections (Jenni et aI. 1986; Sternberg et al. 1988; Gibson 1989), suggested that this would be a normal feature of trypanosome populations in the wild. However, analysis by Cibulskis (1988) showed that the apparent variation observed in natural trypanosome

- by Susan Welburn
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- Genetics, Microbiology, Parasitology, Reproduction

Trehalose, a non-permeating cryoprotective agent (CPA), has been documented as less toxic and highly efficient at cryopreserving different kinds of cells or organisms. In the present study, trehalose was evaluated for its application in cryopreservation of both Trypanosoma brucei procyclic and bloodstream form cells. The cryopreservation efficiency was determined by the motility of trypanosomes after thawing, as well as a subsequent recovery and infectivity assessment. The viability of trypanosomes from cultivation that were frozen in a serial concentrations of trehalose showed similar results to classical CPAs of glycerol and DMSO. Nevertheless, trypanosomes cryopreserved in 0.2M trehalose showed the best growth characteristic during subsequent cultivation. In addition, CPA cocktails with trehalose and permeating CPA glycerol or DMSO were developed and evaluated. Interestingly, trypanosomes in host (mouse) blood cryopreserved in 0.4M trehalose plus 5% glycerol showed higher infecti...

- by Geoff Hide
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- Cryopreservation, Biological Sciences, Mice, Trehalose

The most rapid method for the generation of conditional mutants in Trypanosoma brucei is the use of RNA interference. A single copy of the target sequence is cloned between two opposing T7 promoters bearing tet operators, and the resulting plasmid is integrated into the genome of cells expressing both the tet repressor and T7 RNA polymerase. Upon addition of tetracycline, double-stranded RNA is synthesised from the two T7 promoters. Unfortunately, repression of T7 promoter activity may sometimes be insufficient to prevent expression of toxic amounts of double-stranded RNA. We describe here cell lines in which the expression of T7 polymerase is under tetracycline control, and show that regulation of polymerase expression can modulate transcription from a constitutive T7 promoter. In addition we describe a construct containing two copies of the tn10 Tet repressor for easy creation of repressor-expressing trypanosomes, and an RNA interference vector which allows "TA" cloning of unmodified PCR products and blue/white selection.

- by Vincent Alibu
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- RNA polymerase, Biological Sciences, RNA interference, Cell line

CITATIONS 26 READS 52 2 authors: Some of the authors of this publication are also working on these related projects: Study of Yemeni medicinal plants rich volatile oils. View project Multidisciplinary approach in the search of new molecules of natural origin with antiparasitic and antitumoral activity, mainly sesquiterpene lactones View project

- by William Setzer
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- Organic Chemistry, Drug Discovery, Computer Simulation, Natural Product

In Trypanosomu brucei the enzyme glucose-6-phosphate isomerase, like most other enzymes of the glycolytic pathway, resides in a microbody-like organelle, the glycosome. Here we report a detailed study of this enzyme, involving a determination of its kinetic properties and the cloning and sequence analysis of its gene. The gene codes for a polypeptide of 606 amino acids, with a calculated M, of 67280. The protein predicted from the gene sequence has 54 -58% positional identity with its yeast and mammalian counterparts. Compared to those other glucose-6-phosphate isomerases the trypanosomal enzyme contains an additional 38 -49 amino acids in its Nterminal domain, as well as a number of small insertions and deletions, The additional amino acids are responsible for the 5-kDa-larger subunit mass of the T. brucei enzyme, as measured by gel electrophoresis. The glucose-6phosphate isomerase of the trypanosome has no excess of positive residues and, consequently, no high isoelectric point, in contrast to the other glycolytic enzymes that are present in the glycosome. However, similar to other glycosomal proteins analyzed so far, specific clusters of positive residues can be recognized in the primary structure.

- by Ulco Kooystra
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- DNA, Enzyme, Genes, European

A series of new isothiocoumarin-3-carboxylic acids derivatives had been obtained based on the 5arylidenerhodanines hydrolysis. Anticancer activity screening allowed identification of 7,8-dimethoxy-1-oxo-1H-isothiochromene-3-carboxylic acid (4-phenylthiazol-2-yl)-amide (30) with the highest level of antimitotic activity (GI 50 NCI-H322M/NSC Lung Cancer ¼ 1.28 mM). Evaluation of the antitrypanosomal activity against Trypanosoma brucei brucei showed that investigated compounds did not exhibit significant antiparasitic effects. Additionally, the most pharmacologically attractive compounds were nontoxic and well tolerated by the experimental animals.

- by Danylo Kaminskyy and +1
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- Organic Chemistry, Coumarins, Trypanosoma brucei brucei, Neoplasms

Essential oils obtained by hydrodistillation of the fruit rinds of Citrus jambhiri Lush. (Rough lemon) and C. pyriformis Hassk (Ponderosa lemon) were analyzed by capillary gas chromatography (GLC/FID) and gas chromatography-mass spectrometry (GLC/MS). A total of 94 compounds were unambiguously identified from the oils and the (hexane/ether) extracts of the rind and juices representing 98.55% and 97.98% of the total oil composition. The main component of both oils was D-limonene (92.48% and 75.56% respectively). The antioxidant, anti-inflammatory, antitrypanosomal, antimicrobial and cytotoxic activities of the essential oils were evaluated. Whereas Citrus jambhiri and C. pyriformis have antioxidant activity with IC 50 ± SD 37.69 ± 0.21 mg/ml and 28.91 ± 0.09 mg/ml, respectively, ascorbic acid a known potential inhibitor for DPPH * free radical and commonly used antioxidant has a value of 16.32 ± 0.16 g/ml. Both oils inhibited the activity of 5-lipoxygenase (5-LOX) with an IC50 of 40 ± 1.63 and 38 ± 0.82 g/ml, respectively, and could be considered as interesting candidates for antiinflammatory agents. The essential oils of both species showed substantial antimicrobial activity against all tested Gram positive bacteria and yeasts. The essential oil of C. pyriformis shows higher cytotoxic activity against tested cell lines than that of C. jambhiri. The IC 50 values were 374.36 ± 43.95 g/ml and 588.06 ± 27.12 g/ml in case of HepG2 cells and 213.87 ± 18.50 g/ml and 512.45 ± 61.46 g/ml in case of MIA-PaCa-2 cells respectively.

- by Dalia Hamdan
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- Mass Spectrometry, Antioxidants, Fruit, Distillation

We examined the induction of 4 chemokines during early experimental African trypanosomiasis using in situ hybridization and immunocytochemistry. mRNA expression and protein production of Rantes, MCP-1, MIP-1a and MIP-2 were studied in splenocytes obtained at 0 h, 4 h and 12 h post-infection. Splenic denervation was performed to study the role of the central nervous system in early infection. The mRNA for Rantes increased at 4 h and declined at 12 h, but the protein level was high at both time-points. MCP-1 and MIP-1a had elevated mRNA and protein levels at 12 h post-infection. MIP-2 mRNA was high at both 4 h and 12 h, but the protein level was only increased at 12 h. Splenic denervation, but not sham operation, suppressed these responses. The upregulation of these chemokines during very early infection suggests a chemokine role in the developing immunopathology. The sympathetic nervous system may, however, participate in modulation of such early immune responses.

The complex life cycle of Trypanosoma brucei provides an excellent model system to understand signalling pathways that regulate development. We described previously the classical functions of TOR (target of rapamycin) 1 and TOR2 in T. brucei. In a more recent study, we described a novel TOR kinase, named TOR4, which regulates differentiation from the proliferative infective form to the quiescent form. In contrast with TOR1 loss-of-function, down-regulation of TOR4 triggers an irreversible differentiation process through the development of the insect pre-adapted quiescent form. TOR4 governs a signalling pathway distinct from those controlled by the conventional TOR complexes TORC1 and TORC2. Depletion of TOR4 induces all well-known characteristics of the quiescent developmental stage in trypanosomes, including expression of the PAD (proteins associated with differentiation) surface proteins and transcriptional down-regulation of the VSG (variant surface glycoprotein) gene. TOR4 kinase forms a structurally and functionally distinct complex named TORC4. TOR4 associates with LST8 (lethal with sec-13 protein 8) and other factors including an armadillo-domain-containing protein and the major vault protein, which probably serves as a scaffold for this kinase. Research in T. brucei, a protozoan parasite that diverged from the eukaryotic tree early in evolution, may help to uncover new functions of TOR kinases.

A 24 h polysomnographic recording was performed in a patient with sleeping sickness presenting an atypical neurological syndrome. Trypanosoma gambiense was found in a lymph gland puncture and the CSF, and a serologic immunofluorescence test was positive. The scoring technique of the polygraphic traces had to be adapted because of the presence of a permanent EEG delta wave activity during the NREM sleep stages, and the method used by Schwartz and Escande (1970) was applied. REM sleep and wakefulness presented normal polygraphic characteristics. The patient had 8 sleep episodes throughout the recording period, occurring during the daytime and at night, forming the classical diurnal sleepiness and nocturnal restlessness of sleeping sickness. All but one episode represented 1-3 complete REM-NREM sleep cycles. On all occasions, REM latency was short and 2 SOREM episodes were observed. The nychthemeral organization of the stages of vigilance differed from one state to another. Wakefulness...

- by Alain Buguet
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- Electroencephalography, Circadian Rhythm, Sleep disorders, Sleep

A single copy gene, encoding a protein highly similar to transketolase from other systems, was identified in the Trypanosoma brucei genome. The gene was expressed in E. coli and the purified protein demonstrated transketolase activity with K m values of 0.2 mM and 0.8 mM respectively for xylulose 5-phosphate and ribose 5-phosphate. A peroxisomal targeting signal (PTS-1) present at the C-terminus of the protein suggested a glycosomal localisation. However, subcellular localisation experiments revealed that while the protein was present in glycosomes it was found mainly within the cytosol and thus has a dual localisation. Transketolase activity was absent from the long slender bloodstream form of the parasite and the protein was not detectable in this life cycle stage, with the RNA present only at low abundance, indicating a strong differential regulation, being present predominantly in the procyclic form. The gene was knocked out from procyclic T. brucei and transketolase activity was lost but no growth phenotype was evident in the null mutants. Metabolite profiling to compare wild type and TKT null mutants revealed substantial increases in transketolase substrate metabolites coupled to loss of sedoheptulose 7-phosphate, a principal product of the transketolase reaction.

- by Jane Hubert
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- Kinetics, Gene expression, Biological Sciences, Escherichia coli

African trypanosomiasis (AT), also known as sleeping sickness in humans and Nagana in animals, is a disease caused by the protozoan parasite Trypanosoma brucei. AT is an extremely debilitating disease in human, cattle, and wild animals, and the treatment is difficult with frequent relapses. This work shows that BALB-c mice immunized intramuscularly with a single dose (100 μg) of a plasmid DNA encoding the 5′-terminal region of the transsialidase (nTSA) gene of T. brucei brucei are able to produce IgG antibodies that bind to the bloodstream form of T. brucei-protein extract and recognize the recombinant nTSA protein, expressed in Escherichia coli. Furthermore, this DNA vaccination process was able to protect 60% of mice submitted to a challenge assay with the infective form of T. brucei brucei parasites. These results demonstrate that a DNA vaccine coding for trans-sialidase from T. brucei is potentially useful in the prophylaxis of AT.

- by Duarte Miguel Prazeres and +3
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- Microbiology, Parasitology, Medical Microbiology, Survival Analysis

Troeberg, L., Morty, R. E., Pike, R. N., Lonsdale-Eccles, J. D., Palmer, J. T., McKerrow, J. H., and Coetzer, T. H. T. 1999. Cysteine proteinase inhibitors kill cultured bloodstream forms ofTrypanosoma brucei brucei. Experimental Parasitology91, 349–355.Trypanosoma brucei bruceiis a causative agent of bovine trypanosomiasis (nagana), a disease of considerable economic significance in much of Africa. Here we report investigations on

- by Theresa Coetzer and +1
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- Microbiology, Western blotting, Experimental parasitology, Enzyme

Sterol biosynthesis inhibitors are promising entities for the treatment of trypanosomal diseases. Insect forms of Trypanosoma brucei, the causative agent of sleeping sickness, synthesize ergosterol and other 24-alkylated sterols, yet also incorporate cholesterol from the medium. While sterol function has been investigated by pharmacological manipulation of sterol biosynthesis, molecular mechanisms by which endogenous sterols influence cellular processes remain largely unknown in trypanosomes. Here we analyse by RNA interference, the effects of a perturbation of three specific steps of endogenous sterol biosynthesis in order to dissect the role of specific intermediates in proliferation, mitochondrial function and cellular morphology in procyclic cells. A decrease in the levels of squalene synthase and squalene epoxidase resulted in a depletion of cellular sterol intermediates and end products, impaired cell growth and led to aberrant morphologies, DNA fragmentation and a profound modification of mitochondrial structure and function. In contrast, cells deficient in sterol methyl transferase, the enzyme involved in 24alkylation, exhibited a normal growth phenotype in spite of a complete abolition of the synthesis and content of 24-alkyl sterols. Thus, the data provided indicates that while the depletion of squalene and post-squalene endogenous sterol metabolites results in profound cellular defects, bulk 24-alkyl sterols are not strictly required to support growth in insect forms of T. brucei in vitro.

- by carlos Rodrigues Poveda
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- Microbiology, Parasitology, Zoology, Biology

The nuclear lamina is a filamentous structure subtending the nuclear envelope and required for chromatin organization, transcriptional regulation and maintaining nuclear structure. The trypanosomatid coiled-coil NUP-1 protein is a lamina component functionally analogous to lamins, the major lamina proteins of metazoa. There is little evidence for shared ancestry, suggesting the presence of a distinct lamina system in trypanosomes. To find additional trypanosomatid lamina components we identified NUP-1 interacting proteins by affinity capture and mass-spectrometry. Multiple components of the nuclear pore complex (NPC) and a second coiled-coil protein, which we termed NUP-2, were found. NUP-2 has a punctate distribution at the nuclear periphery throughout the cell cycle and is in close proximity to NUP-1, the NPCs and telomeric chromosomal regions. RNAi-mediated silencing of NUP-2 leads to severe proliferation defects, gross alterations to nuclear structure, chromosomal organization and nuclear envelope architecture. Further, transcription is altered at telomere-proximal variant surface glycoprotein (VSG) expression sites (ESs), suggesting a role in controlling ES expression, although NUP-2 silencing does not increase VSG switching. Transcriptome analysis suggests specific alterations to Pol I-dependent transcription. NUP-1 is mislocalized in NUP-2 knockdown cells and vice versa, implying that NUP-1 and NUP-2 form a co-dependent network and identifying NUP-2 as a second trypanosomatid nuclear lamina component.

- by Miguel Navarro
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- DNA damage, Transcriptome, Biological Sciences, Environmental Sciences

A comparative evaluation of the serum biochemical parameters was carried out in groups of young pigs aged 3-5 months experimentally infected with single infection of Trypanosoma brucei, Trypanosoma congolense, and a mixed infection of the two species. All the parameters studied (alanine amino transferase, aspartate amino transferase, albumin, globulin, cholesterol and creatinine) with the exception of total protein and urea varied significantly (p < 0.05) between the infected groups and uninfected control group. Serum concentrations of alanine amino transferase, aspartate amino transferase, creatinine and globulin were increased whereas albumin and cholesterol decreased, except for the T. congolense group that had similar cholesterol levels as the control group. There was no significant variation (p > 0.05) in the parameters within the infected groups except that creatinine was elevated in the T. brucei group. Administration of diminazene aceturate by day 42 PI restored alanine amino transferase, aspartate amino transferase and albumin to normal values unlike the other parameters. It was thus concluded that trypanosome infection in pigs could lead to some significant alterations in the serum biochemical values and that this was neither influenced by individual parasite species nor their mixed infection.

- by Jude Ndy Omeje
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- Microbiology, Creatine, Veterinary Parasitology, Lipids